MICHIGAN STATE UNNERS TY LIBRAR ES WW!W"!WWII/21W!!!WIN“WW 3 1293 01027 61 5 UBRARY Michigan State niversity fl PLACE ll RETURN BOX to roman this checkout from your rooord. TO AVOID FINES Mum on or before date duo. DATE DUE DATE DUE DATE DUE RECOMBINANT NUCLEASES AS PROBES FOR DNA CONFORMATION IN VITRO AND IN VIVO by Aristides N. Economides A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1993 ABSTRACT RECOMBINANT NUCLEASES AS PROBES FOR DNA CONFORMATION IN VITRO AND IN VIVO by Aristides N. Economides Two recombinant endonucleases, 17.3 and a Lac repressor/17.3 hybrid (H144), were used to probe DNA for unusual structures. T7.3, a single-stranded DNA (ssDNA) endonuclease encoded by bacteriophage T7, has been previously used to study Holliday junctions and cruciforms, and more importantly, to demonstrate the existence of cruciforms on plasmids in viva. H144 maintains the endonucleolytic properties of T7.3, and further directs this activity to DNA fragments containing the lac operator (lacO) via the repressor domain. H144 was shown to be a unique enzymatic probe of intrinsically bent DNA and potentially other unusual conformations, which may exhibit single-strandedness. Cleavage by H144 at the physical center of intrinsic bends revealed the presence of a previously unrecognized unusual conformation at that site. The targets were located beyond the reach of lacO-bound H144 on an extended helix, and their recognition depended on the presence of lacO in cis, on their helical phasing with respect to lacO, and on the conformation of the sequence between a target and lacO. These results demonstrated that lacO-bound H144 cleaves at a target when a DNA loop forms to bring the target to the nucleolytic domain. In a related study, 17.3 was used to probe for unusual structures on the E. coli chromosome in viva. Upon induction of T73 synthesis, the host DNA was cleaved and degraded into many fragments, ranging from 250 to 4700 kilobase-pairs (kb), on pulsed-field gels, with a distinct fragment 8“) to 920 kb in size most apparent. The left end of this fragment wasmappedbetween84.6minand843minandtherightendbetween2.45minand4.5min. The release of a specific chromosomal fragment as a result of T73 synthesis strongly suggests that specific structures at these sites exist in viva. ACKNOWLEDGMENTS I would like to acknowledge Regeneron Pharmaceuticals Inc. for supporting this work through a fellowship and also by generously providing me with laboratory space, equipment and supplies. Thanks are extended to Dr. Sam Davis, Dr. James Fandl, Dr. Petros Hantzopoulos, Dr. Czeslaw Radziejewski, Dr. Lenard Schleiffer, Dr. George Yancopoulos, and especially Dr. Mark Furth and my thesis advisor, Dr. Nikos Panayotatos, for their continued interest in and support of my research, as well as the many discussions of this and other related work. General thanks are extended to the overall Regeneron community for their hospitality. Moreover, I would like to thank my Graduate committee members - Dr. Zachary Burton, Dr. Shelagh-Fergusson Miller, Dr. Arnold Revzin, and Dr. Loren Snyder - for their continued support, advice, and guidance from the very early to the final stages of this work. Finally, the administrative assistance provided by Ms. Carol 5. Smith of the Department of Biochemistry is gratefully acknowledged. Numerous individuals provided me with materials, or technical support and expertise. Thanks are extended towards Dr. Michael Bagdasarian, Dr. Walter Messer, Dr. Victor Morales, Dr. Donald Oliver, Dr. Arnold Revzin, Dr. Loren Snyder for providing me with clones of different Escherichia coli genes, Dr. Ian Bancroft for technical advice on pulsed-field gel electrophoresis, Dan Everdeen for sharing with me his technical expertise on reverse phase chromatography, Dr. David Valenzuella and the personel of the DNA Core Facility of Regeneron for preparing and sequencing plasmids and synthesizing the majority of the DNA oligomers utilized in this work, and Dr. James Labdon and Tanuja Chaudhury for mass spectrometry services. iv TABLE OF CONTENTS List of Tables List of Figures List of Abbreviations CHAPTERI LITERATURE REVIEW: UNUSUAL DNA STRUCTURES AND PROBES USED FOR THEIR DETECTION Introduction Section 1. Unusual structures A. Bent DNA a. Intrinsic bending b. Protein-induced bending B. Cruciforms C. 2 or left-handed DNA D. Intermolecular triple helices E. Homopurine—homopyrimidine (pur—pyr) sequences and hinged DNA (I-I-DNA) F. DNA looping C. Other unusual structures Section II: Probes and methods employed in in the study of unusual DNA structures A. Physical methods B. Chemical probes a. Single-strand selective probes b. Double-strand selective probes c. Shape- or structure-selective probes d. Sequence-selective probes C. Enzymes and DNA-binding proteins as probes of DNA structure D. 51 nuclease E. T73 and the LacI/T7.3 hybrid endonuclease Section III. In vivo approaches for examining unusual DNA structures a. Cruciforms b. Z-DNA c. H-DN A d. DNA looping Section N. Biological functions of unusual DNA structures a. Cruciforms b. Z-DNA c. H-DNA d. Bent DNA e. DNA looping Section V. Concluding remarks V viii xfi' ssmthhNh-I 21 B 24 883388883835 Section VI. Literature cited CHAPTER 2 CLEAVAGE OF INTRINSICALLY BENT DNA BY AN ENGINEERED REPRESSOR/ENDONUCLEASE HYBRID PROTEIN ABSTRACT INTRODUCTION MATERIALS AND METHODS Plasmids a. Vectors b. Genetic engineering of plasmids carrying intrinsically bent DNA DNA primers Induction of H144 synthesis in W3110 lach P‘lpCPl44 Purification of H144 a. Extraction b. Chromatography Characterization of H144 preparation Reactions with H144 or T73 endonuclease lambda O-protein Mapping of H144 targets RESULTS H144 cleaves the DNA at two unrelated promoters and at a gyrase contact site H144 cleaves the K-DNA bend at the physical center of bending The bending locus of the lambda origin of replication is also cleaved by H144 lO-protein inhibits cleavage at the Mri DISCUSSION H144 as a probe for structural variability in bent DNA and other sites The physical center of intrinsic bends may exhibit single-strandedness LITERATURE CITED CHAPTER 3 PREFERENTIAL CLEAVAGE OF INTRINSIC BENDS BY A RECOMBINANT‘ NUCLEASE IS MEDIATED BY DNA LOOPING BETWEEN THE BINDING SITE OF THE NUCLEASE AND IT‘S TARGET ABSTRACT INTRODUCTION MATERIALS AND METHODS Plasmids DNA primers Reactions utilizing Lac repressor protein Other materials and enzymes RESULTS Binding of H144 to the lac operator mediates cleavage at an intrinsic DNA bend Comparison with U3 Cleavage at an intrinsic bend is dependent on helical phasing V1 9 $$SE$$$$$$$ 888%33 \l O $8 8 883 gssssssse 100 103 Cleavage mediated by DNA looping is affected by the structure of the looping segment 105 Cleavage at an intrinsic bend is independent of the orientation of lacO, but still dependent on helical phasing 107 DISCUSSION 111 LacO-bound H144 accesses its targets by the formation of a DNA loop which brings the target within reach of the nucleolytic domain 111 Distribution of H144 targets along the helix 112 Comparison of H144 with a A. repressor/ staphylococcal nuclease hybrid 113 LITERATURE CITED 114 CHAPTER 4 PROBING THE E. COLI CHROMOSOME FOR UNUSUAL DNA STRUCTURES 116 ABSTRACT 117 INTRODUCTION 119 MATERIALS AND METHODS 122 Strains and Media 122 Enzymes and biochemicals 122 Induction of T73 endonuclease synthesis in E. coli W3110 recA lach F 124 Preparation of mini-lysates for conventional gel electrophoresis 124 Isolation of intact E. coli genomic DNA in agarose blocks for PFGE 124 Enzymatic reactions of DNA embedded in agarose 125 In situ hybridizations 126 Polymerase Chain Reactions (PCR) 129 Pulsed-Field Gel Electrophoresis (PFGE) 129 RESULTS 131 Fate of genomic and plasmid DNA after induction of T73 endonuclease synthesis 131 a. Analysis by conventional electrophoresis 131 b. Analysis by PFGE 134 Two distinct DNA bands are present in induced W3110 recA lach F'lpCPé9 cells 134 Mapping of Band I on the E. coli chromosome 135 The F' factor in W3110 recA lach F'lpCP69 is not fragmented during induction of T73 endonuclease synthesis 141 DISCUSSION 143 Band I is a genomic DNA fragment that is released as a result of T73 synthesis 143 Band II is a non-linear form of an 1" factor 146 LITERATURE CITED 147 CONCLUDING STATEMENT 152 vii Number LIST OF TABLES Title Alignment of H144 target sequences E. coli strains, their genotype, and source from which they were obtained DNA probes Results of hybridization of E. coli genes with Band I viii page 82 123 128 138 Number GNU-A cow 10 11 538313 16 17 18 19 LIST OF FIGURES Title Unusual DNA structures Physical characterization of H144 Restriction mapping of H144 targets in the Bolactamase promoter region High resolution mapping of the target sites in the B-lactamase promoter region Restriction mapping of H144 targets in fragments containing K-DN A Restriction mapping of a unique H144 target at the K-DNA bend High resolution mapping of targets at the K-DNA bend Preferential cleavage at Lori and at a T7 late promoter by H144 Identification of targets at the kori bend and me T7 late promoter The effect of 1.0 on cleavage at Mri Physical map of the region containing the K-DNA bend and lacO in plasmids pCPZBO, pCP232, pCP233, pCP234, pCP235, pCP236, and pCP237 Binding and cleavage by H144 Comparison of activity of H144 and T73 on K-DNA Effect of helical phasing on cleavage at the K-DNA bend Cleavage at the K—DNA bend in reactions containing both pCP230 and pCPZBZ DNA The effect of the orientation of a small native bend on cleavage at the K-DNA bend Dependence of cleavage at the K-DNA bend on helical phasing, when the orientation of lacO is inverted Induction of T73 endonuclease synthesis in E. coli W3110 recA laclq F/pCP69 CHEF of DNA samples prepared during induction of T73 synthesis in W3110 recA lacl‘l F'lpCP69. Hybridization with selected E. coli K12 genes Aligned physical] genetic map of the E. coli K12 genome PHOGE of DNA samples prepared during induction of T73 synthesis in W3110 recA lach F'lpCP69. Hybridization with an F factor probe (pi/A) ix 102 104 106 108 110 132 136 139 142 Abbreviated name ampicillin AU bP CM cellulose ddeO dsDNA E. coli ECL EDTA FIGE FPLC ¢T4 d>T7 kb kcl MOPS ODn ONPF LIST OF ABREVIATIONS Full name D[-]-a-Aminobenzyl-penicillin absorbance units base pair(s) Carboxy-methyl cellulose doubly-distilled water double-stranded DNA Escherichia coli E. coli lysis ethylenediaminetetraacetic acid field inversion gel electrophoresis fast protein liquid chromatography bacteriophage T4 bacteriophage T7 isopropyl-B-D-thiogalactopyranoside kilobase pairs bacteriophage lambda bacteriophage lambda repressor Low Gelling Temperature 4-morpholinepropanesulfonic acid optical density at wavelength "n" o-nitrophenyl-B—D-fucopyranoside PFGE PHM PHOGE PKS PMSF S. cerevisiae SDS ssDNA TFA Vx X-gal xi pulsed field gel electrophoresis prehybridization mixture pulsed homogeneous orthogonal field gel electrophoresis proteinase K solution phenyl-methyl-sulfonyl-fluoride Saccharomyces cerevisiae sodium dodecyl sulfate single-stranded DNA trifluoroacetic acid volume of solution "x" 5-bromo-4-chloro-3-indolyl- B-D-galactopyranoside CHAPTER 1 LITERATURE REVIEW: UNUSUAL DNA STRUCTURES AND PROBES USED FOR THEIR DETECTION Introduction DNA structural microheterogeneity - i.e., the existence of neighboring DNA segments with different secondary structures (Jaworski et al., 1987) - has been the subject of intense study in recent years. Investigations conducted with purified DNA have shown that small DNA regions can adopt defined configurations which deviate from the standard B-form (B-DNA). These configurations are collectively referred to as unusual DNA structures. Their formation depends upon environmental conditions such as temperature and solvent, as well as primary nucleotide composition, protein or ligand binding, and DNA topology. The simplest configurations include kinks, wedges, and intrinsic bends which are imposed solely by nucleotide composition. Kinks, bends and loops have also been found in connection with specific protein binding. More complicated configurations include left-handed forms (Z-DNA), cruciforms, hinged DNA (H-DNA), and "anisomorphic" forms, as well as other less defined non-B structures. Cruciforms, Z-DNA, H-DNA, and anisomorphic DNA have been found within defined sequences in covalently circular molecules that are under supercoil pressure, and the first three of these structures have also been shown to exist in viva (reviewed in Wells, 1988; Palecek, 1991). The physical and biochemical methods that led to their recognition and analysis have recently been reviewed (Wells, 1988; Wells et al., 1990; Yagil, 1991; Matthews, 1992; Palecek, 1991). Finally, a separate class of secondary structures that deviate from the B-form are recombination intermediates, some of which structurally resemble the four way junction of a cruciform. They have been reviewed elsewhere and are not considered here (Holliday, 1990; Lilley, 1990a; West, 1991). G 3 This chapter provides a review of DNA structural heterogeneity that focuses on the structures most relevant to the studies described in Chapters 2 through 4, as well as on the methods that have been employed in probing these structures. Section I. Unusual structures A. Bent DNA The subject of bent DNA has been reviewed (Crothers et al., 1990; Hagerman, 1990). DNA bending refers to a net (time-averaged) deflection of the helical axis from linearity (Hagerman, 1990). It may be an intrinsic property of a particular DNA sequence (intrinsic bending or sequence—dependent curvature) (Marini and Englund, 1981; Marini et al., 1982) or may result from binding of proteins or other ligands (induced bending) (Wu and Crothers, 1984; and references within). In either case, DNA bends should be distinguished from increased localized flexibility, where there is no net DNA bending in one direction. a. Intrinsic bending Intrinsically bent DNA (fig. 1a) was initially described as the structure forming at the junction of A and B-DNA helices on synthetic molecules (Selsing et al., 1978; Selsing et al., 1979). Naturally-occurring bends were subsequently found on restriction fragments which displayed abnormally slow mobilities on polyacrylamide but not agarose gels (Marini and Englund, 1981; Marini et al., 1982). However, abnormally slow migration in polyacrylamide gels alone should not be equated with DNA bending as other distortions of the helix such as single-stranded (unwound) regions may have the same effect. Rather, a combination of physical methods, such as gel filtration chromatography, differential decay of birefringence, and electron microscopy have been used to determine whether a DNA fragment contains a bend or an unrelated structural perturbation that accounts for reduced mobility (Marini et al., 1982; Hagerman, 1984; Griffith et al., 1986a; Levene et al., 1986). 5 a. Intrinsically bent DNA b. Cruciforms c. AIM B to Z-DNA d. Triplexes B junction 2 e. H-DNA f. DNA loop“ Figure 1. Unusual DNA structures: (a) intrinsically bent DNA; (b) cruciform; (c) alternating right-handed and left handed helix; ((1) intermolecular triple helix; (e) H-DNA; (f) DNA looping. Illustrations (b), (c), and (e) were adapted from Yagil (1991); (d) and (e) were adapted from Dervan (1989) and Hochschild and Ptashne (1986) respectively. Al- .‘9 J‘ J J J‘J‘M‘J) J J‘ J‘ d‘d’d’ d“ d. v.«' V. vv'vvvv. J. - J”‘.’J J J. J 4? _, ‘ I v1 Section VI. Literature cited CHAPTER 2 CLEAVAGE OF INTRINSICALLY BENT DNA BY AN ENGINEERED REPRESSOR/ENDONUCLEASE HYBRID PROTEIN ABSTRACT INTRODUCTION MATERIALS AND METHODS Plasmids a. Vectors b. Genetic engineering of plasmids carrying intrinsically bent DNA DNA primers Induction of H144 synthesis in W3110 lach F'/pCP144 Purification of H144 a. Extraction b. Chromatography Characterization of H144 preparation Reactions with H144 or T73 endonuclease lambda O-protein Mapping of H144 targets RESULTS H144 cleaves the DNA at two unrelated promoters and at a gyrase contact site H144 cleaves the K—DNA bend at the physical center of bending The bending locus of the lambda origin of replication is also cleaved by H144 AO-protein inhibits cleavage at the Aori DISCUSSION H144 as a probe for structural variability in bent DNA and other sites The physical center of intrinsic bends may exhibit single-strandedness LITERATURE CITED CHAPTER 3 PREFERENTIAL CLEAVAGE OF INTRINSIC BENDS BY A RECOMBINANT NUCLEASE IS MEDIATED BY DNA LOOPING BETWEEN THE BINDING SITE OF THE NUCLEASE AND ITS TARGET ABSTRACT INTRODUCTION MATERIALS AND METHODS Plasmids DNA primers Reactions utilizing Lac repressor protein Other materials and enzymes RESULTS Binding of H144 to the lac operator mediates cleavage at an intrinsic DNA bend Comparison with T73 Cleavage at an intrinsic bend is dependent on helical phasing V1 9 28%$%&%%%%& $88$38 3 88 8 8&3 gssasssss 100 103 Cleavage mediated by DNA looping is affected by the structure of the looping segment Cleavage at an intrinsic bend is independent of the orientation of lacO, but still dependent on helical phasing DISCUSSION lacO-bound H144 accesses its targets by the formation of a DNA loop which brings the target within reach of the nucleolytic domain Distribution of H144 targets along the helix Comparison of H144 with a l. repressor/ staphylococcal nuclease hybrid LITERATURE CITED CHAPTER4 PROBING THE E. COLI CHROMOSOME FOR UNUSUAL DNA STRUCTURES ABSTRACT INTRODUCTION MATERIALS AND METHODS Strains and Media Enzymes and biochemicals Induction of T73 endonuclease synthesis in E. coli W3110 recA lach F' Preparation of mini-lysates for conventional gel electrophoresis Isolation of intact E. coli genomic DNA in agarose blocks for PFGE Enzymatic reactions of DNA embedded in agarose In situ hybridizations Polymerase Chain Reactions (PCR) Pulsed-Field Gel Electrophoresis (PFGE) RESULTS Fate of genomic and plasmid DNA after induction of T73 endonuclease synthesis a. Analysis by conventional electrophoresis b. Analysis by PFGE Two distinct DNA bands are present in induced W3110 recA lacI‘l F‘lpCPé9 cells Mapping of Band I on the E. coli chromosome The F' factor in W3110 recA lach F'lpCP69 is not fragmented during induction of T73 endonuclease synthesis DISCUSSION Band I is a genomic DNA fragment that is released as a result of T73 synthesis Band II is a non-linear form of an F‘ factor LITERATURE CITED CONCLUDING STATEMENT vii 105 107 111 111 112 113 114 116 117 119 122 122 122 124 124 124 125 126 129 129 131 131 131 134 134 135 141 143 143 146 147 152 Number LIST OF TABLES Title Alignment of H144 target sequences E. coli strains, their genotype, and source from which they were obtained DNA probes Results of hybridization of E. coli genes with Band I viii page 82 123 128 138 Number O’NH CD“ 10 11 E33635 16 17 18 19 LIST OF FIGURES Title Unusual DNA structures Physical characterization of H144 Restriction mapping of H144 targets in the B-lactamase promoter region High resolution mapping of the target sites in the B—lactamase promoter region Restriction mapping of H144 targets in fragments containing K-DN A Restriction mapping of a unique H144 target at the K-DNA bend High resolution mapping of targets at the K—DNA bend Preferential cleavage at Kori and at a T7 late promoter by H144 Identification of targets at the Mri bend and the T7 late promoter The effect of 1.0 on cleavage at Mri Physical map of the region containing the K-DNA bend and lacO in plasmids pCPZBO, pCP232, pCPZ33, pCP234, pCP235, pCP236, and pCP237 Binding and cleavage by H144 Comparison of activity of H144 and T73 on K-DNA Effect of helical phasing on cleavage at the K-DNA bend Cleavage at the K-DNA bend in reactions containing both pCP230 and pCPZBZ DNA The effect of the orientation of a small native bend on cleavage at the K-DNA bend Dependence of cleavage at the K-DNA bend on helical phasing, when the orientation of lacO is inverted Induction of T73 endonuclease synthesis in E. coli W3110 recA lach F‘lpCPé9 CHEF of DNA samples prepared during induction of T73 synthesis in W3110 recA lach F'lpCP69. Hybridization with selected E. coli K12 genes Aligned physical/ genetic map of the E. coli K12 genome PHOGE of DNA samples prepared during induction of T73 synthesis in W3110 recA lach F'lpCP69. Hybridization with an F factor probe (pifA) ix 95 102 104 106 108 110 132 136 139 142 Abbreviated name ampicillin AU bp CM cellulose ddeO dsDNA E. coli ECL EDTA FIGE FPLC ¢T4 O17 kb ch MOPS ODn ONPF LIST OF ABREVIATIONS Full name D[-]-a.-Aminobenzyl-penicillin absorbance units base pair(s) Carboxy-methyl cellulose doubly-distilled water double-stranded DNA Escherichia coli E. coli lysis ethylenediaminetetraacetic acid field inversion gel electrophoresis fast protein liquid chromatography bacteriophage T4 bacteriophage T7 isopropyl-B-D—thiogalactopyranoside kilobase pairs bacteriophage lambda bacteriophage lambda repressor Low Gelling Temperature 4-morpholinepropanesulfonic acid optical density at wavelength "n" o-nitrophenyl-B-D—fucopyranoside PFGE PHM PHOGE PKS PMSF S. cerevisiae SDS ssDNA TFA Vx X-gal xi pulsed field gel electrophoresis prehybridization mixture pulsed homogeneous orthogonal field gel electrophoresis proteinase K solution phenyl-methyl—sulfonyl-fluoride Saccharomyces cerevisiae sodium dodecyl sulfate single-stranded DNA trifluoroacetic acid volume of solution "x" 5-bromo~4-chloro-3-indolyl- B-D—galactopyranoside CHAPTER 1 LITERATURE REVIEW: UNUSUAL DNA STRUCTURES AND PROBES USED FOR THEIR DETECTION Introduction DNA structural microheterogeneity - i.e., the existence of neighboring DNA segments with different secondary structures (Jaworski et al., 1987) - has been the subject of intense study in recent years. Investigations conducted with purified DNA have shown that small DNA regions can adopt defined configurations which deviate from the standard B-form (B-DNA). These configurations are collectively referred to as unusual DNA structures. Their formation depends upon environmental conditions such as temperature and solvent, as well as primary nucleotide composition, protein or ligand binding, and DNA topology. The simplest configurations include kinks, wedges, and intrinsic bends which are imposed solely by nucleotide composition. Kinks, bends and loops have also been found in connection with specific protein binding. More complicated configurations include left-handed forms (Z—DNA), cruciforms, hinged DNA (H-DNA), and "anisomorphic" forms, as well as other less defined non-B structures. Cruciforms, Z-DNA, H—DNA, and anisomorphic DNA have been found within defined sequences in covalently circular molecules that are under supercoil pressure, and the first three of these structures have also been shown to exist in viva (reviewed in Wells, 1988; Palecek, 1991). The physical and biochemical methods that led to their recognition and analysis have recently been reviewed (Wells, 1988; Wells et al., 1990; Yagil, 1991; Matthews, 1992; Palecek, 1991). Finally, a separate class of secondary structures that deviate from the B-form are recombination intermediates, some of which structurally resemble the four way junction of a cruciform. They have been reviewed elsewhere and are not considered here (Holliday, 1990; Lilley, 1990a; West, 1991). 0 3 This chapter provides a review of DNA structural heterogeneity that focuses on the structures most relevant to the studies described in Chapters 2 through 4, as well as on the methods that have been employed in probing these structures. Section 1. Unusual structures A. Bent DNA The subject of bent DNA has been reviewed (Crothers et al., 1990; Hagerman, 1990). DNA bending refers to a net (time-averaged) deflection of the helical axis from linearity (Hagerman, 1990). It may be an intrinsic property of a particular DNA sequence (intrinsic bending or sequence-dependent curvature) (Marini and Englund, 1981; Marini et al., 1982) or may result from binding of proteins or other ligands (induced bending) (Wu and Crothers, 1984; and references within). In either case, DNA bends should be distinguished from increased localized flexibility, where there is no net DNA bending in one direction. a. Intrimic bending Intrinsically bent DNA (fig. 1a) was initially described as the structure forming at the junction of A and B—DNA helices on synthetic molecules (Selsing et al., 1978; Selsing et al., 1979). Naturally-occurring bends were subsequently found on restriction fragments which displayed abnormally slow mobilities on polyacrylamide but not agarose gels (Marini and Englund, 1981; Marini et al., 1982). However, abnormally slow migration in polyacrylamide gels alone should not be equated with DNA bending as other distortions of the helix such as single-stranded (unwound) regions may have the same effect. Rather, a combination of physical methods, such as gel filtration chromatography, differential decay of birefringence, and electron microscopy have been used to determine whether a DNA fragment contains a bend or an unrelated structural perturbation that accounts for reduced mobility (Marini et al., 1982; Hagerman, 1984; Griffith et al., 1986a; Levene et al., 1986). A l I l 5 i l g ( I l a. Intrinsically belt DNA b. Cruciforms c. Alternating I to Z-DNA d. Triplex» B junctlol Z c. H-DNA !. DNA looping Figure 1. Unusual DNA structures: (a) intrinsically bent DNA; (b) cruciform; (c) alternating right-handed and left handed helix; (d) intermolecular triple helix; (e) H-DNA; (f) DNA looping. Illustrations (b), (c), and (e) were adapted from Yagil (1991); (d) and (e) were adapted from Dervan (1989) and Hochschild and Ptashne (1986) respectively. 6 An important contribution to the analysis of bent DNA was the development of a circular permutation assay for mapping the physical center of bending (Wu and Crothers, 1984). This and subsequent studies established that the intrinsic curvature of at least some sequences is caused by the presence of homopolymeric dA.dT (A-tract) repeats in the DNA sequence occurring with a periodicity of one helical turn ("helically phased") (Hagerman, 1985; Koo et al., 1986). Subsequently, A5 tracts were shown to bend the DNA by 17 to 21° per tract (Koo et al., 1990), and this estimate was used as a standard in calculating the bend angle of a protein- induced bend (Zinkel and Crothers, 1990). The curvature imparted by helically phased A- tracts is generally regarded as a static phenomenon, although anisotropic flexibility (i.e. flexibility of the helix in one direction) has not been totally ruled out as a mechanism of DNA bending by A-tracts (Hagerman, 1990). The relationship between sequence and intrinsic DNA bending has been studied extensively by comparing the electrophoretic mobilities, X-ray diffraction patterns, and circular dichroism spectra of synthetic molecules. However, the exact deformation of helical structure upon bending and how A-tracts give rise to curvature is still unclear. Models for DNA bending fall into two categories: wedge-type (Trifonov and Sussman, 1980; Ulanovsky and Trifonov, 1987) and junction-type (Selsing et al., 1979; Wu and Crothers, 1984). The wedge models postulate that intrinsic bends occur by the additive effect of small wedges at ApA steps. In the junction models, bends arise at the junctions of A-tracts with B—DNA, in order to maximize base stacking interactions between B and non-B (A-tract) helices. Although bending at a particular sequence may be explained better by one or the other of the two models, neither model sufficiently accounts for the physicochemical properties of all intrinsic bends (Hagerman, 1986; Koo and Crothers, 1988; Haran and Crothers, 1989). A unified model of intrinsic curvature has been proposed by K00 and Crothers (K00 and Crothers, 1988) and has recently been updated (Crothers et al., 1990). In this model, intrinsic curvature can be brought about by a variety of sequences, but helically phased A-tracts bring about the highest degree of bending. The direction of bending brought about by A-tracts is equivalent to compression of the 7 minor groove within an A-tract. Bending brought about by A-tracts is attributed to their greater inclination over neighboring segments with respect to the helix axis, as has been observed for the poly(dA)~poly(dT) homopolymer. Inclination is accentuated by cooperative effects between consecutive A-steps. The DNA sequence between consecutive A-tracts may be in the B-form or may contribute to curvature as well but to a lesser extent. In addition, a roll and a tilt component at the 5' end, and a tilt component at the 3' end of the junctions of the A-tracts with neighboring sequence, contributes to bending. Intrinsically bent DNA has been probed with chemical and enzymatic probes. Although it has been postulated that bending of the helix would result in base-pair opening, or alternatively, that bending and base-pair opening are energetically coupled (Ramstein and lavery, 1988), intrinsically bent DNA has been found recalcitrant to cleavage by nucleases that preferentially cleave single-stranded DNA (ssDNA), such as the mung bean and P1 nuclease under physiological conditions, or by $1 nuclease at acidic pH (Marini et al., 1984; Zahn and Blattner, 1985b; Kitchin et al., 1986; Caddle et al., 1990). likewise, bromoacetaldehyde, a chemical probe that preferentially modifies ssDNA did not react with bent DNA (Kitchin et al., 1986). In one instance, another chemical probe, osmium tetraoxide/ pyridine, which preferentially modifies ssDNA and distorted helices, was shown to be reactive towards an intrinsic bend on supercoiled plasmids but not on linear molecules (Palecek et al., 1988). However, since the sequences employed in these experiments were AT-rich, it is not clear whether the observed reactivity was due to intrinsic curvature or merely towards the AT-rich regions. Overall, the evidence available at present suggests that if single-stranded character is displayed by bent DNA it is probably limited to 1-2 bp so that these reagents cannot access it easily (Dodgson and Wells, 1977) and/ or it is transient in nature. b. Protein-induced bending Unlike intrinsic curvature, the helical structure of protein- or ligand-induced bends depends on the manner of interaction of the protein or ligand with the DNA. The best studied example of induced bending is that brought about by the binding of catabolite activator protein 8 (CAP) to its cognate sequence. The angle of the induced bend has been determined by comparative gel electrophoresis (Zinkel and Crothers, 1990) and more recently the overall molecular structure of a CAP-DNA complex has been deduced by X-ray crystallography (Schultz et al., 1991). The estimates of the bend angle from the two studies (100° and 90° respectively) are in good agreement. Many other proteins bend the DNA to varying degrees. They include prokaryotic repressors (Zahn and Blattner, 1985a; Lloubes et al., 1988; Kim et al., 1989; Zwieb et al., 1989; Koudelka, 1991; Perez and Espinosa, 1991; Rojo and Salas, 1991) and eukaryotic transcription factors (Kerppola and Curran, 1991; Verrijzer et al., 1991). The biological implications of induced DNA bending are discussed in Section IV. Induced DNA bending should be distinguished from DNA looping (Dunn et al., 1984; Griffith et al., 1986b), which refers to a topological linkage of two distal DNA regions (of the same molecule) by protein. Although, in both cases the structure of the helix is deformed, induced bending is localized at the region of binding, whereas looping may bring about an overall distortion of the helical segment located between the two protein binding sites. Moreover, this distinction is important as bending may facilitate the interaction necessary for looping mediated by two or more proteins bound at distal cognate sites. B. Cruciforms Cruciforms (fig. 1b) (Gierer, 1966) are stem and loop structures that are formed by inverted repeats (palindromes) on supercoiled molecules (Lilley, 1980; Panayotatos and Wells, 1981; Singleton and Wells, 1982). The single-stranded loops of cruciforms are particularly good targets for ssDNA endonucleases like 51, 17.3, and P1 (Lilley, 1980; Panayotatos and Wells, 1981; Blaho et al., 1988), and it was particularly through the use of 17.3 and S1 that native cruciforms were detected on supercoiled plasmids in vitra, and more recently, with 17.3 in viva (Panayotatos and Fontaine, 1987) (see Section III). However, the four-way junction forming at the base of cruciforms is also a target for 173 and phage T4 endonuclease V11 (de Massy et al., 1984; de Massy et al., 1987; Dickie et al., 1988). In addition, chemical single-strand selective 9 probes have been used to probe for the presence of single strands at the loops (Lilley and Hallam, 1983; Gough et al., 1986; Blaho et al., 1988; Voloshin et al., 1989; Palecek, 1991), as well as for studying the pathway for cruciform formation (Furlong et al., 1989; Voloshin et al., 1989; Bowater et al., 1991). Cruciform formation has also been monitored with restriction enzymes whose recognition sites overlap the sequence that forms the loops of the cruciform. When a palindrome adopts a cruciform configuration, restriction at the region corresponding to the single-stranded loops is inhibited (Courey and Wang, 1983; Blaho et al., 1988). DNA supercoiling provides the energy that drives extrusion of a palindrome to a cruciform. However, depending on primary sequence (Blaho et al., 1988) or physical conditions (Panyutin et al., 1984), certain palindromic sequences can also adopt non-B conformations other than a cruciform, on supercoiled plasmids. Moreover, the sequences flanking the palindrome (Lilley and Hallam, 1983; Lilley et al., 1985; Sullivan and Lilley, 1986; Furlong et al., 1989; Schaeffer et al., 1989; Wang and Sauerbier, 1989), or at the region corresponding to the single- stranded loops of the cruciform (Courey and Wang, 1988), as well as DNA modifications (Murchie and Lilley, 1989), and environmental conditions (Dayn et al., 1991) greatly affect the dynamics of cruciform extrusion. Extrusion is favored when the flanking regions are AT-rich (Furlong et al., 1989; Schaeffer et al., 1989; Wang and Sauerbier, 1989), because AT-rich sequences exhibit transient unstacking and larger amplitude openings on supercoiled molecules, as evidenced by their reactivity towards single-strand selective probes (Lilley and Hallam, 1983; Furlong et al., 1989). Based on these and other observations, melting of the region encompassing the palindrome as a single unit has been proposed as a mechanism for cruciform extrusion, and has been termed C-type extrusion (Sullivan and Lilley, 1986). Although this mechanism is the most probable for palindromes flanked by AT-rich sequences that are not disturbed by GC tracts, evidence has been obtained for an alternative pathway in which cruciform extrusion is nucleated by the formation of a single-stranded bubble near the center of the palindrome (Courey and Wang, 1988). In these experiments, replacement of an all AT loop region by a GC- 10 rich sequence of equal length, resulted in a decrease in the rate of conversion of the palindrome to the cruciform configuration by at least loo-fold. This type of extrusion has been historically termed S—type because the rate of extrusion is also dependent on salt concentration (reviewed in Palecek, 1991). Assuming that there are 10.6 bp per helical turn (Rhodes and Klug, 1980; Rhodes and Klug, 1981; Peck and Wang, 1981), the extrusion of a palindrome to the cruciform configuration results in relaxation of one supercoil per ~10.6 bp involved in the stem and loop regions of the cruciform (Lilley, 1983). However, some synthetic palindromic sequences have been shown to adopt a cruciform configuration on linear (non-supercoiled) molecules during duplex- ' in interconversion (McCampbell et al., 1989; Xodo et al., 1989; Zuo et al., 1990). C. 2 or left-handed DNA Left-handed DNA was discovered while studying artificial homopolymeric molecules by circular dichroism (Mitsui et al., 1970; Grant et al., 1972) and later by X-ray crystallography (Wang et al., 1979; Drew et al., 1980). It was later demonstrated that specific sequences can adopt a Z-DNA conformation on supercoiled plasmids under physiological conditions (Klysic et al., 1981; Singleton et al., 1982; Stirdivant et al., 1982; Peck and Wang, 1983). The conversion of a right handed helical segment to a left-handed form brings about the release of approximately two superhelical turns for every 10.6 bp of B-DNA. The discovery, structural analysis, and the biological functions of Z-DNA have been the subject of several recent reviews (Wells, 1988; Wells et al., 1990; Palecek, 1991). Although it was originally postulated that only alternating purine-pyrimidine sequences could form Z- DNA (for a review, see Wells et al., 1987), it is now clear that some sequences that conform to the above rule are not in a left-handed conformation, whereas others that diverge from it can adopt a Z-DNA configuration. For example, alternating purine-pyrimidine sequences of the (A—T)n or (T-G)n type adopt a cruciform configuration on supercoiled plasmids and not Z-DNA (Greaves et al., 1985; Haniford and Pulleyblank, 1985; Blaho et al., 1988; Dayn et al., 1991). 11 Moreover, a site for the restriction enzyme BamHl (which is not an alternating purine- pyrimidine sequence) adopts a left-handed conformation when embedded within Z-DNA helices, and cannot be cleaved by BamHl (Singleton et al., 1983; Palecek et al., 1987a). Likewise, EcaRl methylase does not modify the EcaRl site when the latter is in the Z- conformation. This last property is important, not only because it indicates that enzymes interact differently with Z-DNA, but also because it was utilized in probing for Z—DNA on supercoiled plasmids in viva (Jaworski et al., 1987). Of special interest are the junctions that form between consecutive segments of B and 2- DNA (B-Z junctions; fig. 1c). They are cleaved by S1 nuclease (Singleton et al., 1984), P1 nuclease (Blaho et al., 1988), and are also modified by single-strand selective chemical probes such as osmium tetraoxide/bipyridine, bromoacetaldyhe, and chloroacetaldehyde (see Section 11.8 and Palecek, 1991, for a review). These data suggest the presence of unpaired bases at 8-2 junctions. The exact number of unpaired bases involved in the junction is not known, although estimates of three bases per strand have been obtained by thermodynamic experiments on artificial oligomers (Doktycz et al., 1990). Naturally occurring sequences that can adopt a Z-conformation on supercoiled plasmids have been found (for examples, see Kilpatrick et al., 1984; Hayes and Dixon, 1985; Muller et al., 1987; Thomas et al., 1990; Pestov et al., 1991). D. Intermolecular triple helices Like Z-DNA, intermolecular triple helices (fig. 1d) were discovered during structural analysis of synthetic homopolymers (reviewed in Palecek, 1991). Triple helices form at polypurine-polypyrimidine duplexes by the association of a third polypyrimidine strand with the duplex. The third strand is complementary to the polypurine strand but in parallel orientation, and ”wraps around" the major groove of the duplex forming Hoogsteen base pairs with the polypurine strand (reviewed in Dervan, 1989). This reaction is favored at lower pH, due to protonation of the cytosines, which stabilizes the Hoogsteen base pairs, but recently, 12 techniques have been developed which extend the formation of intermolecular triplexes to the physiological pH and /or at more varied sequences (Griffin and Dervan, 1989; Povsic and Dervan, 1989; Home and Dervan, 1990; Beal and Dervan, 1991; Sun et al., 1991; Jayasena and Johnston, 1992). The formation of intermolecular triplexes has been probed in vitra by footprinting (Francois et al., 1988), affinity chromatography (Letai et al., 1988), and photochemical methods (Praseuth et al., 1988). Alternatively, sequence-specific strand scission catalyzed by appropriate triplex-forming oligonucleotides carrying at one or both ends an activatable nucleolytic moiety (such as EDTA, which cleaves DNA in the presence of Fe(II) plus dithiothreitol), has been used (Le Doan et al., 1987; Maser and Dervan, 1987). Oligonucleotides that associate with duplex DNA to form triple-stranded helices, are being used as chemical "nucleases" (see Section II.B). A more physiological type of triple helix forms between ssDNA and duplex DNA in the presence of stoichiometric amounts of RecA protein (reviewed in Fox, 1991). One strand of the duplex must be complementary or nearly complementary (some mismatches are tolerated) to the incoming single strand. The crystal structure of RecA protein monomer and polymer, and in a complex with ADP, has been obtained (Story and Steitz, 1992; Story et al., 1992), but the molecular structure of RecA-mediated triplexes has not been deciphered. Since the formation of this type of triplex is sequence-specific, RecA-assisted triplexes can be directed at specific sites on DNA in vitra. This property has been utilized recently in developing a technique for cleaving chromosome-size fragments at single sites of known sequence (Ferrin and Camerini- Otero, 1991). E. Homopurine-homopyrimidine (pur-pyr) sequences and hinged DNA (H-DN A) Naturally occurring pur-pyr tracts and pur-pyr direct repeats have been found in the upstream region of several eukaryotic genes and recombination hot spots, and are statistically overrepresented (see Wells et al., 1988, for a listing of sequences; Wells et al., 1990; Palecek, 13 1991, for reviews; and Kinniburgh, 1989; Pestov et al., 1991). Most of these sequences were identified while examining intergenic regions that contained 51 hypersensitive sites. Early investigations demonstrated that they retained their 81 hypersensitivity when subcloned into supercoiled plasmids (Hentschel, 1982; Mace et al., 1983; McKeon et al., 1984), or on relaxed molecules at low pH (Hentschel, 1982). Several non-B conformations such as slipped DNA (Hentschel, 1982; Mace et al., 1983; McKeon et al., 1984), or a heteronomous duplex in which the two strands have "different backbone conformations" (Evans and Efstratiadis, 1986) were proposed to account for the reactivity of H-DNA with $1 and other enzymatic or chemical probes. However, it is now clear that direct repeats of homopurine-homopyrimidine sequences or repeating copolymers of (dT-dC)n0(dA-dG)n adopt a composite unusual conformation that involves both single and triple strands, as well as a flexible kink which acts as a hinge - viz. hinged or H-DNA (fig. 1e) (Htun and Dahlberg, 1988; Johnston, 1988; and references within). The homopyrimidine strand (donor strand) of a denatured region of the repeat, forms Hoogsteen base-pairs with the homopurine strand (acceptor strand) in the part of the repeat that remains in the duplex conformation. The donor strand wraps around the major groove of the acceptor duplex parallel to the polypurine strand, forming an intramolecular triple helix that resembles the intermolecular triplexes described above. In addition, a kink forms at the boundary of the triple-helical region and the duplex region following the single strand. This kink does not display a fixed angle and thus functions as a flexible hinge between the two regions (Htun and Dahlberg, 1988). The currently proposed structure for H-DNA is supported from studies with $1 nuclease (Htun and Dahlberg, 1989; and references within), chemical probes (Htun and Dahlberg, 1989; Bernues et al., 1990; Pestov et al., 1991; and references within these; Johnston, 1988; Kinniburgh, 1989), hybridization and protection assays (Htun and Dahlberg, 1988), and supercoil relaxation experiments (Htun and Dahlberg, 1989). A pathway for the formation of H-DNA has been 14 presented along with supporting data (Htun and Dahlberg, 1989), and is in agreement with other existing models (Wells et al., 1990). Evidence was obtained for the existence of H-DNA on supercoiled plasmids in viva (Pamiewski et al., 1990), using an approach similar to that employed for Z-DNA (see Section 111). Although, the presence of H-DNA in promoter regions and recombination hot spots is suggestive of a role in transcription and recombination, there is little direct evidence for its postulated biological functions. In one instance, H-DNA interactions with ribonucleoprotein have been reported in the region upstream of the c-myc gene suggesting a regulatory role (Davis et al., 1989; Kinniburgh, 1989). As with other unusual structures, the formation of H-DNA relaxes negative supercoils and may affect biological processes in that manner. In addition to the H—DNA-forming sequences that have been found associated with eukaryotic promoters and recombination hot-spots, pur-pyr sequences capable of adopting an H-DNA conformation have been found in bacteria (Belland, 1991; Lichtenstein and Brenner, 1982), and at a eukaryotic replication origin (Caddle et al., 1990), but have not been characterized as rigorously. F. DNA looping DNA looping (fig. 1f) has been demonstrated in many prokaryotic and eukaryotic systems (reviewed in Schleif, 1988; Travers, 1989; Hochschild, 1990; Matthews, 1992). Here it is reviewed briefly, and primarily from a structural rather than a genetic perspective. DNA loops form when specific DNA-binding proteins (e.g. repressors, or transcription factors) bind at two or more distal cognate sites (e.g. operators, or enhancers) and stably contact each other, topologically linking the two regions of the DNA. DNA looping was first demonstrated in the am operon, in which stable loops form by binding of the Ara repressor (AraC) at appropriately spaced operators (araO) and AraC-AraC contacts (Dunn et al., 1984; Martin et al., 1986). The first direct demonstration of DNA looping in vitra was obtained with the phage lambda repressor (cl) bound at genetically engineered DNA molecules containing two operators (OR1 and 032) separated by an integral number of helical turns (Griffith et al., 15 1986b; Hochschild and Ptashne, 1986). Similar observations have been made with Lac repressor (lacl) bound at lac operator (Kramer et al., 1987). Other than specific protein-DNA and protein-protein interactions between proteins bound at cognate DNA sites, other requirements must be fulfilled for effective loop formation. For proteins that bind on one side of the helix, looping between cognate sites that are separated by a small distance (e.g. between five and seven helical turns for the cI/OR l-OR2 system), exhibits a strong periodic dependence on the helical arrangement of the sites with respect to each other (Hochschild, 1990). More specifically, looping is greatly favored when the sites are located on the same ”face” of the helix (are in phase) as only the formation of a smooth curve between the two sites would be necessary for looping. Instead, for sites separated by non- integral turns, twisting and/ or writhing of the helix would be required, in addition to curving, for loop formation. This is energetically unfavorable for short helical segments as the amount of twisting/writhing must be distributed over a small number of bases. As expected, the requirement for appropriate helical phasing of the cognate sites proportionally decreases with increasing distance between them, as twisting/ writhing can be distributed over a larger number of base-pairs. For example, for looping mediated by cl, the requirement for appropriate helical phasing of 0R1 and 0122 decreased after 8 helical turns and altogether disappeared after 20 helical turns (Hochschild and Ptashne, 1986). Looping between closely spaced cognate sites results in curvature of the helix in- between the sites. DNaseI preferentially cleaves within this curved segment at approximately 5 bp intervals, as expected for exposed sites on the outside of the curve (Hochschild and Ptashne, 1986; Kramer et al., 1987). Moreover, loops have been visualized directly by electron microscopy (Griffith et al., 1986b; Kramer et al., 1987), and loops as large as 800 bp have been visualized by this technique (Amouyal et al., 1989). Evidence for loops as large as 5 kilobase-pairs (5 kb) has been obtained by measuring transcription in viva (Dandanell et al., 1987). 16 Loop formation in viva has also been shown for [ad by measuring repression of transcription of a reporter gene whose promoter region contains the DNA elements necessary for loop formation (Bellamy et al., 1988). However, these experiments were primarily designed to measure the helical repeat between the two lac operators. Interestingly, the helical repeat between the two sites was found to be either 9.0 or 11.7 bp/turn, both of which differ significantly from the 10.6 bp/turn estimate obtained for DNA in solution. Structural deformations such as protein-induced DNA bending, may account for this difference. In a similar set of experiments, looping by AraC was used to determine that the helical repeat exhibited a periodicity of 11.1 bp/turn in solution (Lee and Schleif, 1989). Interestingly, in vitra experiments demonstrated that at low superhelical densities, the helical periodicity of a looped DNA segment is between 10.3 and 10.7 bp/turn, and the expected sinusoidal dependence of looping on the spacing of the operators is observed (Kramer et al., 1988). However, this dependence breaks down at high superhelical densities. Both sets of experimental observations indicate that looping as well as primary sequence and protein binding to the DNA can greatly alter local helix structure and periodicity. G. Otherunusual structures Other less characterized and perhaps less abundant unusual structures have been shown to exist. These include slipped DNA (at direct repeats), anisomorphic DNA (Wohlrab et al., 1987), right handed non-B conformations (e.g. A-DNA), stably unwound regions (Kowalski et al., 1988; Caddle et al., 1990), parallel DNA , and DNA quadruplexes (Kang et al., 1992; Smith and Feigon, 1992). Their structure and biological significance has been reviewed elsewhere (Wells, 1988; Palecek, 1991). Section II: Probes and methods employed in the study of unusual DNA structures A variety of physical, chemical, and enzymatic probes, as well as combinations of these have been used to probe for unusual structures, and have been the subject of several recent reviews (Wells, 1988; Wells et al., 1988; Crothers et al., 1990; Hagerman, 1990; Palecek, 1991). A. Physical methods A great number of physical methods have been used to decipher and probe DNA structure, its deviations from the B-form, as well as in association with proteins. Nuclear Magnetic Resonance (NMR), X—ray crystallography, circular dichroism (CD), electron microscopy (EM) and scanning tunneling microscopy (STM), and gel electrophoresis are suitable for analysis of purified DNA or protein-DNA complexes, but their usefulness becomes very limited when it comes to probing small structural perturbations within a large DNA, and much more so for direct probing in the cell. Crystallography has been employed in deciphering the structure of large DNA, DNA oligomers of defined sequence, and DNA-protein complexes. Early studies employing CD revealed that the structure of certain homopolymers was not in the B-form. These observations together with later X-ray diffraction data, not only led to the discovery of left-handed helices, but, more importantly, established that DNA can exhibit considerable structural polymorphism depending upon primary sequence (for reviews, see Amott et al., 1983; Drew et al., 1990). NMR has also provided detailed structures for small synthetic oligonucleotides (for examples, see Crothers et al., 1990; Drew et al., 1990; Hagerman, 1990; and references within). 17 18 Electron microscopy has been used extensively in the study of the bacterial chromosome (reviewed in Kellenberger, 1988; Kellenberger, 1990), euchromatin, and purified DNA by itself or complexed with proteins (reviewed in Le Cam et al., 1991). Electron microscopy is prone to artifacts, due to the reagents employed in fixation of the samples. A promising new technique that bypasses the problems associated with sample fixation, is scanning tunneling microscopy, which has been used to examine purified DNA and protein-DNA complexes without fixation, in aqueous solutions (reviewed in Lilley, 1990b). Gel electrophoresis has been employed widely in analyzing the structure of purified DNA and protein-DNA complexes. Mobility of linear dsDNA on polyacrylamide gels has been widely employed in the study of intrinsic and induced DNA bending (Crothers et al., 1990; Hagerman, 1990, for recent reviews), and of protein binding (reviewed in Ceglarek and Revzin, 1989). Two dimensional gels (Wang et al., 1983) are used to measure supercoil relaxation brought about by the formation of cruciforms (Blaho et al., 1988), Z or left-handed DNA (Wang et al., 1983; McLean et al., 1986), H-DNA (Htun and Dahlberg, 1988; and references within), anisomorphic DNA (Wohlrab et al., 1987), and stably unwound regions (Kowalski et al., 1988). B. Chemical probes The use of chemicals in probing DNA structure has been extensively reviewed by Palecek (1991). There are four major categories of chemical probes: single-strand selective, double-strand selective, shape- or structure-selective, and sequence-selective. Early studies employing chemical probes mostly failed to detect unusual DNA structures. Instead, the existence of unusual conformations in vitra and in viva was first demonstrated through the use of physical and enzymatic methods (see below). With that knowledge available, traditional chemical probes and the newly developed shape-selective chemical "nucleases" have been used with increasing success in recent years. Since most of these chemicals can diffuse into cells, they are finding increasing use in probing for unusual structures in viva. 19 a. Single-strand selective probes: Single-strand selective probes are chemicals that preferentially react with unpaired bases or at ”distorted" regions of dsDNA. A listing of these, their chemical reaction with DNA, and their use in probing DNA structure has been presented (Palecek, 1991). Upon reaction with DNA, some agents form stable adducts that can be cleaved chemically, or can be detected enzymatically or immunologically, whereas others result in direct strand scission. Their reaction with B-DNA is minimal as long as the reactions are performed under appropriate conditions. The most commonly used single-strand selective probes are bromoacetaldehyde (BAA) and chloroacetaldehyde (CAA), which react preferably with bases that are not hydrogen-bonded, as well as osmium tetraoxide complexes, potassium permanganate, and diethylpyrocarbonate (DEPC), which detected unpaired bases as well as other helix distortions. BAA, CAA, and osmium tetraoxide complexes have been used in probing B-Z junctions in vi tro, establishing the presence of unpaired bases at the junction. More recently, osmium tetraoxide has been used to detect B-Z junctions on supercoiled plasmids in E. coli cells (Palecek et al., 1987b; Rahmouni and Wells, 1989; Rahmouni and Wells, 1992). Single-strand selective chemical probes have also been used to decipher the molecular structure of H-DNA (Htun and Dahlberg, 1988; Johnston, 1988; Htun and Dahlberg, 1989), to distinguish between alternate structures that can be adopted by the same sequence (Pestov et al., 1991), and to study the pathways of cruciform formation (Furlong et al., 1989; Bowater et al., 1991). b. Double-strand selective probes: Two useful double-strand selective probes are dimethyl sulfate (DM5) and nitrosourea. They have been particularly useful in probing triplex DNA regions (e.g., Johnston, 1988; Voloshin et al., 1988; and Palecek, 1991, for a review). 20 c. Shape- or structure-selective probes: These probes are transition metal complexes that have been developed to bind preferentially local DNA structures that differ from B-DNA (reviewed in Barton, 1986; Barton, 1988; Sigman and Chen, 1990). These molecules bind to DNA stereospecifically and strand scission is activated by light. Complexes specific for A-DN A (Mei and Barton, 1988) and Z-DNA (Barton and Raphael, 1985; Barton, 1986) have been developed. They have been used in demonstrating that structural variability in the SV-40 genome is associated with promoter and enhancer sites (Muller et al., 1987), and in finding A and B-DNA junctions at a DNA binding site for transcription factor IIIA (Huber et al., 1991). A complex developed to recognize cruciforms was found to cleave instead at AT-rich sites next to cruciforms (Kirshenbaum et al., 1988) . Thus, it appears that this probe recognizes sequences that are unwound or unstacked during the process of cruciform extrusion (Furlong et al., 1989; Schaeffer et al., 1989; Bowater et al., 1991), rather than the cruciform itself. A transition metal complex that appears to cleave at or near sites occupied by proteins that distort the helix (Barton and Paranawithana, 1986), has been used to cleave DNA in mammalian cells, and showed that sequences recognized were interspersed throughout the genome (Chapnick et al., 1988). Although shape-selective probes have been useful in demonstrating structural variability, the specificity of these probes is not as geat as that of the P1, 51, and 17.3 nucleases or the LacI/ 17.3 hybrid nuclease (reviewed below). Moreover, they are prone to "induced fit" artifacts when used in high concentrations. d. Sequence-selective probes: Sequence-selective chemical probes are derived from oligonucleotides that form intermolecular triple helices at defined duplex sequences. Their conversion to nucleases has been accomplished by modifying one of their ends with an activatable nucleolytic moiety such as EDTA (Moser and Dervan, 1987; Strobe] et al., 1991), 1,10-pherranthroline (Sun et al., 1988; Francois et al., 1989), elipticine (Perrouault et al., 1990), or even an enzyme such as staphylococcal nuclease (Pei et al., 1990). 21 DNA-binding-protein sequence-selective chemical endonucleases (also reviewed in (Sigman and Chen, 1990) have been constructed in vitra as hybrids consisting of a protein DNA- binding domain coupled to a nucleolytic moiety. For example, the DNA-binding domain of the Hin recorrrbirrase has been coupled with EDTA and was shown to cleave at Hin recombination sites upon addition of Fe(II) (Sluka et al., 1987). In a similar approach, two other DNA- binding proteins - the tryptophan repressor and CAP - have been converted to nucleases by site- specific covalent attachment of a 1,10-phenanthroline-copper complex (Chen and Sigman, 1987; Ebright et al., 1990). Strand scission by 1,10-phenanthroline-modified tryptophan repressor was restricted to the operator regions of aroH and trpEDCBA, and the reaction was dependent on the presence of the corepressor L-tryptophan, which is necessary for effective binding of tryptophan repressor to the operators (Chen and Sigman, 1987). Likewise, the CAP-derived nuclease cleaved exclusively at CAP-binding sites on plasmid (Ebright et al., 1990) and 1. DNA (R. H. Ebright; personal communication). So far, sequence-selective probes have only been used like restriction enzymes to cleave small plasmids, phage 1 DNA and in some cases, intact chromosomes (Strobel et al., 1991). Their potential use as structural probes has not been investigated. Since these molecules cannot be expressed in the cell, they can only be used in viva by micro-injecting into Xenapus oocytes, or by treating active isolated nuclei. C. Enzymes and DNA-binding proteins as probes of DNA structure A separate class of structural probes are enzymes and other proteins that interact with DNA. They include natural and genetically engineered nucleases, DNA modifying enzymes, as well as antibodies raised to specific structures (anti-cruciform or anti-Z-DNA antibodies). The use of antibodies in the study of unusual DNA structures has been reviewed (Drew et al., 1990; Palecek, 1991). The nucleases can be divided into two basic categories: sequence-specific endonucleases, i.e. restriction enzymes, and structure-selective, i.e. those that recognize and cleave at 22 particular structures irrespective of the particular sequence. Restriction enzymes and their respective metlrylases have been employed in establishing the formation of Z-DNA in vitra and in viva (see Wells, 1988, for a review; and Jaworski et al., 1988; Jaworski et al., 1989). 8-2 junctions have been shown to display enhanced reactivity towards restriction endonuclease Mbal (Winkle et al., 1991), but decreased reactivity towards BamHl (Singleton et al., 1983) endonuclease and towards the EcaRl methylase (Jaworski et al., 1987). The formation of cruciforms has also been monitored by differential methylation/restriction of restriction enzyme sites located at the loop of the cruciforms (Courey and Wang, 1983; Lilley and Hallam, 1983; Blaho et al., 1988; Troster et al., 1989). Endonucleases that recogrize and cleave particular structures have been widely employed in deciphering the structure of selected sequences. The single-strand specific nucleases include 51 nuclease (51; see below), 17 endonuclease 1 (173; the product of gene 3 of phage 17; see below), T4 endonuclease VII (Picksley et al., 1990), P1 nuclease (Blaho et al., 1988), Bal31, and mung bean nuclease, and have been reviewed extensively elsewhere (Shishido and Ando, 1985). DNaseI and micrococcal nuclease which cleave preferentially dsDN A have had limited use in probing unusual structures. Other less characterized nucleases have also been employed in examining DNA structural heterogeneity. They include an endonuclease from Chrithidia fasciculata which nicks bent DNA (Linial and Shlomai, 1987; Linial and Shlomai, 1988), a cruciform DNA resolving endonuclease (Endo X3) from S. cerevisiae (Jensch et al., 1989), endonuclease G from calf thymus nuclei which cleaves at (dG)n.(dC)n tracts (Cote et al., 1989), a resolvase (Rqu) from E. coli (Connolly et al., 1991; Dunderdale et al., 1991; lwasaki et al., 1991; Sharples and Lloyd, 1991), a herpes simplex virus-induced nuclear endonuclease that cleaves anisomorphic DNA (Wohlrab et al., 1991), and two putative eukaryotic resolvases (Elborough and West, 1990; Jeyaseelan and Shanmugam, 1988). Several DNA-binding proteins which do not display catalytic properties have been employed in deciphering DNA structure. They include lacl (Bellamy et al., 1988) and AraC 23 (Lee and Schleif, 1989). Their capacity to form stable DNA loops between appropriately spaced operator sites has been used in demonstrating that the number of bp / helix tum within a looped segment in viva, can vary considerably from that observed in solution in vitro (Bellamy et al., 1988; Kramer et al., 1988; Lee and Schleif, 1989). These experiments have been presented above (see Section IF). A new class of enzymatic probes has been developed recently, by fusing a repressor with a nuclease. The first, was a genetically engineered hybrid between of LacI and T73 (Panayotatos et al., 1989) and is described more extensively in the section devoted to 17.3. Another repressor/nuclease hybrid was made in vitro by chemically connecting the A repressor with staphylococcal nuclease (Pei and Schultz, 1990). It has been developed as a sequence- specific endonuclease, and its use has been limited to cleaving AT-rich sequences cloned right next to the 1. repressor binding site. Three enzymatic probes deserve special attention: the $1 nuclease, the T73 endonuclease, and a lad/173 hybrid protein. D. S1 nuclease The $1 nuclease preferentially cleaves ssDNA (Vogt, 1980). It has been used in probing for cruciforms on supercoiled plasmids in vitro (Lilley, 1980; Panayotatos and Wells, 1981). In these experiments, incubation of $1 with supercoiled DNA bearing the colEl palindrome resulted in specific cleavage at the position of the sequence that corresponds to the loops of the cruciform configuration that can be adopted by that sequence, providing the first evidence that naturally occurring palindromes can adopt the cruciform configuration on supercoiled plasmids. Subsequently, SI - in conjunction with modeling, supercoil relaxation experiments and chemical probes - has been used to demonstrate that several other unusual structures exhibit single—strandedness. For example, 81 has been used in mapping the single-stranded regions present in 8-2 junctions (Singleton et al., 1982; Singleton et al., 1983; Singleton et al., 1984), H- DNA (Lyamichev et al., 1985; Johnston, 1988; Pestov et al., 1991; and Wells et al., 1990; 24 Palecek, 1991, for reviews), and slipped DNA (Gama et al., 1988). 81 has also been used to differentiate between the formation of cruciforms or Z-DNA by sequences which can adopt either conformation (Blaho et al., 1988; Naylor et al., 1988; Panyutin et al., 1985). However, as with other enzymatic probes, strand scission by $1 alone does not yield conclusive information about a structure. For example, the reactivity of S1 towards pur-pyr sequences was erroneously interpreted (see Section LE). E. 17.3 and the Laclfl'73 hybrid endonuclease Like S1, 173 also cleaves preferentially ssDNA (Sadowski, 1971; Pham and Coleman, 1985), and has been used in probing for cruciforms on supercoiled plasmids in vitra in parallel with $1 (Panayotatos and Wells, 1981), confirming the reactivity of cruciform loops towards ssDNA endonucleases. More importantly, it was later utilized as a probe of cruciforms in viva (Panayotatos and Fontaine, 1987). Other investigators have used T73 to probe synthetic branched molecules which resemble recombination intermediates (Holliday structures). T73 has been shown to bind Holliday junctions selectively by gel retardation assays, and footprinting of the binding site revealed that the base of the junction rather than the stems of these molecules were contacted by 17.3 (Parsons and West, 1990). Upon addition of magnesium, the junctions are resolved to dsDNA molecules bearing single nicks (Dickie et al., 1987; Muller et al., 1990; Picksley et al., 1990; Lu et al., 1991). More recently, a recombinant hybrid protein (Hl44) consisting of the first 339 amino acids of lacI fused in frame with the full length 173 was constructed and expressed in bacteria (Panayotatos et al., 1989). The purified protein maintained the properties of [ad and 173 in viva and in vitro. However, it displayed a novel property, cleaving at highly preferred sites on linearized dsDNA that carried the lac operator (lacO) (Panayotatos and Backman, 1989). H144 is the topic of the studies described in Chapters 2 and 3. Section III. In vivo approaches for examining unusual DNA structures Although the majority of studies on unusual DNA structures has been conducted with purified DNA in vitra, evidence has also been obtained for the existence of such structures in prokaryotic and eukaryotic cells (see Wells, 1988; Palecek, 1991, for reviews). The most effective approaches, and indeed those to unequivocally demonstrate the existence of cruciforrrrs (Panayotatos and Fontaine, 1987) and Z-DNA (Jaworski et al., 1987) on supercoiled plasmids in viva employed enzymes as biological probes. These two studies were performed in E. coli, as it is a very well understood organism biologically, and has been used extensively in expressing native and recombinant proteins in a tighfiy controlled manner. It should be noted that the early reports of localizing Z-DNA on fruit fly polytene chromosomes utilizing anti-Z- DNA antibodies, were an artifact of the process used in preparing the chromosomes for antibody binding (see Drew et al., 1990). a. Cruciforms: Indirect evidence for fire existence of cruciforms in viva had originally been obtained by measuring the superhelical density of plasmids that contained d(AT)n . d(AT)n tracts (Haniford arnd Pulleyblank, 1985), which have been shown to adopt cruciform configurations upon supercoiling. It has been shown that the decrease in apparent superhelical density brought about by a structural transition (such as palindrome extrusion to cruciform, or conversion of B-DNA to Z-DNA) is compensated for by fire action of DNA gyrase, which reintroduces fire "missing" supercoils. This process results in a net increase in the absolute number of supercoils in comparison to firose displayed by a plasmid in which fire transition did not take place. This 25 26 effect is geafiy increased when protein synfiresis is inhibited, presumably because DNA gyrase remains active (Dayn et al., 1991). Indeed, plasmids containing d(A1')n . d(AT)n tracts exhibited a higher absolute superhelical density than plasmids of equal size firat did not have such tracts (Haniford and Pulleyblank, 1985; Dayn et al., 1991). However, in these experiments ofirer structural transitions (e.g. unwinding) could not be ruled out. This method is referred to as "in viva supercoil relaxation". Direct evidence for fire existence of cruciforms in viva, came later, through fire use of recombinant 17.3 endonuclease (Panayotatos and Fontaine, 1987). These experiments were based on the observation firat 17.3 cleaves fire colEl and ofirer cruciforms wifir high specificity in vitra (see Section LB). A high copy number plasmid carrying the colEl palindrome, as well as the 17.3 endonuclease gene under fire control of a lacUVS promoter, was transformed into an E. coli recA host (which is highly deficient in repairing DNA damage). Upon induction of 17.3 synthesis, firere was progressive nicking/linearization of fire supercoiled plasmid at a site corresponding to fire loop of the cruciform configuration adopted by fire palindrome. The target site was identical to firat cleaved in vitra. Thus, fire first direct evidence was obtained that an unusual structure such as a cruciform can exist on supercoiled plasmids in the cell (Panayotatos and Fontaine, 1987). The adoption of cruciform configuration by inverted repeats in E. coli has been verified indirectly by supercoil relaxation (Blaho et al., 1988), or by monitoring transcription from a promoter capable of forrrning a cruciform (Horwitz and Loeb, 1988), and directly, by preferential modification of fire bases comprising cruciform loops by single-strand selective chemical probes, such as CAA (Dayn et al., 1991), and osmium tetraoxide (McClellan et al., 1990). Since cruciform extrusion is dependent on supercoiling, monitoring cruciform extrusion in viva wifir chemical probes has provided a means to measure changes in the superhelical density of plasmids in fire cell, as a response to changes in gowth conditions (McClellan et al., 1990; Dayn et al., 1991; Zheng et al., 1991a). 27 In eukaryotes, cruciform formation has been demonstrated using anti-cruciform antibodies (Ward et al., 1991). b. Z-DNA: As with cruciforms, indirect evidence for the presence of Z-DNA on supercoiled plasmids in viva came from supercoil relaxation measurements (Haniford and Pulleyblank, 1983). Direct evidence was obtained by taking advantage of the fact firat in vitro, EcoRl mefirylase does not modify EcoRl sites when they are located within Z-helices (see Section I.C). To probe for Z-DNA formation in E. coli, plasmids containing sequences firat had been shown to adopt a Z-form in vitra, were placed in a host that has a temperature sensitive EcaRl methylase (which is inactive at 42°C) (Jaworski et al., 1987; Jaworski et al., 1988). The plasmids were gown at 42°C in order to avoid mefirylation during replication, and the cells were switched to 22 or 5°C, to activate the methylase. As a last step, plasmid DNA was isolated, and restricted wifir EcaRl. Since only unmethylated sites would be cleaved, only sites firat were within Z-DNA regions would be reactive. lrrdeed, sequences that adopted a Z- conformation in vitra, were highly recalcitrant to methylation in viva and firus, were not cleaved by EcaRl. Moreover, in vitro methylation of fire same plasmids under conditions in which the above sequences are in the Z-conformation, gave fire same results as fire in viva experiments. The adoption of a left-handed conformation by certain sequences on supercoiled plasmids in viva, was also demonstrated by site-specific modification of 8-2 junctions by osmium tetraoxide (Palecek et al., 1987b). Thus, the existence of Z-DNA in prokaryotes has been demonstrated by two different direct mefirods. In eukaryotic cells, Z—DNA helices have been probed with anti-Z-DNA antibodies. As mentioned above, early reports of binding of these antibodies to fixed polytene chromosomes were erroneous (see Drew et al., 1990; Palecek, 1991). However, techniques firat allow direct binding and detection of antibodies to chromatin 28 inside metabolically active nuclei have been utilized to localize Z-DNA in eukaryotic cells (Wittig et al., 1989). Like cruciform extrusion, fire conversion of a right-handed segment to fire Z-form, can provide a measure of fire degee of overall as well as localized supercoiling (Zheng et al., 1991b). The formation of Z-DNA has been used to follow localized changes in (or domains of) supercoiling as a result of transcription. It had been postulated firat the translocation of a transcription complex would generate positive supercoils ahead of and negative supercoils behind fire transcription ensemble (Wang and Giaever, 1988; and references within). This hypothesis has been verified in vitro, by measuring fire accumulation of positive supercoils in a plasmid containing a single transcribing gene in fire presence of a prokarytotic topoisomerase I which selectively removes negative supercoils (Tsao and Liu, 1989). The formation of bofir negative and positive localized supercoils in vivo, has been verified by measuring fire adoption of a left-handed conformation by particular sequences placed before promoters or near terminators by site-specific chemical modification of B-Z junctions (Rahmouni and Wells, 1989; Rahmouni and Wells, 1992). An immunological approach has also been used in isolated but metabolically active nuclei, to demonstrate the dependence of Z-DNA formation on torsional tension (Wittig et al., 1989) and transcription (Wittig et al., 1991). c. H-DNA: Very little evidence has been obtained for fire existence of H-DNA in the cell. Whereas direct chemical probing has failed to detect H-DNA formation on supercoiled plasmids in viva under physiological conditions, sequences that are located in fire single- stranded loop of an H-DNA form in vitra, were found to be undermethylated in viva (Parniewski et al., 1990). Interestingly, when protein synthesis was inhibited, firese sequences were fully methylated, suggesting that fire single-stranded loop is contacted by DNA-binding protein(s). More recently, an intramolecular dG.dG.dC triplex was detected on supercoiled plasmids in E. coli by probing with chloroacetaldehyde after treatment with clrloramphenicol 29 (Kohwi et al., 1992). Treatment wifir chloramphenicol increases fire superhelical density of fire plasmids, firereby favoring extrusion to fire H-form. d. DNA looping: Evidence of fire existence of loops in viva comes primarily from indirect experiments, by measuring repression of transcription as a result of loop formation, and has been verified wifir in viva footprinting in some systems. The evidence obtained from firese types of experimmts has already been presented (Section I.F). Section IV. Biological functions of unusual DNA structures The demonstration that cruciforms, Z-DNA, H-DNA, and loops exist in viva, strongly suggests that finese and ofirer unusual structures participate in biological processes. The structure for which fire most evidence for a biological functions has been obtained is DNA looping. Its roles in transcriptional regulation, genetic recombination, and DNA restriction have been reviewed extensively and will not be presented further (see Matthews, 1992; and references wifirin). The biological functions of cruciforms, Z-DNA, and H—DNA are less well established. The conversion of a right-handed sequence to any of firese unusual conformations results in a reduction in apparent superhelicity. For finis reason, these structures have been postulated to regulate processes, such as transcription, firat are affected by fire superhelical density of the substrate. Alternatively, unusual conformations may simply function as a recognition signal for DNA-binding proteins. a. Cruciforms: Because cruciforms structurally resemble Holliday junctions, their interaction wifir DNA resolvases - i.e. enzymes firat resolve recombination intermediates to duplex DNA - has been studied very actively. Indeed, cruciforms are cleaved at fireir base by DNA resolvases - such as Rqu (lwasaki et al., 1991) — that cleave Holliday junctions. However, very little evidence firat cruciforms play a direct role in recombination has been obtained (reviewed in Blaho and Wells, 1989). Cruciforms have been reported to have an effect on transcription. Negative regulation of transcription by cruciforrrns has been postulated to occur by two different mechanisms. In fire 30 31 first mechanism, cruciforms function as negative regulators by directly altering the conformation of a promoter (Horwitz and Loeb, 1988; Horwitz, 1989). An artificial promoter capable of adopting a cruciform configuration on supercoiled plasmids was utilized. As fine superhelicity of fire template increased, transcription from firat promoter was repressed, as it was no longer recognized by RNApol in fire cruciform configuration. In fine second mechanism, cruciforms act as blocks within a transcribed region (Bagga et al., 1990; Waga et al., 1990a; Waga et al., 1990b). In fine latter case, transcriptional blocking could be overcome, when the eukaryotic protein HMG-l, which has been shown to interact wifir cruciforms (Bianchi et al., 1989), was included in the transcription reaction (Waga et al., 1990a; Waga et al., 1990b). However, firere are examples in which cruciform extrusion wifirin a transcribed sequence had no detectable effect on transcription at least in vitra (Morales et al., 1990). Cruciforms have also been implicated in positive regulation. Evidence has been obtained firat some eukaryotic enhancers can adopt a cruciform configuration which is important for binding of transcription factors and subsequent transcriptional activation (McMurray et al., 1991). More recently, evidence was obtained for fire involvement of cruciforms in DNA replication by demonstrating fireir presence at active mammalian origins of DNA replication (Bell et al., 1991; Ward et al., 1991), and at a prokaryotic origin (Noirot et al., 1990). However, there is no evidence firat cruciforms play a direct role in replication, and are not just fortuitously extruded during firis process. b. Z-DNA: Z-DNA has been implicated to play a role in homologous recombination as bases within a Z-helix are exposed to solvent and finus become more accessible for the formation of heteroduplexes. The evidence firat supports firis role for Z-DNA has been reviewed recerrfiy (Blaho arnd Wells, 1989). 32 The demonstration that Z-DNA is not cleaved by restriction enzymes in vitra or modified by a methylase in vitra and in viva (Jaworski et al., 1987; and references within) suggested firat Z-DNA may also react differently with other enzymes, or DNA-binding proteins. Although Z-DNA has been postulated to play a role in transcription, no direct evidence has been obtained supporting finis hypofiresis (Hayes and Dixon, 1985; Pestov et al., 1991). In one instance, a Z-DNA forming sequence was shown to repress transcription of fire 8- galactosidase gene when inserted within firat gene or when it was used to replace fire lacO region (which is located immediately upstream of fire promoter) of the lac promoter-operator region (Horbach and Muller-Hill, 1988). c. H-DNA: The presence and statistical overrepresentation of H-DNA-forming sequences in fire promoter regions of many eukaryotic genes (see Wells et al., 1988, for a listing) implies a function for H-DNA irn transcriptional control. Indeed, negative regulation of transcription of fire human c-myc gene finrouglr intermolecular triple helix formation has been demonstrated in vitra, with an oligonucleotide directed to an upstream region of firat gene (Cooney et al., 1988). However, only indirect evidence has been obtained for fire putative function of H-DNA in transcriptional regulation. Negative regulation by H-DNA-forming (dG)n-(dC)n tracts has been observed in vivo, and is perhaps due to inhibition of binding of a transcription factor at fine (dG)n-(dC)n tract when it forms H-DNA (Kohwi and Kohwi, 1991). A transcription (‘2') factor firat binds to an H-DNA-formimg oligopyrimidine repeat has also beer isolated but has not been characterized furfirer (O'Neill et al., 1991). d. Bert DNA: Bert DNA is fire probably fire most ubiquitous of fire unusual structures, but has not been studied in viva to any appreciable extent. Both intrinsic and induced bends have been associated wifir basic biological functions such as replication, transcription, and recombination in many different systems. However, in rrrost of firese cases, it is still unclear whefirer bending 33 (i.e. fire secondary structure) is important for function, rafirer firan fire particular primary sequence. Unfortunately, mutagenesis studies to address firis problem are not entirely effective because changing fire primary sequence alters fire degee of or altogether abolishes DNA bending. The best example in which DNA bending appears to play a direct biological role is probably fine replacement of an induced bend brought about by integration host factor (IHF) with an intrinsic bend (Goodman and Nash, 1989). IHF binds at specific sites on DNA and induces a sharp bend which brings distal DNA segnents in proximity for recombinafion to occur. Replacement of the IHF binding site with an intrinsic bend led to IHF-independent recombination at fire same distal sites, indicating firat bending rafirer than DNA-protein interactions were required for function. e. DNA looping: DNA 100ping has been shown to play a role in transcriptional regulation, genetic recombination, DNA replication, and DNA restriction. The manner in which DNA looping participates in firese biological processes has been reviewed (Schleif, 1988; Hochschild, 1990; Matthews, 1992). Section V. Concluding remarks It is clear from this review firat DNA can possess significant localized structural heterogeneity, depending eifirer solely on primary sequence or, in addition, on protein binding, supercoiling, DNA topology, and environmental conditions. The demonstration firat several of the unusual conformations that have been studied in vitro can also exist in vivo, has revitalized fine search for biological functions of finese structures. One approach for finding out how finese structures carry out biological functions is by looking for ofiner factors (e.g. proteins) that interact wifir firern. This approach has been used successfully in cloning DNA resolvases - i.e. enzymes that convert recombination intermediates to duplex DNA - in both eukaryotes and prokaryotes. It was in finis manner firat an E. coli resolvase, Rqu, was discovered and used irn conjunction wifir RecA protein to reconstitute a homologous recombination system in vitra (Dunderdale et al., 1991). This approach has also been used in cloning a specific class of proteins firat bind cruciforms (Wright and Dixon, 1988; Bianchi et al., 1989; Waga et al., 1990a; and references wifinin firese). Another approach is to look for native sequences that exhibit structural microheterogeneity in vitra and in viva. Indeed, it was by firis approach firat homopurine- homopyrirnidirne sequences were found to exhibit an unusual conformation, which was later deciphered in most cases to be H-DNA. It is also firis approach finat was utilized in fine work presented in fire upcoming chapters. Inseparable to finis approach, is fine development of new mefirods and tools to examine or look for unusual DNA structures. To finis effect, fire ssDNA endonuclease 17.3 and a novel lad/T73 hybrid (H144) were developed as structural probes, in viva and in vitra, respectively. 34 35 173 was used to probe for native unusual conformations on fine E. coli chromosome, by taking a similar approach as firat used for probing for fire colEl cruciform in viva (see Section 111). By finis mefinod, specific sites were localized on fine bacterial chromosome firat are cleaved as a result of 17.3 synfiresis. The putative unusual structures present at finese sites have :notlxxnnCharacuntueifurfiun: H144, originally designed as a sequence-targeted nuclease, was found to be a unique enzymatic probe for intrinsic bends on linear dsDNA. As such, H144 provided fire first evidence of structural heterogeneity wifinin intrinsic bends, as well as the first evidence for transient DNA loop formation between two non-identical functional sites by a single protein. 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CHAPTER 2 CLEAVAGE OF INTRINSICALLY BENT DNA BY AN ENGINEERED REPRESSORIENDONUCLEASE HYBRID PROTEIN 51 ABSTRACT A recombinant hybrid protein (I-ll44), consisting of fine lac repressor (lacI) and fire 17.3 endonuclease (17.3), has been shown to cleave DNA preferentially at specific target sites located on restriction fragments carrying the lac operator (IacO) (Panayotatos and Backman, 1989). By determining fine size of radioactively-labeled cleavage products with base-pair resolution, fire targets were mapped to fire transcription start site and -35 element of fire 8- ]actamase promoter, and at a site cleaved by gyrase in vitro. Since fine target sites were localized in a region finat appeared to display overall intrinsic curvature, H144 was tested for reactivity towards intrinsically bent DNA. Restriction fragnents were generated carrying fine lacO sequence and one or fine ofirer of two well characterized sequences known to display sequence-dependent curvature (intrinsic bending). Eifiner fragment was cleaved by H144 prefeertially at fine bendirng locus. Determination of fine target sites wifin basepair resolution indicated finat cleavage was directed neifiner towards a common primary sequence not fire A- tract repeats arrnong fire targets but, rafirer, towards fine physical center of each bend. These results indicated finat intrinsically bent DNA sequences expose an unusual conformation at fire center of fire bend. Since fire nucleolytic (17 3) domain of H144 exhibits a strong preference for cleaving at single-stranded DNA regions wifirin duplex DNA molecules, fine target sites may expose single-stranded character. The recognition of a localized unusual conformation wifinin a well-studied unusual structure (intrinsically bent DNA) demonstrates fine powe of H144 as a probe of structural microheterogeneity on non-supercoiled duplex DNA. Moreover, fine preferential cleavage exhibited towards two intrinsic bends finat have litfie oveall sequence similarity, indicated firat H144 is a unique enzymatic probe for bent DNA. 52 INTRODUCTION A recombinant endonuclease consisting of the Lac repressor (lacI) and the 17.3 endonuclease (173), fine product of gene 3 of phage 17, has been constructed and characterized (Panayotatos et al., 1989). The hybrid protein (H144) was originally engineered in order to direct fire T73 endonuclease to specific regions of double-stranded DNA (dsDNA), and was fournd to display fine properties of its components. In viva, H144 functioned like lac], since it could repress its own expression from a lac Protrroter, and fire repression could be overcome by fine addition of inducers (lactose or IPTG). In addition, after induction of H144 synfinesis, fine genomic DNA of fire host strain was degraded, suggesting firat H144 retained fine nucleolytic functions of fire T73 endonuclease (Panayotatos et a1., 1989; A. N. Ecornornides and N. Panayotatos, unpublished results). In vitro, purified H144 preferentially bound restriction fragnents finat carried fine lac operator (lacO), and cleaved cruciforms on supercoiled plasmids, wifin specificity and kinetics essentially identical to finose of 17.3. However, unlike T73, H144 cleaved linear dsDNA fraglrrents finat carried lacO, and strand scission was localized at higlnly preferred arnd specific sites (Panayotatos et al., 1989). Restriction mapping indicated that fire targets were located in a l‘Qgiorr that displayed overall intrinsic curvature, and contained a promoter and a gyrase cot\tact site. Cleavage at firose sites was largely independent of fine orientation of lacO, suggesting firat fine structure of fire sites and not fineir particular distance from lacO determined cleitrvage. Moreover, fire specificity of cleavage was lost when fine operator was not present on “‘9 same fragnent as fine targets. Likewise, IPTG which reduces fine affinity of H144 for fire t)I>Qrator, decreased fine relative amount of cleavage at firese targets, and increased cleavage at Quiet, less specific sites. This suggested that binding of H144 at lacO was important for 53 54 preferential cleavage at nearby targets. The targets were located between 130 and 200 bp away from lacO, i.e. at a distance geater than fire estimated effective radius (90 A) of even an H144 tetrarrrer (Panayotatos arnd Backman, 1989). Thus, fire question arose of whefiner DNA looping was rnecessary in order to bring fine targets wifinin fine reach of H144 bound to IacO. The recognition of higlnly preferred targets on non-supercoiled dsDNA by a nuclease that has a marked preference for single-stranded DNA (ssDNA), suggested fine presence of unusual structures at fine targets. In order to examine fine structure of fine targets, fireir precise location was determined at fine DNA sequence wifin base-pair resolution. Moreover, since fine targets were located in a region firat appeared to be intrinsically bent (Panayotatos and Backman, 1989), fire possibility firat intrinsically bent DNA was a structure preferentially cleaved by fine nuclease was tested. Two well-characterized sequences finat display sequence- dependert curvature (intrinsic bends) were subcloned near lacO. The precise localization of fine cleavage sites on fire origirnal targets described by Panayotatos and Backman (1989), and on two intrinsic bends is presented here. The molecular mechanism by which H144 locates its targets is presented in Chapter 3. MATERIALS AND METHODS Plasmids a. Vectors: All plasmids utilized were pBR322 derivatives except for pCP64. Plasmid pCP144 carries fine gene encoding H144 under the control of fine lacUVS promoter (Panayotatos et al., 1989). Plasmid pCP82 is a high copy number (copl) derivative of pBR322, carrying fire lacUV5 promoter cloned at fire unique EcoRI site of pBR322, as well as a 17 late promoter (Panayotatos and Backman, 1989). Plasmid pCP64 carries a kanamycin resistance gene and a region encompassing base-pair coordinates 37401 and 41740 of bacteriophage lambda (A) (N. Panayotatos, personal communication). This region includes fine It origin of replication (Lori). Plasmid pCP207 was derived from pCP82 by replacing fine region between fine unique Aat2 and EcoRI sites wifin an insert cantairning an Nhel site, according to standard genetic engineering techniques (Sambrook et al., 1989). This insert was obtained by palynnerase chain reaction (PCR) amplification (Innis and Gelfand, 1990) from pCP82, utilizing primers TRH-l and 82.6. Prior to subcloning, fire 124 bp PCR product was restricted wifin Aat2 and EcoRI to generate compatible cohesive ends. The presence of fine correct insert was verified by restriction analysis arnd DNA sequencing. b. Genetic engineering of plasmids carrying intrinsically bent DNA: The K-DNA bend region from Leishmania tarentalae (L. tarentalae) (Wu and Crofirers, 1984; Koo et al., 1986) and a region encompassing hori (Zahn and Blattner, 1985b), were subcloned into pCPZUI. 55 56 The K-DNA bend insert was prepared by PCR utilizing finree primers. First, an original PCR reaction was set up wifin Bent 1 and 82.5 primers and pCP82 as fine template. The 314 bp amplified DNA was reamplified wifin primers 82.5 and RB1.2, and subcloned direcfiy into vector pCR1000 (Invitroger). Subsequenfiy, fine insert in pCR1000 was moved as a 322 bp Afl2 - filled-in HindlII fragnent into pCP207 that had been cleaved wifin EcaRl, filled-in, and then cleaved again by Afl2. The resulting plasmid was named pCP230. The correctness of fine construct was determined by DNA sequencing of fine region containing fire insert, as well as by restriction analysis. The Aori fragment was subcloned between fine Swal and Eagl sites of pCP207 as a 482 bp Ssp l-Eael fragment from pCP64. The resulting plasmid was restriction-mapped to verify fire presence of fire insert, and was narrred pCP212. DNA primers Primers were fine kind gift of fine DNA Core Facility, Regeneron Pharmaceuticals, Inc. Primer nan-1 (sucrc: TAG ACG T‘CT TTC CTC TCT m TCC ccr CTA AGA AAC CAT TAT TAT C-3') contains a region homologous to a sequence downstream fine Aat2 restriction site in PCP82, and has a non-homologous "tail" finat carries an Aat2 site. Prinner 82.6 (5'-GT'T CGA ATI‘ CTA CCT TGC AAG CTA GCG AAG ACG AAA GGG CCT CGT G-3') is honnologous to a Se(II-aence direcfiy upstream of fine EcoRl site of pCP82, and contains a non-homologous region Carlymg a Nhel site arnd an EcoRl site. Primer Bent] (5'-CGT ACG ACG TCC AAA AAT GTC AAA AAA TAG GCA AAA AAT GCG AAA AAT arr CCG CGC ACA m ccc-s') contains fire K-DNA bernd and a region l-‘°‘1'I(rlogous to pCP82 upstream of fine -35 element of fine B-lactamase promoter. Primer 82.5 (5'- QQT CTT AAA GTT AAA CCT TA-3') is homologous to a region of pCPSZ located near fire 17 late promoter. Primer 1131.2 (5'-ACI' AGT crc GTA CGA car CCA AAA ATG TC-3'), contains part of the 5' end sequence of Bent 1, and a tail with restriction enzyme sites. 57 Induction of H144 synthesis in W3110 lach F'lpCP144 E. coli W3110 lacI‘l F' competent cells were transformed with plasmid pCP144. The transformation reactions were plated on LB agar plates supplemented with 0.1 mg/ml ampicillin and 2 mM o-nitrophenyl-B-D-fucopyranoside (ONPF), to select for cells that harbored fire plasmid. ONPF was included in order to maximize repression from fine lacUVS promoter. Four 2.4 liter Fernbach flasks each containing 1 liter of LB supplemented as above were inoculated wifin W3110 lach F' /pCP144 and were gown at 37°C, wifir shaking at 250 rpm. When fine culture reaclned OD590 = 0.3 - 0.6, fine cells were harvested by centrifugation, in a Sorvall RC-3B centrifuge at 4,000 rpm (2,500 RCF), for 30 rrnin, at 4°C. Each pellet was resuspended in 1 liter of LB supplemented wifir lactose to 1%, to induce synfinesis of H144, and incubamd as above. Three hours after induction, fine cells were harvested by certrifugation as above, and fire pellets were resuspended in storage buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1 mM DT'I') at a concentration of 1 g of cells per ml. The suspensions wee stored at -70°C, in 50 ml polypropylene tubes (Falcon). P‘tlrificatiorn of H144 H144 had been previously purified by ammonium sulfate fractionation of a cleared 13’ Sate of induced E. coli harboring pCPl44, followed by ion exchange chromatography on S/P Sl"elhharose (Panayotatos et al., 1989). A modified purification protocol was utilized in finis Work arnd is described below. a- Extraction: A suspension of 40 g of induced W3110 lach F' [pCP144 cells was thawed and its volume adj‘-lsted to 200 m] wifin storage buffer. The cells wee lysed by passing firrough a Stanstead c':)1"‘tinuous flow lysis cell, twice. The fluid lysate was transferred into 50 rrnl polycarbonate 0ath'idge tubes (Nalgene), and was centrifuged at 16000 rpm (37,000 RCF) in an SS-34 rotor (sorxmll), for 30 min, at 4°C, to pellet the bacterial debris. The cloudy supernatant was 58 transferred irnto 30 ml polycarbonate tubes and centrifuged at 42,500 rpm (183,000 RCF) in a 1865 rotor for 1 hour, at 4°C, to pellet any remaining particulate material. The nucleic acids present in fine lysate were precipitated by addition of an aqueous 30% streptomycin sulfate (Sigma) solution to a final concentration of 3%, and stirring on ice for 30 min. The rrnixture was transferred into 50 ml polycarbonate Oakridge tubes, and fire precipitated nucleic acids were separated from fine lysate by centrifugation in an SS-34 rotor, 16,000 rpm (37,000 RCF), 10 min, at 4°C. Proteins in fine supernatant were precipitated by addition of ganular ammonium sulfate (Mallinckrodt) to 55% saturation (Scopes, 1982). The protein pellet was dissolved in cold buffer A (50 mM MOPS pH 7.2, 5 mM EDTA, 0.5 mM DTT, 5% glycerol), and fine volume adjusted so finat fire conductivity of fine suspension would be approximately equal to fine conductivity of fine starting buffer CM (buffer A with 20 mM NaCl). The solution was passed finrough a 0.45 mm HV filter (Millipore), and finer finrough a 0.2 mm GV filter (Millipore) to remove particulate material. b. Chromatography: H144 was purified from fine filtered suspension by liquid chromatogaphy. Fractions cOntaining H144 were identified by assaying for preferential and specific binding and cleavage 0‘ DNA fragment carrying IacUV5 in a mixture of restriction fragments, as described (Parnayotatos et al., 1989). As a first step, fast protein liquid chromatogaphy (FPLC) on a CM cellulose cartridge (CM1010; Millipore) was performed. After fine solution was loaded, a salt gradient in buffer A was applied to elute bound proteins. H144 eluted at 200 mM NaO m a sit‘gle peak arnd was 50% pure as judged by SDS-PAGE stained by coonrassie brilliant blue R-250 (BiOrad) or by silver (Merril, 1990). Fractions containing H144 wee pooled, and were loaded direcfiy onto a phosphocellulase P11 column (Whatman), equilibrated in buffer P11 (40 mM sodium phosphate ph 7.0, 40 mM NaO, 5 mM EDTA, 05 mM arr, 5% glycerol). When a NaO g‘radient was applied, H144 eluted at approximately 450 mM NaO and was approximately 9090 pure by fire above criteria but still contained a contaminating nuclease activity. For ad(litional purification, hydrophobic interaction chromatogaphy was performed on a 1 m] 59 Phenyl Superose FPLC column (Pharmacia). Pooled active fractions were adjusted to 1 M (NH4)2SO4 by progressive addition of granular ammonium sulfate in buffer A, and after loading. a linear reverse gradient was applied to 20 mM NaCl. H144 eluted at 0.5 M ammonium sulfate, and was better tlnan 90% pure, as judged by SDS-PAGE followed by coomassie staining (fig. 2, panel a). The peak fractions were dialyzed against 500 volumes of H144 storage buffer (50 mM Tris pH 7.0, 50% glycerol, 20 mM NaCl, 2 mM EDTA, 0.1 mM DTT), at 4°C, overnight. Subsequently, fine preparation was divided into 100 ml aliquots in polypropylene eppendorf tubes and stored at -20°C. By this method, 3 mg of H144 were obtained from 40 g of induced cells. Characterization of H144 preparation The concentration of the H144 preparation used was 0.84 mg/ml (16 uM) as determined by the Bradford protein assay (Bradford, 1976) utilizing a commercially available kit (Biorad). In order to find whether H144 was a monomer, or a higher order multimr in solution, it was analyzed by gel filtration chromatography on a Superdex-75 column (Pharmacia) in buffer GP (50 mM Tris pH 8.0, 100 mM NaCl). H144 eluted as a single peak, displaying an apparent molecular weight of 58 kd (fig. 2, panel b). This figure is close to 52.5 kd calculated for an H144 monomer from the amino acid composition. More importantly, higher order multimers or aggregates were not detected by this mefinod. Thus, in the absence of lacO DNA, it appears that H144 is a monomer in solution, urnlike Lacl protein which is believed to be primarily a dimer or tetrarner (Pace et al., 1990; and references within). H144 was also analyzed by reverse phase chromatography. H144 (24 ug) was loaded on a 1 ml C8 column (Poros [IR-M), in 0.1% trifluoroacetic acid (TFA), 10% acetonitrile, and was eluted with an acetornitrile gradient at 1 ml/ min. H144 eluted as a single peak at 18.978 min, wlnich corresponds to 42% acetonitrile (fig. 2, panel c). A non-protein absorption peak at 24.198 min was seen with fine 70% acetonitrile wash. This is due to a change in the refractive index of 60 Figure 2. Physical characterization of H144. Panel a: 12.5% SDS-PAGE gel stained witln coomassie-blue R250. Lane M: Molecular weiglnt protein standards, sizes 97.4, 66.2, 45.0, 31.0, 21.5, and 14.4 kd respectively, 0.5 ug/ protein (Biorad). lanes 1 and 2: 1 and 20 ug of H144 respectively. Panel b: Molecular weight determination of H144 by gel filtration chromatography. K" has been plotted as a function of logMW (Stellwagen, 1990). Kay = (V., - Voy (Vt - V0), where Km, is fine fraction of stationary gel volume accessible to fine protein, Va is fine elution volume of the protein, V0 is the void volume of fine column, and Vt is fine total volume of fine gel bed. The position at which H144 elutes is marked. Panel c: Elution of H144 on reverse phase chromatography. Acetonitrile gradient (dashed line); A230 (contirnous line). .4 . >21 2c: r333. re: 1‘ l; i l i g E i’ i E E i! 1 a , Cr“=‘»‘aovw~f~f.,—-g‘vnc 442‘. 4 61 b. M 1 2 j, 1 o— 97 0.8 66 - 45. . 0.6 ; RNase1 x O 31 o 0 4 Chymotrypsinogen 21' 0.2 Ovalbumin . AH144 ‘4 BSA 0.0 V ' I 1 l l Yfi'fi 10 100 MW (kDa) C. 1% 0.02 18.978 “‘ r“1 2 — I I a? 5 I I T E 60- II I e E— I 8 - ’J I —o.on § , / Il “’ 40— // 3‘ /’ I _ l// | /,/ I 20- //’ || / —/// 24.198 L _____’____ 5 1O 15 20 25 30 (min) Figure 2 62 the solution. No other peaks were detected, indicating finat H144 is more finan 95% pure and essentially intact. The H144 peak from this C-8 column was analyzed by mass spectrometry at fine Protein Core Facility of Regeneron Pharmaceuticals, Inc. The MW obtained by mass spectrometry was 52,430 i: 104. This figure is within experimental error of fine 52,511 value calculated from fine amino acid composition (data not shown). Reactions with H144 or T73 endonuclease Reactions wifin H144 or 17.3 endonuclease and DNA were carried out at 37°C, in reaction buffer (50 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgC12, 1 mg/ ml BSA), unless ofinerwise specified. Reactions were initiated by the addition of MgClz, and were stopped eifiner by fine addition of 0.2 M EDTA pH 8.0 to 20 mM final concentration, or by fine addition of 1/10 reaction volume of Stop buffer (50 mM Tris pH 8.5, 50 mM borate, 125 mM EDTA, 43.5% glycerol, 0.05 mg/ ml bromophenol blue). When desired, stopped reactions employing H144 were heated to 75°C for 5 min, to abolish binding of the hybrid protein to lacO-DNA. The T7.3 endonuclease was better than 90% pure and has been used in previous studies (Panayotatos and Wells, 1981; Panayotatos and Backman, 1989; Panayotatos et al., 1989). Fifty nanograms (1 ul) of T73 converted 3 ug of supercoiled pVH51 to approximately 50% nicked arnd linear forms in 6 min at 37°C in reaction buffer. Lambda O-protein lambda O-protein (10; fine gift of Dr. R. Inmann) was at a concentration of 0.733 mg/ ml (21.7 M) as calculated from densitometry of SDS-PAGE gels stained with coomassie blue. The preparation was more finan 70% pure. No contaminating endonucleolytic or exonucleolytic activity was detected when DNA was incubated with 10 under fine conditions employed in H144 or 17.3 assays (data not shown). Mapping of H144 targets In order to map H144 target sites wifin base-pair resolution, restriction fragments were specifically radiolabeled at one end by filling-in wifin DNApol large fragment (Klenow; NEB) in fine presence of fine appropriate (II-35$ dNTP (NEN) or wifin polynucleotide kinase (NEB) in fine presence of 7-32P ATP (NEN). Fragments labeled wifin polynucleotide kinase were first dephosphorylated with alkaline phosphatase (Boehringer Mannheim). Filling-in with Klenow labels fine 3' end of the molecule, whereas labeling wifin polynucleotide kinase labels fine 5' end of fine molecule (Sambrook et al., 1989). The radiolabeled DNA was reacted with H144, and fine reactions were brought to 10 mM EDTA and 100 mM sodium acetate. The DNA was ethanol precipitated, and fine pellet was resuspended in sequencing stop buffer (63% formamide, 13 mM EDTA, 0.03% bromophenol blue, 0.(B% xylene cyanol FF). The samples were loaded onto 6% polyacrylamide-7 M urea strand- denaturing gels cast for fine Model 52 sequencing apparatus (BRL). An M13 sequencing ladder size marker was generated as recommended (Sequenase 2.0; United States Biochemicals). Bands were visualized by autoradiography on XAR-5 film (Kodak) or on a Fuji phosphorimager. The latter allowed direct quantitation of fine amount of radioactivity present in each band. RESULTS H144 cleaves the DNA at two unrelated promoters and at a gyrase contact site The approximate location of the two preferred targets for H144 on linear dsDNA fragments finat carried lacO and part of plasmid pBR322, was mapped by restriction analysis within fine region of fine fl-lachmase promoter (Panayohtos and Backman, 1989). In order to determine fine nature of fine sequences cleaved by H144 in finese molecules, fineir precise location was determined wifin base-pair resolution. An ApaLl restriction digest of plasmid pCP82 was specifically radiolabeled at the ApaLl site by filling-in with Klenow in fine presence of a-355 dCTP. Subsequently, fine labeled DNA was treated with Ale. In this manner, only fine left end of fine lacO fragment was labeled (fig. 3, panel a). This DNA was reacted with H144 and examined by electrophoresis on native 6% polyacrylamide gels. Efinidium bromide shining of fine DNA fragments revealed fine appearance of four distinct major bands (fig. 3, panel b, lane 2). Two double-strand brakes wifinin fine lacO fragment can account for fine appearance of finese four bands, as fine added sizes of fine pairs - 350 plus 160, or 280 plus 200 bp - approximately equal fine size of fine lacO fragment (466 bp), considering finat fine lacO fragment migrates abnormally slowly on polyacrylamide gels (Panayotatos and Backman, 1989; also see below). Confirmation of finis assignment and more accurate determination of fine sizes of finese fragments was obhined from experiments wifin labeled DNA. Only two of fine bands, migrating at 160 and 200 bp respectively were radiolabeled (fig. 3, panel c, lane 2). Thus, fine two major hrgets mapped 160 and 200 bp away from fine ApaLl site. This result placed fine hrgets wifinin fine region of fine B-lachmase promoter (fig. 3, panel c), in agreement with previous experiments (Panayohtos and Backman, 1989). 64 Figure 3. Restriction rrnapping of H144 hrgets in fine B-lachmase promoter region. Panel a: Map of lacO ApaLl-Aflz restriction fragment of pCP82. The positions of the transcription shrt site (+1), -10 and -35 elements of fine B-lachmase promoter, fine gyrase site (GYR), lacO, and a T7 late promoter (T7) have been marked. The orienhtion of 1000 is indicated by a horizonhl arrow. Vertical arrows (A) irndicate fine positions of fine two strong hrgets on the lower strand. An asterisk marks fine radioactively labeled ApaLl site on fine lower strand. The scale above fine map is in bp. Panel b: 6% polyacrylamide gel shined with ethidium bromide. Lane M: size markers (¢X174 Haelll digest), 0.15 pg. Lanes 1 and 2: pCP82 ApaLl-AflZ restriction digest, 0.2 ug (0.1 pmole) and 1.0 ug (0.5 pmole) labeled at fine 11le site wifin a- 355 dCT'P, reacted with 0 or 252 ng (0.48 pmole) H144, respectively, for 10 min at 37°C, and heated before loading. The positions of fine lacO substrate fragment (S) and four new bands in lane 2 have been marked (350 plus 160 bp, arnd 280 plus 200 bp apparent molecular sizes). Notice finat fine lacO substrate fragment (466 bp) migrates abnormally slowly. Apparent molecular sizes are placed in brackets. Panel c: Autoradiogram of fine gel in panel b. The positions of two new fragnents in lane 2 are marked. bp 50 1 90 1 50 290 250 35:0 B-lactamascpromotelegion [ApaLI lT2140 -35 GYBI * A A 160 200 b. M 1 2 1353 -—— 603 *—* 310 ———— 234 m...— 194 ~—— 118 ——- a 1.3» Q a. _i.. .. I 490 450 ._ [200] .__ [160] Figure 3 67 The exact size of each radiolabeled band was determined by separating fine DNA on 6% polyacrylamide-7 M urea strand-denaturing gels, next to an M13 sequencing ladder, which served as a high resolution size marker (fig. 4, panel a). The two nicks were placed 157 and 198 bp away from fine ApaLl site on the lower strand. This type of experiment was repeated, selectively labeling the upper or the lower strand at eifiner end of the lacO fragment (see Materials and Mefinods). In finis nnanner, fine exact position of fine hrgets was determined wifin base-pair resolution. The DNA sequence of the hrget regions shows that H144 preferably cleaves at the transcription shrt site and wifinin fine -35 element of fine B-lachmase promoter (Brosius et al., 1982), as well as a gyrase contact site (Kirkegaard and Wang, 1981) (fig. 4, panel b). The prominent nick rendered by H144 at position 207 on fine upper strand of fine sequence, is in fine same position as finat introduced by gyrase. In addition to fine five prominent nicks mapped, several rrnirnor nicks have been mapped (fig. 4). All of finese rrnap wifinin fine -35 element of fine B-lachmase promoter, except two that flank that element, and one finat maps at fine lower strand rnext to fine major nick localized at fine transcription shrt site. The finree minor nicks observed on fine upper strand flank the major nick wifinin fine -35 element (fig. 4, panel b). Subsequent experiments wifin ofiner fragments identified a prominent cleavage site finat mapped wifinirn a T7 late promoter. Three major nicks were seen at finat site, one of which mapped at fine transcription shrt site of finat promoter (see below, fig. 9). Interestingly, T7 late promoters differ greafiy from bacterial promoters, as finey are comprised of one contiguous 22 base-pair recogrnition sequence, and lack -35 and -10 elements (Panayohtos and Wells, 1979; Rosa, 1979). Thus, finree functional sites - a bacterial promoter, a phage promoter, and a gyrase conhct site - are targets for H144. Alfinough finey exhibit no apparent sequence homology, fineir recogrnition by H144 indicates fine presence of an unusual structure finat may be comrrnon among these sites. Since fine nucleolytic moiety of H144 preferentially cleaves at single- stranded regions, finis structure may exhibit single-strandedness. A common property of finese Figure 4. High resolutiorn mappirng of fine hrget sites in fine B-lachmase promoter region. Panel a: Autoradiogram of 6% polyacrylamide-7 M Urea gel. Lane C: pCP82 ApaLl-AflZ restriction digest, 0.05 pg (0.025 pmole) labeled at the ApaLl site with a-358 dCI'P. Lanes 1 and 2: as irn lane C, reacted wifin H144 as described in figure 3, lane 2. 0.05 arnd 0.20 ug loaded respectively. Lanes 3 finrough 6: M13 sequencing ladder, A, C, G, and T respectively. The positions and precise size of fine two labeled bands shown on figure 3 have been marked wifin large arrows. Panel b: Identification of hrgets on fine sequence of pCP82. Major nicks on both strands have been marked wifin large arrows (*), whereas mirnor nicks have been marked wifin small arrows (-+). The positions of lacO (solid line), fine transcription shrt site (+1) and fine -10 (P3) and -35 elenents of fine B-lachmase promoter are also shown. The site cleaved by gyrase is marked (gyr) and fine nicks introduced by finat enzyme are marked wifin small bold triangles (A). - .- fi HINAM‘x, IL“ WA~D.‘3'&“W . .‘ . A ‘- ' '- * ‘ " .efl’ '1' .. i - KI. I‘TTr-lsl 1 «1.1.7. 5:55:36.‘ ‘. ‘ ’z“,‘z'§‘.‘§lfihflfiht a y. m MM’A M’m l 'r lllll | 20 4O 60 80 l i a it n * a t t WMWWQWACWWWWMW AW ACI‘AGAAGT‘C GTAGAAAATG AAAGI‘GGI‘CG CAAAGACCCA CTCGT'I'I'I'N TCCT‘TCCGI'I‘ TT'ACGGIG'IT 100 120 140 160 t n AAAAGGGAAT AAGGGOGM‘A CGGAAATGT'; GAATACICAT‘ ACT‘CI'I‘CCIT T'I'PCAA'MT'I‘ AT'IGAAGCAT‘ T'I'AT‘CAGGG; Tm T'I‘CCCGCTGI‘ GOCT'I'I‘ACAA CT'I'ATGAGTA T'GAGAAGGAA MAGT‘T'ATAA TAACI'I‘CGTA Mmc: 180 200 a a - n t TAT'I'GI’CTCA TGAGCGGAT‘A CATAT'I'I‘GAA TGI‘AT'I'I'AGA AATAGGGGIT OCGCGCACAT‘ T'I‘CCCCGAA; ACTCGCCT'AT GTA TAAACTT ACATAAA mamas} TEATCCCCAA GGCGCGTGTA AAGGGGCI'I‘I‘ P3 ‘ ‘ ‘f —35 300 320 AGTGCCACCI‘ GACG'I‘CTAAG MACCAT'IAT TA TTAACCIATA AMATAGGCG TATCACGAGG CCCTTI‘CG'I‘C TCACGGTGGA CT‘GCAGAT‘I‘C T'I'I'GGI‘AATA ATAGI‘ACI‘GI' AATI‘GGAT‘AT T'l'I‘TATCCGC ATAGI‘GCT‘CC GGGAAAGCAG‘ 340 380 400 t t Lac0* a «k a t MAT; C'l‘G'l'I'i’CC'rG TGTGAAA'I'I‘G ”AW ACAATTCCAC ACA'I'IIATAOG AGCCGGAAGC AWR WM GACAMGGAC AQC'I'PI‘AAC MTAGGCGAG TGTTAAGGN TGTMTATGC TCGGCC‘I'I'CG TA'I'I'I‘CACAT >Af12 | 420 440 460 | t t i it I AAGCCT‘GGGGTGCCI‘AA TGAGTGAGAAT'I‘A AT‘TCGGT'I‘AA TACGACICAC TATAGGAGAA 0C TT‘CGGACOCC ACGGAT'I‘ACI CACI‘CTTAAT‘ TAAGCCAA‘I'I‘ ATGCTGAGIC ATA'I‘CCTC‘I‘T GGAAT'I‘ Figure 4 70 sites is finat finey are accessed by enzymes (bacterial or phage T7 polymerase, bacterial gyrase). An unusual conformation(s) at finese sites may serve as a recognition signal / entry point for fine relevant proteins (see Discussion). The hrgets mapped above were located on a fragment finat migrated abnormally slowly on polyacrylamide gels (Panayohtos and Backman, 1989) (fig. 3, panel a, lanes 2 and 3). Retarded electrophoretic mobility has been correlated with fine presence of intrinsic DNA bends on linear dsDNA molecules (Marini et al., 1982; Wu and Crothers, 1984). Thus, it was hypothesized that intrinsic curvature might contribute to preferential cleavage or, alternatively, might constitute a hrget in itself. H144 cleaves fine K-DNA bend at the physical center of bending In order to test whether H144 preferentially cleaves intrinsically bent DNA on lirnear dsDNA molecules, a 40 bp fragment encompassing an intrinsic bend from kinetoplast DNA (1(- DNA) (Wu and Crofiners, 1984; Koo et al., 1986) was chosen. It is comprised of four A545 tracts, finat occur approximately every 10 bp and are finus placed on fine same side of fine helix (i.e. in phase with each ofiner; for example see fig. 7, panel b) (Wu and Crofiners, 1984). The intrinsic curvature displayed by finis sequernce has been attributed to finese phased A-tracts (Koo et al., 1986), at an estimate of 17 to 21° per A6 tract (Koo et al., 1990). However, fine exact molecular structure of intrinsically bent helices is not known (see Clnapter 1). The K-DNA fragment was subcloned near lacO, to generate plasmid pCP230. The plasmid was first incubated wifin eifiner one of finree different sets of restriction enzymes - Sspl plus Pvu2, Nhel plus Pvu2, and Nhel plus Afl2 plus Afl3 - and subsequently with H144 for irncreasing amounts of time. In all three digests fine K-DNA bend and lacO were located on fine same fragment. As expected, fine apparent molecular sizes of finese fragments as determined on 6% polyacrylarrnide gels, wee greater finan fine sizes predicted from fineir sequence due to fine presence of fine K-DNA bend (fig. 5). 71 Figure 5. Restriction mapping of H144 hrgets in fragments conhining K—DNA. Panel a: Restriction map of fragments carrying fine K-DNA bend and lacO. The positions of fine K-DNA bend, lacO, and a T7 late promoter (T7) have been marked. Verical bold triangles (A) indicate fine position of a highly preferred hrget on fine lower strand wifinin fine K—DNA bend. Ofinersymbolsasonfigure3. Panel b: 6% polyacrylarrnide gel shined wifin ethidium bromide. Iane M: 100 bp ladder, 0.1 ug. Lane 1: pCPZBO Sspl-Pvu2 restriction digest, 0.2 ug (0.1 prrnole). lanes 2 finrough 5: as in lane 1, but reacted with 8.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 min respectively. Lane 6: pCPZ30 Nhel-PvuZ restriction digest, 0.2 pg. Lanes 7 finrough 10: as in lane 7, but reacted wifin 8.4 ng of H144 for 0, 15, 30, and 60 rrnin respectively. lane 11: pCP230 Nhel-AflZ-AflS restriction digest, 0.2 ug. Lanes 12 finrough 15: as in lane 11, but reacted wifin 8.4 ng of H144 for 0, 15, 30, and 60 mirn respectively. Reactions in lanes 3 finrough 5, 8 finrough 10, and 13 through 15 wee heated before loading. The position and apparent rrnolecular size for fine two bands resulting from cleavage at K—DNA in each set have been marked (650 plus 340, 650 plus 80, and 320 plus 80, respectively). m M m W I. A 1“!) 1". a ..o.I.'I .A( trail II .. / {deI’rFrJIJI .rr Erni|\i}afl.l '1' fol-taller. rt a...:l.r.r.alil|nr|trnol riots-salads" areal .- Jr. I» .' nl. 1' Is tfi a H—’ W“ I 'I— k. ’.. z.... I I a. hp 0 100 200 300 400 500 600 700 800 900 g I l l 1 l 1 l I Ssp1 Nhe1 EcoFiI A112 Pvu2 888 l—l Nhel EcoFH AII2 Pvu2 681 A / K-DNA “0 T7 348 Nhe1 Ecom A02 A K-DN A IacO T7 In. Figure 5 — [650] [340] [320] 73 Preferential cleavage of the lacoscontaining fragments was evidenced by the progressive decrease in fine intensity of finese fragments with increasing incubation time wifin H144, and fine corncorrnihnt appearance of two new bands. Cleavage of fine Nhel-Pvu2 laco- cornhining fragrrnent by H144 gave rise to two predominant products, migrating at 80 and 650 bp on 6% polyacrylarrnide gels by comparison to fine size markers (fig. 5, panel b, lanes 8 finrough 10). Likewise, fine Nhel-AflZ fragment (348 bp actual size; 450 bp apparent size) was cleaved at a single position giving rise to two fragments 320 and 80 bp each (apparent size). In each digest, fine sum of fine sizes of fine two products approximately equaled the size of fine respective substrate fragments. Sirnce fine 80 bp band was common in bofin sets of reactions (fig. 5, panel b, lanes 10 and 15), it must originate from cleavage 80 bp from fine Nhel site, finus placing fine cleavage wifinin the K-DNA bend. By finese criteria, fine K-DNA bend appeared to be a highly preferred target for H144. By similar analysis, fine single most pronounced cleavage observed wifin fine Sspl-PvuZ fragment (888 bp actual size; apparent size 1200 bp) was also localized wifinin fine K-DNA bend. In finese reactions, anofiner faint band was present migrating at approximately 700 bp (fig. 5, panel b, lanes 4 and 5). The cleavage site generating finis band corresponds to cleavage at fine pBR322 hrgets which are located between fine K-DNA bend and fine Sspl site. Thus, fine K-DNA bend appeared to be a highly preferred hrget for H144 even over fine ofiner preferred hrgets mapped in fine B-lachmase promoter region. To determine the exact DNA bases at which cleavage occurred wifinin the K-DNA bend, pCP230 was labeled at the NheI site by filling-in wifin Klenow in fine presence of a-3SS dCTP. The DNA was reacted wifin H144 as before, and loaded on native and denaturing polyacrylarrnide gels. Autoradiography of the native gels confirmed that fine 80 bp band originated from fine Nhel site (fig. 6, lanes 3 finrough 5). Autoradiography of fine denaturing gel and comparison wifin fine M13 sequencing ladder which served as a size marker, revealed finat fine exact size of fine fragment was 85 bp, and placed fine major cleavage at fine physical center of fine bend, as defined by Wu and Crofiners (1984) on fine lowe strand (fig. 7, panel a, 3.“ “m-.- m- ‘-4_ “Aw. ._.-,. ‘Q A 4""!- P ' n‘k‘- . A a r .. . -. I _..-. J I». ,s- ,j a].-. ._. __a . 74 a. b. M12345 M12345 t‘.’ 2200 ----—- 1000 600 <—[650] 100 “U r— [80] , — -*—[80] Figure 6. Restriction rrnapping of a unique H144 hrget at fine K-DNA bend. Panel a: 6% polyacrylarrnide gel shined wifin efinidium bromide. Lane M: 100 bp ladder, 0.1 pg. Lane 1: pCP230 NheI-Pvu2 restriction digest, 0.2 pg (0.1 pmole) labeled at fine Nhel site with tit-355 dCTP. lanes 2 through 5: as in lane 1, but reacted with 3.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 min respectively. Reactions in lanes 3, 4, and 5 were heated before loading. (S) indicates fine labeled lacO substrate fragment. The positions of fine 650 and 80 bp fragment that result from cleavage at fine K-DNA bend have been marked. Panel b: Autoradiogram of fine above gel. Of fine two fragments resulting from cleavage at fine K-DNA bend, only fine 80 bp bp fragment should be labeled, and has been marked. 75 Figure 7. High resolution mapping of hrgets at fine K-DNA bend. Panel a: Autoradiogram of a 6% polyacrylamide-7 M Urea gel. lane C: pCP230 Nhel-Pvu2 restriction digest, 0.05 pg (0.025 pmole) labeled at fine Nhel site wifin a-355 dCTP. Lane 1: as irn lane C, but reacted wifin 2.1 ng (0.04 pmole) of H144 for 60 rrnin. The position and size of fine labeled band resulting to cleavage at the K-DNA bend as shown on figure 6, has been marked. Ianes 3 finrough 6: M13 sequencing ladder, A, C, G, and T respectively. Panel b: Identification of hrgets at fine K-DNA bend sequence. K-DNA bend (dashed line); lacO (solid line). Two major nicks have been marked on fine upper and lower strand wifin large arrows (fi). In addition, four less promirnent nicks are marked on fine upper strand wifin small arrows (4). 75 Figure 7. High resolution mapping of hrgets at fine K-DNA bend. Panel a: Autoradiogram of a 6% polyacrylamide-7 M Urea gel. Lane C: pCP230 Nhel-Pvu2 restriction digest, 0.05 pg (0.025 pmole) labeled at fine Nhel site wifin a—35S dCTP. Iane 1: as irn lane C, but reacted wifin 2.1 ng (0.04 pmole) of H144 for 60 min. The position and size of fine labeled band resulting to cleavage at the K—DNA bend as shown on figure 6, has been marked. Lanes 3 finrougln 6: M13 sequencing ladder, A, C, G, arnd T respectively. Panel b: Identification of hrgets at fine K-DNA bend sequence. K-DNA bend (dashed line); lacO (solid line). Two major nicks have been marked on fine uppe and lowe strand wifin large arrows (fi). In addition, four less promirnent nicks are marked on fine upper strand wifin small arrows (4). ‘ 76 C12345 4‘ 7: r; [f r.‘ a ”co 4 one--- mum“ but“ v ,-. 20 40 60 a n a a a CTAGC'I‘TGCA AGG'I‘AGAA'I‘T‘ AGC'I'I‘A’I‘CGG CCGAGGTGAG AAGGGT'I‘ACT‘ AG‘I‘C‘I‘CG‘I‘AC GATCGAACGT‘ TCCATCT'I‘AA TCGAA’I‘AGCC GGCT‘CCACTC T'I‘CCCAAT'GA TCAGAGCA‘IG i ..‘~ .._-- - 1 100 120 a x‘lll n it it nt- GACGT‘ggAAA AATG'I‘CAAAA AATAGGCAAA AAA'I‘GCCAAA AATGT'I‘CCGC GCACAT'I'I‘CC C‘I‘GCAGGT'I‘T T'I‘ACAGT'I‘TT T'I‘ATCCG‘I'I‘I‘ T'I'I‘ACGGT'I‘T T'I‘ACAAGGCG CG'I‘GTAAAGG 140 160 i i t t k CCGAAAAGT’G CCACCT‘GACG T‘CTAAGAAAC CAT'I‘A’I'I‘ATC A’I‘GACAT'I‘AA CC‘I‘AT‘AAAAA GGCT'I'I'I‘CAC GGT'GGACTGC AGATTC'I'I‘TG GTAATAA’I‘AG TAC’I‘GT‘AA'I'I‘ GGATA'I'I'I'I'I' 180 it 200 220 I t I . TAGGCGTATC ACGAGGCCCT‘ T'I‘CG'I‘C‘I‘I‘CA AGAA‘I‘I‘C‘I‘GT T'I‘CCIEIEIE AAA'I'I‘GT'I‘AT ATCCGCATAG T‘GCTCCGGGA AAGCAGAAGT 'I‘C'I'I‘AAGACA AAGGACACAC T'I'I‘AACAATA Laco ‘ 269 t 28:) ‘ 309 CCGCTCACAA TTCCACACAT TATACGAGCC GGAAGCATAA AGTGTAAAGC CTGGGGT‘GCC GGCGAGTG‘I'I‘ AAGGT‘G’I‘GTA ATA’I‘GCTCGG CCT‘I‘CGTAT'I‘ TCACAT'I‘I‘CG GACCCCACGG 240 at t 320 340 360 i i t i I i TAA’I‘GAGT‘GA GAAT'I‘AA'I'I‘C GG'I'I‘AAT‘ACG ACTCAC'I‘A’I‘A GGAGAACCT'I‘ AAG'I‘G‘I'I‘AA’I‘ AT'I‘ACTCACT C'I'I‘AA'I'I‘AAG CCAAT'I'ATGC T‘GAGTGATAT CCTC'I'I‘GGAA T'I‘CACAA'I'I‘A Figure 7 .‘Q‘n-Q.-‘uu‘.4--.n-u..-a-o4-A g.."~‘-“--‘ J .. ”-o-o-O-O-WQ .0 09-49-01 lean-«sew. ‘g r .. 75 Figure 7. High resolution mapping of hrgets at fine K-DNA bend. Panel a: Autoradiogram of a 6% polyacrylamide-7 M Urea gel. Lane C: pCPZ30 Nhel-Pvu2 restriction digest, 0.05 pg (0.025 pmole) labeled at the Nhel site with o-355 dCTP. lane 1: as irn lane C, but reacted wifin 2.1 ng (0.04 pmole) of H144 for 60 mirn. The position and size of fine labeled band resulting to cleavage at fine K—DNA bend as shown on figure 6, has been marked. lanes 3 through 6: M13 sequencing ladder, A, C, G, and T respectively. Panel b: Identificafion of hrgets at fine K-DNA bend sequence. K-DNA bend (dashed line); lacO (solid line). Two major nicks have been marked on fine upper and lowe strand wifin large arrows (*). In addition, four less promirnent nicks are marked on fine upper strand wifin small arrows (4). :e‘h all! Him 1W 15mlS I 20 t 40 a t i 60 t C'I‘AGC'I'I‘GCA AGG'I‘AGAAT‘T‘ AGCT'I‘A’I‘CGG CCGAGG'IGAG AAGGGT'I‘ACT‘ AGT'CT‘CGTAC GATCGAACGT TCCATCT‘TAA TCGAATAGCC GGCT‘CCACTC TTCCCAAT‘GA 'I'CAGAGCAT'G i CCGAAAAGTG GGCT'I'I'I‘CAC i TAGGCGT‘AT‘C AT‘CCGCATAG LacO . CCGC'I‘CACAA GGC G'I‘G'I'I‘ i 100 120 x 1 1 i t i . GACGPCCAMMMMMMTAGGCAMAWGCCAMMWWCGC GCACA'I'l'rcc C'IGCAGG'I'I‘T T‘T GT‘TTT‘ T'I‘AT‘CCG'I'I'I‘ T'I'I‘ACGGT’I'I‘ TT‘ACAAGGCG CGT‘GTAAAGG 140 160 180 i t I I ' CCACC’I‘GACG TC‘I'AAGAAAC CAT'I‘A'I'I‘ATC ATGACA'I'I‘AA CC‘I‘A'I‘AAAAA GG'ICGAC‘I‘GC AGAT'I‘CI‘T'I‘G GTAATAAT‘AG TAC'I‘G'I‘AAT'I‘ GGATAT'I'I'I'I‘ 200 220 240 i i fi I ‘ ACGAGGCCCT 'I'I‘CGT‘CTT‘CA AGAATTCT‘GT T'TCCT‘G MAT'I‘GTTAT TGCTCCGGGA AAGCAGAAGT TC’I'I‘AAGACA AAGGACACAC T'I'TAACAATA 260 280 300 i I i I . T'I‘CCACACAT TATACGAGCC GGAAGCATAA AGTGTAAAGC TGCC GG'I‘GT‘GTA ATATGC’I‘CGG CCT‘TCGT‘AT'I‘ T‘CACATTTCG GACCCCACGG 320 340 360 t i I t t GAA’I'I‘AA'I'I‘C GG'I'I‘AAT‘ACG ACT‘CAC'I‘ATA GGAGAACCT'I‘ AAGT‘GT'I‘AAT‘ 'I‘AAT‘GAGT‘GA AT'I‘ACT’CACT‘ CT'I‘AAT'I‘AAG CCAAT‘T‘ATGC 'I‘GAGT‘GATAT‘ CCT‘CT'I‘GGAA Figure 7 T'I‘CACAAT'I‘A l . «a. an a; c MH-"“”—.”“.‘..~. “*"U‘ W“#-~MQ~4---~a u. ,- as... u a... g - .- .. - \5 75 Figure 7. High resolution mapping of hrgets at fine K-DNA bend. Panel a: Autoradiogram of a 6% polyacrylamide-7 M Urea gel. Lane C: pCP230 Nhel-Pvuz restriction digest, 0.05 pg (0.025 pmole) labeled at fine Nhel site wifin 0:358 dCTP. lane 1: as in lane C, but reacted wifin 2.1 ng (0.04 pmole) of H144 for 60 mirn. The position and size of fine labeled band resulting to cleavage at fine K—DNA bend as shown on figure 6, has been marked. lanes 3 finrough 6: M13 sequencing ladder, A, C, G, and T respectively. Panel b: Identification of hrgets at fine K-DNA bend sequence. K-DNA bend (dashed line); lacO (solid line). Two major nicks have been marked on fine upper and lower strand wifin large arrows (’). In addition, four less prominent nicks are marked on fine upper strand wifin small arrows (-*). It CTAGCT'IGCA GATCGAACG'I‘ * CCGAAAAGTG GGCT'I'I‘TCAC I TAGGCGTA’I‘C ATCCGCATAG LacO . CCGC'I‘CACAA GGCGAGTGTT i TAA’I‘GAGTGA AT‘T‘AC'I‘CAC’I‘ 76 C12345 20 a 40 s 60 it it a AGGTAGAATT AGC'I'I‘A'I'CGG CCGAGG'I’GAG AAGGG'I'I‘AC'I‘ AGTCTCG'I‘AC TCCATCTTAA TCGAAT‘AGCC GGCTCCAC'I'C 'I'I‘CCCAATGA T‘CAGAGCATG 100 120 xbll 1 t t a a AATGTCAAAA AATAGGCAAA AAATGCCAAA AAT‘G’I'I‘CCGC GCACAT‘T‘T‘CC --- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- G'I'TT‘T‘ T'I‘ATCCGT'I'I‘ T‘T‘T‘ACGGT‘T‘T‘ T‘T‘ACAAGGCG CGTGT‘AAAGG 140 160 180 i i I t * CCACCTGACG ’I‘C'I‘AAGAAAC CAT'I‘A'I'I‘ATC AT‘GACA'I'I‘AA CC'I‘A'I‘AAAAA GG‘ICGACTGC AGAT'I‘CT'I‘TG GTAATAATAG TAC‘I‘GTAAT'I‘ GGATA'I'I'I'I'I‘ 200 220 240 i i i t 1 ACGAGGCCCT ’I‘T‘CGTCT'I‘CA AGAATTCTGT‘ T‘T‘CC‘TEIETQ AAAT'I‘G'I'I‘AT TGCTCCGGGA AAGCAGAAGT T‘CT'I‘AAGACA AAGGACACAC T'I'I‘AACAATA 260 280 300 i t i t I 'I'I‘CCACACAT TATACGAGCC GGAAGCAT‘AA AGTG’I‘AAAGC CTGGGGT‘GCC AAGGT‘GT‘G'I‘A ATAT'GC’I‘CGG CCT‘T‘CGTATT TCACA’I'I'I‘CG GACCCCACGG 320 340 360 i t t i i GAAT'I'AA’I'I‘C GGT'I‘AATACG ACTCACTATA GGAGAACC'I'T AAG'PGT'I‘AAT C'I'I‘AA’I'I‘AAG CCAA’I'I‘A’I‘GC 'I‘GAG’I‘GATAT CC'I‘CT'I‘GGAA T'I‘CACAAT'I‘A Figure 7 77 lane 1, arnd panel b). By labeling fine top strand wifin polynucleotide kinase and repeating this experiment (dah not shown), fine precise location of fine cleavage sites at fine top strand was localized 6 bp to fine left of fine physical center of bending (fig. 7, panel b). In contrast to the lower strand where only a single major cleavage (nick) could be detected, four additional rnicks of decreasing intensity flank fine strorngest nick on fine upper strand. Thus, H144 was shown to cleave fine K-DNA bend at its physical center wifin high prefeence, when fine bend is located on fine sarrne linear dsDNA fragment as lacO. Interestingly, of the four essentially identical and helically phased A-tracts, which have been shown to account for most of fine curvature displayed by finis molecule, only one is cleaved by H144 (fig. 7). Therefore, it appears finat a localized unusual structure at fine center of fine bend, rafiner than fine primary sequence of fine bend dictates cleavage. In finis respect fine center of fine K-DNA bend appears to be structurally different finan fine rest of fine sequences comprising finis bend. The bending locus of the lambda origin of replication is also cleaved by H144 In order to establish finat H144 recogrnizes an unusual structural feature particular to intrinsically bent DNA rafiner finan a specific primary DNA sequence, an unrelated sequence which has also been shown to display intrinsic curvature was tested. The bending locus of finis sequence, fine lambda origin of replication (Aori), was chosen because it does not share great sequence similarity wifin fine K-DNA bend, except for several A4.5 tracts, four of which are in phase wifin each ofiner. Moreover, four binding sites for 7. O-protein, called iterons, are present (Zahn arnd Blattrne, 1985a), and fine physical center of bending for finis sequence is known to be located between iterons N and III (Zahn and Blattrner, 1987). As previously, a plasmid (pCP212) was constructed conhirning fine Mri about 200 bp downstream of lacO (fig. 8, panel a). Restriction fragments of pCP212 were generated such finat fine Lori was located on fine same fragment as lacO. Reaction wifin H144 for increasing amount of time resulted in fine appearance of four prominent bands originating from fine fragment finat 78 Figure 8. Preferential cleavage at lori and at a T7 late promoter by H144. Panel a: Restriction map of fine Nhel-EcoR5 fragment carrying lacO, a T7 late promoter, and Md. The positions of lacO, fine T7 late promoter (T7), and fine iterons comprising tori are irndicated. The numbering of fine iterons is according to Zahn and Blattrner (1987). The Nhel site was labeled with a-3SS dCTP and is marked wifin an asterisk. The positions of strong hrgets has been marked wifin bold triangles (A ). A less preferred hrget is marked wifin a smaller bold triangle (A). Panel b: 6% polyacrylarrnide gel shined wifin efinidium bromide. lane M: 100 bp ladder, 0.1 pg. Lane 1: pCP212 Nhel-PvuZ-ECORS restriction digest, 0.2 pg (0.1 prrnole), labeled at fine Nhel site. Lanes 2: as irn lane 1, but reacted wifin 8.4 ng (0.16 pmole) of H144 for 60 min, stopped and heated before loading. The positions of fine lacO/kori-conhining substrate fragment (S) and four new bands in lane 2 have been marked (450 plus 150 bp, and 290 plus 280 bp apparent molecular sizes). Notice finat fine lacO substrate fragment (577 bp) rrnigrates abnormally slowly. Panel c: Autoradiogram of fine gel in panel b. 79 3. bp 0 100 200 3100 4100 590 600 l l l J Nhe1 Af|2 7L origin ECOR1 T7 Afl2 VI "I II I [ECORl [ECORS lacO A A A b. c 1.2 2200 ~- 1000 600i ~—s— - .__[450] [290] ‘—[280 [ ”[2801 +— 150 ; +— 150' 100 l l I [ l l .I... Figure 8 80 carded lacO (fig. 8, panels b and c, lane 2). By restriction analysis of end-labelled molecules and soufinern hybridization, it was determined finat fine bands migrating at 280 and 290 bp resulted from cleavage at Md. The ofiner two promirnent bands (at 150 and 450 bp) resulted from cleavage at a T7 late promoter which happened to be present in fine vector in which fine phage A sequence was subclorned (Panayohtos and Backman, 1989). The exact DNA bases at which cleavage occurred within fine T7 late promoter and at Md were determined wifin basepair resolution on denaturing gels (dah not shown). By finis analysis, fine T7 late promoter was nicked at two positions approximately one helix turn apart, at coordinates 139 and 149 on fine top strand, and a unique position, at coordinate 145 on fine lower strand (fig. 9). The two preferred hrgets wifinin Md were mapped at positions 265 and 278 on fine upper strand and positions 268 and 278 on fine lowe strand. These positions are approximately one helical turn apart and fall wifinin fine GC-dch region of iterons III and IV, but outside fine A-tract repeats wifinin Md. This region coincides wifin fine physical center of bending at Md. An alignment of fine primary DNA sequence surrounding the hrget sites at Md, 1(- DNA, T7 late promoter, B-lachmase promoter, and gyrase site, revealed no sequence identity at any particular position wifin eight out of fourteen hits being fine most frequent occurrence (table 1). Five of the fourteen sequences encompassing the hrgets share the degenerate pentanucleotide C'I‘AlA/TliA/T} immediately 3' to fine nick (positions 11 finrough 13), but finis sequence also exists in several ofiner locations where it is not a preferred hrget. Anofiner corrnrnon sequence, fine A-tract repeats at K-DNA and Md, were not uniformly recognized by H144. Therefore, sequence homology does not appear to play a major role in hrget recognition, even finougln fine CI‘A{A/THA/T} penhnucleotide may be a preferred scission site wifinin fine context of a hrget. To test fine possibility finat DNA modification may be involved in hrget recognition, experiments were carded out wifin substrate fragnents generated by PCR, and finus devoid of modified bases. Identical results were obtained wifin finese synfinetic substrates, w ‘..§‘:iIlIito . . I III...rrfrfv'lfsitrfieraIiisfiitivrfflr 81 '20 40 a . * * * Ljfl(:() * CTAGCTTGCA AGGTAGAATT CTGTTTCCTG TGTGAAATTG TTATCCGCTC GATCGAACGT TCCATCTTAA GACAAAGGAC ACACTTTAAC AAIAGGCGAG 60 80 100 a a t a * ACAAITCCAC ACAITRIACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGTTAAGGTG TGTAAIEEGC TCGGCCTTCG TATTTCACAT TTCGGACCCC 120 . 'k * T7 * mac-marsh mam arrcccma raggagrcacrara ACGGATTACT CACTCTTAAI TAAGCCAATT ATGCTGAGTG ATATC 1k 200 a a * CCTTAAGGTT TAACTTTRAG ACCCTTAAGT GTTAATTAGA GATTTATTAT GGAAITCCAA.AITGAAAITC TGGGAATTCA CAATTAATCT CTAAATAAIA 160 180 * t 220 240 a n * 'k * CAAGCAGCAA GGOGGCAIGT TTGGACCAAA TAAAAACATC TCAGAATGGT GTTCGTCGTT COGCCGTACA.AACCTGGTTT ATTTTTGTAG AGTCTTACCA . 300 w .. i. m . . GCATCCCTCA AAAC GGGA AAATCCCCTA AAACGAGGGA TAAAACATCC CGTAGGGAGT TTTGCTCCCT TTTAGGGGAT TTTGCTCCCT ATTTTGTAGG i 320 340 II * t t I * * CTCAAATTGG GGGATTGCTA TCCCTCAAAA CAGGGGGACA CAAAAGACAC GAGTTTAACC CCCTAACGAT AGGGAGTTTT GTCCCCCTGT GTTTTCTGTG 360 380 400 t a t e * TATTACAAAA GAAAAAAGAA AAGATTATTC GTCAGAGAAT TCTGGCGAAT ATAATGTTTT CTTTTTTCTT TTCTAATAAG CAGTCTCTTA AGACCGCTTA ' -"' v-4 A .4 ,4 -0‘J—J.’-J—J-J..-44 Figure 9. Identificatiorn of hrgets at fine Mri bend and fine T7 late promoter. The positions of lacO, fine T7 late promoter, and Md are shown. The four iterons of Md are marked by a dashed line and numbered wifin Latin clnaracters (Zahn and Blattrner, 1987). The major cleavages on both strands of iteron IV are marked by large arrows (’). A medium strengtln cleavage in iteron III is marked wifin small arrows (—*). .4' a ‘4 .a‘_J .4 .a J .4 .4‘-4 -4' Q-Q-I-IT""‘ ~r-‘aec' .a-JJ . J 82 Table 1 Alignment of H144 hrget sequences i i 1 S'GAAGCATTTA/TCAGGGTTAT 2 5'GACAATAACC/CTGATAAATG 3 5'ATTTGAATGT/ATTTAGAAAA 4 S'GTTTATTTTT/CTAAATACAT 5 S'ATACGACTCA/CTATAGGAGA 6 5'CTATAGGAGA/ACCTTAAGGT 7 S'TAAGGTTCTC/CTATAGTGAG 8 5'AGAAAAATAA/ACAAATAGGG 9 S'AAATGTCAAA/AAATAGGCAA 10 5'CATTTTTTGC/CTATTTTTTG 11 5'CCTCAAAACC/AGGGAAAATC 12 5'GGGGATTTTC/CCTCGTTTTG 13 5'GGAAAATCCC/CTAAAACGAG 14 5'CTCGTTTTAG/GGGATTTTCC #ofA 4573764435/5185846463 #ofT 1545277842/1726455443 #ofG 5314411032/1232252436 #ofC 4122102245/7411001212 Key: Major H144 hrget sequences are shown 5' to 3'. Ten nucleotides on each side of fine nick (/ ) are shown. Code numbers refer to sequences from fine following hrget sites: (1) B-lactamase promoter transcription shrt site - upper strand; (2) B-lachmase promoter transcription shrt site - lower strand; (3) B-lachmase promoter -35 element - upper strand; (4) B-lactamase promoter -35 element - lower strand; (5) T7 late promoter - upper strand (hrget at coordinate 139 on fig. 9); (6) T7 late promoter - upper strand (hrget at coordinate 149 on fig. 9); (7) T7 late promoter - lowe strand (hrget at coordinate 145 on fig. 9); (8) gyrase site - uppe strand; (9) 1(- DNA - upper strand; (10) K-DNA - lower strand; (11) Md iteron IV - upper strand; (12) Mri iteron IV - lower strand; (13) Md iteron III - upper strand; (14) Md iteron III - lower strarnd. # of A, T, G, or C refes to fine tohl number of each particular base occurdng at each position in fine 14 ssDNA sequences. 83 irndicating finat DNA modification does not play a role in hrget recognition. Therefore, unusual DNA structure(s) must be fine major feature recognized at fine hrgets. . The hrgets for H144 in bofin Md and K-DNA coincide wifin fine physical center of fine bend. Therefore, fine physical center of fine bend must display fine structural feature recognized by H144. This structural vadability is a novel feature of intrinsically bent DNA. Furfinermore, because of fine nature of fine T7.3 domain of H144, finis unusual structural feature most likely displays single-stranded character (see Discussion). 20-protein inhibits cleavage at fine Md To test fine possibility finat protein-induced bending could also serve as a hrget for H144, 20-protein (20; fine gift of Dr. Ross B. Inrrnan) was bound to Md (fig. 10, lane O). It has been shown finat when 20 birnds to fine iterons wifinin Md, fine site is bent furfiner (Zahn and Blattrner, 1987). Thus, it was expected finat irncreased bending by 20 at Md would provide a better target for H144. However, inclusion of 20 in finese reactions specifically inhibited cleavage at Md (fig. 10, lane H/O). 20 did not inhibit H144 action on anofiner hrget, a T7 late promoter, located on fine same fragment. Therefore, inhibition of cleavage at Md when 20 is present is most probably due to stedc hindrance by 2.0 bound at Md. Mcshho 2200 1 500 1000 s 600 ._[450] =[290] [280] ~—[1 50] 1 00 Figure 10. The effect of 2.0 on cleavage at Md. 6% polyacrylamide gel shined wifin efinidium bromide. Lane M: 100 bp ladder, 0.1 pg. Lane C: pCP212 Nhel-AatZ-ECORS restriction digest, 0.2 pg (0.1 pmole). Lane B: as in lane 2, but incubated with 25.2 ng (0.48 pmole) of H144 wifinout MgClz. lane H: as in lane 3, but reacted in the presence of MgClz for 15 min, stopped and heated before loading. Lane H/O: as in lane 4, except that 14.6 ng (0.438 pmole) 20 was included in fine reaction. lane 0: as in lane 2, except finat fine DNA was incubated with 14.6 ng 20 in reaction buffer for 15 min before loading. The positions of fine lacO/Mri-containing substrate fragment (S), fine 280 and 290 bp bands resulting from cleavage at Md, and fine 150 and 450 bp bands resulting from cleavage at the T7 late promoter, have been marked. DISCUSSION H144 as a probe for structural variability in bent DNA and other sites The strong prefeence exhibited by H144 towards cleavage at intrinsic bends, makes H144 a unique enzymatic probe for intrinsically bent DNA. The requirement for fine physical presence of lacO on fine same fragnent imposes some restrictions on fine usefulness of H144. Moreove, a requirement for appropdate phasing of the K-DNA bend wifin respect to fine opeator has been observed (see Chapter 3). These two requirements are direcfiy related to fine mecharnism by which H144 accesses its hrgets and are presented in fine next chapter. In addition to fine two intrinsic bends, four ofiner sequences - fine transcdption shrt site and -35 elenent of fine B-lachmase promoter, a T7 late promote, and a site recognized by gyrase in vitro - wee also cleaved prefeentially. Overall, thee is litfie pdmary sequence homology among fine hrgets. Thus, fineir reactivity must arise from fine presence of a structural element(s) which constitutes a target(s) for H144. At least in the case of intrinsic bends, fine unusual configuration postulated to be present at fineir physical center seems to be fine structure recognized. This conclusion is consistent wifin fine observation finat fine B-lactamase promoter and gyrase site wee located wifinin fragments finat displayed rehrded electrophoretic mobility on polyacrylamide gels, a property that has been associated with the presence of intrinsic curvature. Thus, it appeared finat at least part of finese sequences could be intrinsically bent. This obsevation is furfiner supported by fine fact finat irntrirnsic bends have been found in fine upstream region (between -40 to -150 bp) of several E. coli promoters (Plaskon and Wartell, 1987), and have been postulated as a possible conformation of TATA boxes (Koo et al., 1986). In contrast, however, fine region encompassing fine T7 late promoter, which is also a good hrget for 85 86 H144, appeared to be straight bofin in gel rrnobility assays, and by computer modeling (dah not shown), alfinougln kinking of fine helix could not be ruled out. Nonetheless, all the sequences hrgeted by H144 share a common property: finey interact wifin DNA-binding proteins. More specifically, fine Md interacts with O-protein, fine B-lactamase and T7 late promoters interact wifin fineir respective polymerases, and fine gyrase conhct site interacts wifin a bactedal gyrase. In addition, intrinsic bends are finought to be preferred sites for topoisomease action (Caserh et al., 1989; Camilloni et al., 1991), and fine related K—DNA bent region of Chrithidia fasciculata mirnicircles is nicked by a nuclease isolated from finat trypanosorne (Lirnial and Shlomai, 1987; Linial and Shlomai, 1988). Altered (non-B) sequence-dependent conformations at finese furnctional sites may serve as recognition signals and/ or entry points for relevant proteins. The physical center of intrinsic bends may exhibit single-strandedness The nucleolytic domain of H144 is known to exhibit high preferernce towards ssDNA or single-stranded regions wifinin duplex molecules regardless of fineir pdrrnary sequence. Thus, cleavage by H144 at fine physical center of intrinsic bends, suggests fine presence of a localized single-stranded conformation in finat region, or some ofiner structural feature finat renders fine hrget a preferred cleavage site for finis nuclease. Ofiner endonucleases, which like T7.3 (or H144) preferentially cleave ssDNA are the mung bean, P1, and SI nucleases (see Chapter 1). Interestingly, thee was no appreciable cleavage of intrinsically bent DNA by T7.3, or by fine 51, P1, and mung bean nucleases (Marini et al., 1984; Zahn and Blattrner, 1985b; Kitchin et al., 1986; Caddle et al., 1990). It is possible finat under fine conditions employed for reactions wifin 81 (low pH, Zn”), bent DNA adopts a different conforrrnation finat renders it a poor hrget for 51. Alternatively, 81, P1, or mung bean nuclease may have been unable to cleave bent DNA prefeentially, much like T73, or H144 in fine absence of lacO (see Panayohtos and Backman, 1989; and Chapter 3). The chemical probe 87 brorrnoacehldehyde, finat preferably reacts with ssDNA, also failed to modify an intrinsic bend from C. ' fasciculata (Kitchin et al., 1986). Howeve, fine presence of single-stranded regions in bent DNA has been postulated by fineoretical molecular modeling (Ramstein and lavery, 1988). This model proposes finat DNA bending facilihtes base-pair opening, or alternatively, finat bending and base-pair opening are enegetically coupled. In finis model only single base-pair opening, which may not be detected by a ssDNA endonuclease (Dodgson and Wells, 1977), was studied, but cooperative interactions between sequences finat contribute to bending and may give dse to larger scale or more shble opening, wee not ruled out. Nonefineless, cleavage at fine center of intrinsic bends by a ssDNA endonuclease is consistent wifin finis model. LITERATURE CITED Bradford, M. M. (1976). A rapid and sensitive mefinod for the quantitafion of microgram quantities of protein utilizing fine pdnciple of protein-dye birnding. Anal. Biochem. 72, 248-254. Brosius, J., Cate, R. L., arnd Pelmutter, A. P. (1982). Precise location of two promoters for fine 8- lachmase gene of pBR322. J. Biol. Chem. 257, 9205-9210. Caddle, M. S., Lussie, R. H., and Heintz, N. H. (1990). Intramolecular DNA triplexes, bent DNA and DNA unwinding elements in fine initiation region of an amplified dihydrofolate reductase replicon. J. Mol. Biol. 211, 19-33. Camilloni, G., Caserta, M., Amadei, A., and Di Mauro, E. (1991). The conformation of constitutive DNA interaction sites for eukaryotic DNA topoisomerase I on intrinsically curved DNAs. Biochem. Biophys. Acta 1129, 73-82. Caserta, M., Amadei, A., Di Mauro, E., and Camilloni, G. (1989). In vitro preferential topoisorrnerization of bent DNA. Nucleic Acids Res. 17, 8463-8474. Dodgson, I. B., and Wells, R. D. (1977). Action of single-strand specific nucleases on model DNA heteroduplexes of defined size arnd sequence. Biochemistry 16, 2374-2379. Innis, M. A., and Gelfand, D. H. (1990). Optimization of PCRs. In PCR Protocols. M. A. Innis, D. H. Gelfand, I. I. Sninsky and T. J. White, eds. (San Diego, California 92101: Academic Press, Inc.). 3-12. Kirkegaard, K., and Wang, 1. C. (1981). Mapping fine topology of DNA wrapped around gyrase by nucleolytic and clnerrnical porbing of complexes of unique DNA sequences. Cell 23, 721-729. Kitchirn, P. A., Klein, V. A., Ryan, K. A., Gann, K. L., Rauch, C. A., Kang, D. S., Wells, R. D., and Englund, P. T. (1986). A highly bent fragment of Cdfinidia fasciculata kinetoplast DNA. J. Biol. Chem. 261, 11302-11309. Koo, H. S., Drak, J., Rice, 1. A., and Crofiners, D. M. (1990). Determination of fine extent of DNA bending by an adenirne-finymirne tract. Biochemistry 29, 4227-4234. Koo, H. S., Wu, H. M., and Crofines, D. M. (1986). DNA Bending at adenine-finymine tracts. Nature 320, 501-506. Linial, M., and Shlomai, I. (1987). The sequence-directed bent structure in kinetoplast DNA is recognized by an enzyme from Crithidia fasciculata. J. Biol. Clnerrn. 262, 15194-15201. Linial, M., and Shlomai, I. (1988). A unique endonuclease from Chrithidia fasciculata which recognizes a bend irn fine DNA helix. Specificity of fine cleavege reaction. J. Biol. Chem. 263, 290-297. 88 89 Marini, I. C., Effron, P. N., Goodman, T. C., Singleton, C. K., Wells, R. D., Wartell, R. M., and Englund, P. T. (1984). Physical characterization of a kinetoplast DNA fragment wifin unusual properties. J. Biol. Chem. 259, 8974-8979. Marini, I. C., Levene, S. D., Crofiners, D. M., and Englund, P. T. (1982). Bent helical structure in kinetoplast DNA. Proc. Nafi. Acad. Sci. U.S.A. 79, 7664-7668. Merril, C. R. (1990). Gel-shining techniques. Mefinods Enzymol. 182, 477-488. Pace, H. C., Lu, P., and Lewis, M. (1990). lac repressor: Cryshllization of inhct tetrarrner arnd its complexes wifin induce and operator DNA. Proc. Nafi. Acad. Sci. U.S.A. 87, 1870-1873. Panayohtos, N ., and Backman, S. (1989). A Site-hrgeted Recombinant Nuclease Probe of DNA Structure. J. Biol. Clnem. 264, 15070-15073. Panayohtos, N ., Fonhine, A., and Backman, S. (1989). Biosynfinesis of a repressor/ nuclease hybdd proteirn. J. Biol. Chem. 264, 15066-15069. Panayohtos, N., and Wells, R. D. (1979). Recognition and initiation site for four late promoters of phage T7 is a 22-base pair DNA sequence. Nature 280, 35-39. Panayotatos, N., and Wells, R. D. (1981). Cruciform structures in supercoiled DNA. Nature 289, 466-470. Plaskon, R. R., and Wartell, R. M. (1987). Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters. Nucleic Acids Res. 15, 785-796. Ramstein, J., and Lavery, R. (1988). Enegetic coupling between DNA bending and base pair opening. Proc. Nafi. Acad. Sci. U.S.A. 85, 7231-7235. Rosa, M. D. (1979). Four T7 RNA polymerase promoters conhin an identical 23 bp sequence. Cell 16, 815-825. Sambrook, J., thsch, B. F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory). Scopes, R. K. (1982). Protein Pudfication. Principles and Practice. (New York: Springer-Verlag New York, Inc.). Stellwagen, E. (1990). Gel filtration. Mefinods Enzymol. 182, 317-328. Wu, I-L-M., and Crofiners, D. M. (1984). The locus of sequence-directed and protein-induced DNA bending. Nature 308, 509-513. Zahn, K., and Blattrner, F. (1987). Direct evidence for DNA bending at fine lambda replication origin. Science 236, 416-422. Zahn, K., and Blattrne, F. R. (1985a). Binding and bending of fine lambda replication odgin by fine phage 0 protein. Embo J. 4, 3605-3616. Zahn, K., and Blattrne, F. R. (1985b). Sequence-induced DNA curvature at fine bacteriophage 2 odgin of replication. Nature 317, 451453. CHAPTER3 PREFERENTIAL CLEAVAGE OF INTRINSIC BENDS BY A RECOMBINANT NUCLEASE IS MEDIATED BY DNA LOOPING BETWEEN THE BINDING SITE OF THE NUCLEASE AND ITS TARGET 90 ABSTRACT The mecharnism by which the recombinant nuclease H144 cleaves linear double- stranded DNA at intrinsic bends was examined, and compared wifin T7.3 endonuclease. Bofin fine physical presence of fine lac operator (lacO) on fine same fragment as fine bent DNA and direct binding of H144 to lacO were found to be required for preferential cleavage to occur at fine bend. Eifine physical separation of 1:100 from fine hrgets or fine inclusion of Lac repressor in fine reaction resulted in inefficient cleavage of bent DNA. In contrast, fine T7.3 endonuclease was found to exhibit no particular preference for bent DNA ove ofiner hrgets regardless of fine presence of lacO. H144 preferentially cleaved bent DNA alfinough fine dishnce between fine bent DNA target and lacO was beyond fine physical reach of lacO-bound H144, assuming finat fine DNA helix is extended. However, cleavage was strongly dependent on fine exact dishnce between fine bend and lacO. Varying fine number of base-pairs (bp) separating fine bent DNA from lacO by multiples of 5 bp, altemately promoted or greafiy reduced cleavage, indicating a strong dependence of cleavage on helical phasing. These results are consistent wifin a mechanism by which H144 binds at lacO finrough its repressor domain, and cleaves when DNA looping brings a hrget wifinin reach of fine nucleolytic domain. 91 INTRODUCTION A recombirnant hybdd protein (Hl44), consisting of fine lac repressor (LacI) and fine 17.3 endonuclease (T73), cleaves linear double-stranded DNA (dsDN A) preferentially at hrget sites finat coincide wifin fine B-lachmase promoter, a phage T7 late promoter, a gyrase conhct site, arnd two non-horrnologous intrinsic bends (see Clnapter 2). The mechanism by which H144 locates its targets is unknown, but H144 binds at lacO, like lacl, and fine presence of lacO on fine same restriction fragment as fine hrgets is required for appreciable cleavage to occur (Panayohtos et al., 1989). However, fine dishnce of fine operator from fine nearest hrget is several fold greater than the effective radius of H144 even if the protein binds at lacO as a tetramer. This observation raised fine possibility finat looping of fine DNA region between 1:100 and fine hrgets is the mechanism by which lacO-bound H144 accesses its hrgets (Panayohtos arnd Backman, 1989). Protein-induced DNA loops form when two or more molecules of a DNA-binding protein bind sirnulhneously and bddge two specific sites (cognate sites) on fine DNA (Dunn et al., 1984; Hochsclnild and Ptashne, 1986). Looping, which was first demonstrated for prokaryotic repressor/ operator systems, is now known to play imporhnt mechanistic roles not only in transcdptional regulation, but also in recombination, replication, and DNA restriction (reviewed in Matthews, 1992). When fine dishnce between two cognate sites is less finan 20 helical turns, and when fine DNA-binding protein pdrrnadly conhch one face of the DNA helix, fine cognate sites must be on fine same helical face of fine DNA helix ("in phase") in orde for effective looping to occur (Hochsclnild, 1990) . This necessity for phasing is due to fine fact fiht loop formation between two sites finat are in phase requires only curving of fine helix. In contrast, looping between sites finat are out of phase requires not only curving but also twisting 92 93 and / or wrifining of fine helix, which are enegetically unfavorable, and particularly so for small DNA segnents. It is difficult to distinguish looping from an alternative nnechanism in which a DNA- binding protein (e.g. a repressor) first binds at its cognate site (e.g. an Operator) and then polymerizes along the DNA helix. Conclusive evidence for DNA loop formation in repressor/operator systems such as LacI/lacO (Kramer et al., 1987), AraC/araO (Dunn et al., 1984), or lambda repressor/0R (Hochschild and Phshne, 1986), has been obtained by "phasing expedments". In these expedments, the distance between two operators was varied by increments of approximately one-half turn of fine helix (5 bp), and then repression of transcdption was measured in viva (Dunn et al., 1984). As mentioned above, for looping to occur fine two operators must be in phase when fine dishnce between finem is small. Indeed, effective repression was achieved only when fine two operators where in phase. The same dependence on phasing was observed in vitro, by derrnonstrating finat DNaseI cleaves fine curved segnent of fine loop formirng between two operators wifin a periodicity of approximately 5 bp as would be expected for bases present on fine outside of fine curve (Hochschild and Phshne, 1986). Direct observation of loops with electron microscopy (EM) verified fine necessity for appropriate phasing for loop formation (Gdffifin et al., 1986). If looping is also necessary for cleavage at dish] hrgets by H144 bound at lacO, finen finis reaction should exhibit a dependence on fine helical phasing of fine operator wifin respect to a hrget. If, however, H144 binds at lacO, and finen polyrrnerizes along fine helix until it can locate a hrget, finen helical phasing should not affect fine reaction. To test the above hypothesis, it was first necessary to establish that cleavage at a hrget near lacO, is cahlyzed by H144 bound at lacO. Subsequenfiy, fine effect of phasing was tested wifin molecules in which the dishnce of the K-DNA bend from lacO differed by intervals of 5 bp. MATERIALS AND METHODS Plasmids Plasmids pCPSZ, pCR1000 (Invitrogen), pCP207 and pCPZ30 have been descdbed (see Chapter 2). In order to change fine phasing of fine K-DNA bend wifin respect to lacO, fine bend was subcloned into pCP207 or pCP230. The relevant regions encompassing fine K-DNA bend and IMO are shown diagrammatically (fig. 11). Plasmid pCP232 carries fine K-DNA bend placed 5 bp further away from lacO finan in pCPZBO. In addition, pCP232 has 14 bp between its unique Nhel site and K-DNA finat are different from fine pCPZBO sequence, which has an Spel site in fine corresponding position. Plasmid pCP232 was constructed by employing primers Bent 2 (see below), 82.2, and R812, as descdbed for pCP230 in Chapter 2. Plasmid pCPZ33 was dedved from pCP232 by restricting with EcoRI, filling in and religating. Plasmid pCP234 was constructed using pdmers Bent 6 and 82.7, and plasmid pCP82 as a template for PCR. The amplified fragment was exchanged as an Spel-BcoRl fragment wifin fine corresponding region in pCPZ30. In finis construct, fine K-DNA bend was placed 5 bp closer to lacO in comparison to pCP230. Plasmid pCP235 was constructed by fine same method as pCP234, except that primer Bent 7 was utilized instead of Bent 6. In pCP235, fine K-DNA bend was placed 10 bp close to lacO irn comparisorn to pCP230. Two additional plasmids, pCP236 and pCP237, were designed, carrying lacO in revese orienhtion wifin respect to fine K-DNA bend in compadson to pCP230. The operator in pCP237 is 5 bp farfiner away from fine bend than in pCP236. Plasmid pCP236 was constructed by 94 95 bp PO 1 00 200 300 400 500 600 700 8010 888 [Ssp‘l [NheL1 [EcoFH [Afl2 [Pvuz l__l BJac P K-DANA I860 T7 Nhe 1 EcoR 1 All 2 Pvu 2 681 I E [Iran—lI I A 17 K-DNA ”“0 Nhe 1 EcoFl 1 Atl 2 Pvu 2 348 [ E [H J A 17 K-DNA [“0 I -10 El -35 0 GYR El IacO I T7 1.p. -K-DNA A target Figure 11. Physical map of fine region conhirning fine K-DNA bend and 1:100 in plasmids pCPZBO, pCPZ32, pCP233, pCPZ34, pCP235, pCPZ36, and pCP237. The position of fine K-DNA bend (K-DNA), lacO, a T7 late promoter (T7), and fine position in which insertions or deletions (in bp) have been made in each plasmid in order to alter the helical phasing of fine K-DNA bend wifin respect to lacO, are shown. The odentation of lacO is indicated by a bold lnorizonhl arrow. 96 exchanging an EcoRl-Afl2 fragment that carries lacUVS in pCP230 wifin an amplified fragment. Two pdrrners, lacl and lac2, were used to amplify fine operator region. The amplified fragment was restricted wifin Afl2 and EcoRI, and ligated into pCP230 restricted wifin fine sarrne enzymes. Plasmid pCP237 was constructed by fine same mefinod, except finat primer lac3 was used instead of lac2. Plasmid pCPZZ6 (fig. 12, panel a) was constructed by deleting fine region encompassing fine lacUVS promoter from a pCP230 dedvative. The constructs were checked by DNA sequencing of fine regions conhining inserts, as well as by restriction analysis. DNA primers Pdmers were fine gift of fine DNA Core Facility, Regeneon Pharmaceuticals, Inc. Primer Bent 2 (5'-CGT ACG ACG T'CC AAA AAT GTC AAA AAA TAG GCA AAA AAT GCC AAA AAT TAG GGG TI‘C CGC GCA CAT TT-3') conhins fine bending locus and a region homologous to plasmid pCP82 upstream of the -35 element of fine B-lactamase promoter. Pdrrners Bent 6 (5'-AGG GTT ACT ACT CTC GTA CGA CGT CCA AAA ATG TCA AAA AAT AGG CAA AAA ATG CCA AAA ATG CGC ACA TIT CCC CGA AAA G-3') and Bent 7 (5'-AGG GTTACTAG’TCTCGTACGACGTCCAAAAATGT‘CAAAAAATAGGCAAAAAAT’GCCA AAA ATC ATT TCC CCG AAA AGT GCC-3') encompass fine Spel site in pCP230, followed by fine K-DNA bend and pCP82-dedved sequence from fine region near fine B-lachmase promoter. Alfinough fine K-DNA bend sequence is identical in finese two primers, fineir pCP82-dedved sequence differs in a manrner such that Bent 7 hybddizes close to lacUV5 in pCP82 by 5 bp. Pdmer 82.7 (5'-AAC CIT AAG GTT CTC GAG TAG TGA GTC-3') is homologous to a region of pCP82 located near fine T7 late promoter except for finree nucleotides at fine 5' end of fine sequence. Primers lacl (5'-CIT CAA CIT AAG T‘GT TTC CTG TGT GAA ATT-3'), and lac2 (5'-CGG CTCGAATI‘CTGI‘GTGGAATTGT‘GAGCG—B')andlac3(5'-GCI‘TCCGAATTCTATAATGTG 97 Figure 12. Binding and cleavage by H144. Panel a: Physical map of fine Nhel-PvuZ fragment of pCP230, and Sspl-PvuZ fragment of pCP226. Bold triangle (A) marks fine position at which H144 cleaves K-DNA. Ofiner symbols as in figure 11. Panel b: Effect of lacO on cleavage at fine K-DNA bend by H144. 6% polyacrylamide gel shined wifin efinidium bromide. Lane M: 100 bp ladder, 0.1 pg. lane 1: pCP230 Nhel-Pvu2 restriction digest, 0.2 pg (0.1 pmole). Lanes 2 finrough 5: as in lane 1, but reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 min respectively. Lane 6: pCPZZ6 Sspl-PvuZ restriction digest, 0.2 pg (0.1 pmole). lanes 7 finrough 10: as in lane 6, but reacted wifin 8.4 ng of H144 for 0, 15, 30, and 60 min respectively. Reactions in lanes 3 finrough 5 and 8 finrough 10 were heated before loading. The positions of fine 650 and 85 bp fragnents resulting from cleavage at fine K- DNA bend are shown. Brackets indicate apparent size. (S) pCP230 Nhel-Pvuz lacO/K-DNA fragment; (S') pCP226 Sspl-Pvu2 K-DNA fragment. The right half of fine gel has been oveexposed to better show fine fragnents resulting from treatment wifin H144. Panel c: Effect of LacI on cleavage at fine K-DNA bend by H144. 6% polyacrylamide gel shined wifin efinidium bromide. Lane M: 100 bp ladde, 0.1 pg. Lane 1: pCP230 Nhel-Pvuz restriction digest, 0.2 pg (0.1 pmole). Lane 2: as in lane 1, plus 16.3 ng (1.1 pmole) lacI was included. lane 3: as in lane 1, plus 16.3 ng (1.1 prrnole) LacI and 16.8 ng (0.32 pmole) H144 were incubated wifin fine DNA for 60 min wifinout MgClz, and finen heated at 75°C for 10 rrnin. Lanes 4 finrough 8: as in lane 1, except finat 0.21, 0.42, 1.1, 2.2 and 0 pmole LacI was prebound to fine DNA. Subsequenfiy, 0.32 pmole H144 was added, and incubated for 60 min at 37°C. Reactions in lanes 3 finrough 8 were heated before loading. Symbols as in panel b. 2200 1 000 600 98 a. 0 100 200 300 400 500 600 700 I l l l l I l I Nhe1 EcoR1 A112 [Pvu2 K—DNA 1300 T7 S 1 Nh1 Nhe1 P 2 sp 6 I vu I Jr. J K-DNA b. M2468 I I III IIIIIIII 100 ' signs 12 .._4..4‘. ..a-<.4-—4.v r.vlr|.rl['."t'-'l'tfrrrrr['n (I 'I. v 98 a. P 100 200 300 400 500 600 700 Nhe1 EcoR1 Af|2 [Pvu2 K-DNA [360 T7 Nhe1 [Ssp1 Nhel I [Pvu2 KENA b. 2200 M 2 4 M6'810 1000 600 f 100 - M 2 4 6 8 -~U~HHHUO— H... Figure 12 99 TGG AA TTG T-3'), are homologous to sequences flanking M60, and carry an Afl2 or EcoRl hil respectively. Reactions utilizing Lac repressor protein lac repressor protein (lacI) was fine gift of Drs. H. C. Pace and P. Lu (Pace et al., 1990) . It was provided at a concentration of 32.6 pg / pl in 1 M Tris-HCl, 40% glycerol, 28 mM 2- rrnercaptoefinanol, pH 7.6 at 4°C. Less concentrated solutions of [ad were prepared in 50 mM Tris pH 7.5, 50 mM NaCl, 10% glycerol, 0.1 mg/ml acetylated BSA by serial dilutions. This preparation of lacI was more finan 90% pure (H. C. Pace, personal communication) and did not conhin detechble levels of conhminating endonucleolytic or exonucleolytic activities unde fine reaction conditions employed (dah not shown). The same preparation has been utilized in transcription inhibition assays (Lee and Goldfarb, 1991; and A. Goldfarb, personal communication). Reactions wifin H144 and Lacl were performed as descdbed in Clnapter 2, except finat glyceol was included to a final concentration of 5%. After fine reactions wee stopped finey were heated at 75°C for 10 rrnin to denature fine proteins arnd reverse binding. Other matedals and enzymes All ofine materials, enzymes and mefinods utilized in finis work have been described in fine previous chapters. Bindi niece Iowa DN/ 5). exp pan the aPl in ad at Ia. int: RESULTS Binding of H144 to the lac operator mediates cleavage at an intrinsic DNA bend In orde to determine whefiner fine presence of lacO on fine same restriction fragment is necessary for fine efficient cleavage of intrinsically bent DNA, fine activity of H144 was tested towards restriction fragments from two plasmids (pCP226 and pCP230), finat carry fine sarrne K- DNA bend and are nearly identical except for a 127 bp deletion of lacO in pCPZZ6 (fig. 12, panel a). After 15 min of incubation, H144 preferentially cleaved fine Nhe1-Pvu2 fragment that carded lacO at fine K-DNA bend, as evidenced by fine appearance of fine two bands (650 and 85 bp) expected from cleavage at fine center of fine K-DNA bend (fig. 12, panel b, lanes 3 finrougln 5). In contrast, fine fragment carrying K-DNA but not lacO was not cleaved appreciably into fine expected size fragments (220 bp and 710 bp) even after 60 rrnin of irncubafion wifin H144 (fig. 12, panel b, lanes 8 finrougln 10). In ofiner experiments where fine K-DNA bend was separated from the operator by restriction, the fragment carrying fine K-DNA bend was not cleaved appreciably (dah not shown). These results indicate finat lacO must be present on fine same fragment as fine bend for efficient cleavage at fine bend by H144, and suggest finat cleavage is mediated by enzyme bound to lacO. To test finis possibility, lac repressor proteirn (lacI) was mixed wifin fine DNA before addition of H144. Under finese conditions, LacI competed wifin H144 for birnding at lacO. LacI at 1.1 pmole was sufficient to bind most of fine 0.1 pmole of lacO fragment (fig. 12, panel c, lane 2), and approximately 50% inhibition of specific cleavage was observed when finat amount of Led competed wifin 0.32 pmole H144 for binding at lacO (fig. 12, panel c, lane 6). The inhibition irncreased as fine amount of [ac] increased. This was evidenced by a decrease in fine intensity of fine 650 bp fragment (fig. 12, panel c, lanes 4 finrough 7). Thus, occupancy of lacO by 100 101 LacI inhibited specific cleavage at fine K-DNA bend by H144. The same pattern of inhibition was also observed for cleavage at ofiner prefered hrgets (dah not shown). These results confirm finat fine requirement for 11100 on fine same fragment as a preferred hrget is due to fine fact finat cleavage at fine hrgets is mediated by H144 bound at lacO. Lacl also resulted in an increase of non-specific cleavage, as evidenced by fine stronger appearance of several new bands. This is not surpdsing, because in fine presence of LacI, a higher fraction of H144 not bound to lacO is available to cleave at other targets. No conhminating endonucleolyfic or exonucleolyfic activity could be detected in fine preparation of [ad employed (dah not shown). Comparison with T7.3 Consistent wifin finese results was fine observation finat pudfied T73, which does not have a lacO—binding domain, could not cleave at fine K-DNA bend efficienfiy. When fine Nhe1-Pvu2 restriction fragments of pCP230 were reacted with T73, many products were obhined in addition to fine fragnents odginating from cleavage at fine K-DNA bend (fig. 13, lanes 7 through 9). As would be expected from a random interaction of an endonuclease with a substrate, the larger fragnents were cleaved first. Thus, fine K-DNA bend was not a preferred target for T73 endonuclease. This result indicates an imporhnt role for lacO instrand scission by H144 nuclease. Further compadson of fine activity of H144 and T73 points to an interesting difference in fineir activity towards linear dsDNA fragments not containing lacO. H144 appears to generate a pattern of fewer and more distinct cleavage products finan T7.3 (compare fig. 12, panel b, lanes 8 finrough 10, wifin fig. 13, lanes 7 finrougln 9). In addition, approximately finree tinnes more T73 finan H144 must be used to yield a similar amount of cleavage on linear dsDNA, even though fine rate of cleavage at cruciforms by fine two enzymes is essentially identical (dah not shown; and Panayohtos et al., 1989). Thus, H144 appears to cleave linear dsDNA 102 M2468 2200 - -::::l:l! 600 f ‘—[650] 100 _ 85 Figure 13. Comparison of activity of H144 and T73 on K-DNA. 6% polyacrylamide gel shined wifin efinidium bromide. Lane M: 100 bp ladder, 0.1 pg. lane 1: pCP230 Nhe1-Pvu2 restriction digest, 0.2 pg (0.1 pmole). lanes 2 finrough 5: as in lane 2, reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 nnin respectively. lanes 6 finrough 9: as in lane 2, reacted with 10 ng (0.5 pmole) T73. Reactions in lanes 3 finrougln 5 and 7 througln 9 were heated before loading. Symbols as on figure 12. The presence and identity of the 85 bp band in reactions wifin T73 has been confirmed with end-labeled DNA (not shown). ,'.‘"'7“"Vwr'v'~,q‘cv-—AII ...‘ . . . . . r, ‘. -4 .4 4-.4._4-._.,_.A,_. .0 I. - II- N vole- -. ‘-‘~tud ‘. C. -.m .4! 0 am w... .-.-d- M “-4“. non-w. “Sure 13. c 6% POIyaCry With 8.4 ng ( e 2’ reacte heated before rEaC‘tions Wit} 102 M2468 2200 -::::l:l! Z ’1 600 ‘3' “"' r—[650] 100 *—85 Figure 13. Comparison of activity of H144 and T73 on K-DNA. 6% polyacrylamide gel shined wifin efinidium bromide. Lane M: 100 bp ladder, 0.1 pg. lane 1: pCP230 Nhe1-Pvu2 restriction digest, 0.2 pg (0.1 pmole). lanes 2 finrough 5: as in lane 2, reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 min respectively. Lanes 6 finrough 9: as in lane 2, reacted wifin 10 ng (0.5 pmole) T73. Reactions in lanes 3 finrough 5 and 7 through 9 were heated before loading. Symbols as on figure 12. The presence and identity of fine 85 bp band in reactions with T7.3 has been confirmed wifin end-labeled DNA (not shown). 1.444..t«-n...- JilIl-IA“J.4_"-l a 103 more selectively, and at higlner rates finan T73. The LacI domain of H144 must be responsible for finis difference. Cleavage at an intrinsic bend is dependent on helical phasing Alfinough fine above expeiments show finat binding of H144 to lacO is required for preferential cleavage at targeted sites present on fine same fragment as lacO, they do not explain how lacO-bound H144 accesses hrgets located at dishnces greater finan its physical reach. DNA looping, to bring fine hrgets in conhct wifin fine cahlytic domain, has been postulated as a possible mechanism (Panayohtos and Backman, 1989). One prediction of finis mechanism is finat cleavage at a hrget by lacO—bound H144 would be dependent on fine helical phasing of a hrget wifin respect to lacO. In contrast, location and cleavage of fine hrget by H144 polymedzing along fine helix, should not be affected by helical phasing. These predictions were tested in order to distinguish between fine two possible mechanisms. To that purpose, the position of fine K—DNA bend was helically phased relative to lacO by approximately one-half helical turn intervals (5 bp), in constructs where fine dishnce from fine operator was approximately 130 bp. In pCP232, pCP230, pCP234, and pCP235 fine dishnce between fine target nucleotide on fine lower strand of center of fine K-DNA bend (5'... TGC/ CT A ...3'), and fine nucleotide at fine center of symrrnetry of the taco inverted repeat sequence (5'... ATCC I GCTC ..3') (Sadler et al., 1983) was 162, 157, 142, and 147 bp respectively. Restriction fragments (Nhe1-P0142) of finese plasmids were reacted wifin H144 and examined for cleavage at fine K-DNA bend, by fine appearance of a new band at approximately 650 bp. Such a band was detected only wifin restriction fragments from pCP230 arnd pCP235 (fig. 14, lanes 3 and 4, or 11 and 12, respectively), but not from pCP232 or pCP234 (fig. 14, lanes 7 and 8, or 15 and 16, respectively). Thus, preferred cleavage of fine K-DNA bernd occurred when fine dishnce (as defined above) between fine bend and lacO was 157 and 147 bp, but not 162 and 142 bp. Therefore, prefeential cleavage at fine K- DNA bend is strongly dependent on fine exact dishnce between fine hrget and lacO wifin respect 104 l M246810121416 2200 1 000 600 s— [650] 1 00 — 85 pCP phase 0 +5 -1 O -5 cleavage yes no yes no Figure 14. Effect of helical phasing on cleavage at fine K—DNA bend. 6% polyacrylamide gels stairned wifin efinidium bromide. lane M: 100 bp ladder, 0.1 pg. lane 1: pCP230 Nhel-Pvu2 restriction digest, 0.2 pg (0.] pmole). lanes 2 finrough 4: as in lane 1, reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 30, and 60 min respectively. lane 5: pCP232 NheI-Pvu2 restriction digest, 0.2 pg. lanes 6 finrougln 8: as in lane 5, reacted wifin 8.4 ng of H144 for 0, 30, and 60 min respectively. lane 9: pCP235 Nhe1-Pvu2 restriction digest, 0.2 pg (0.1 pmole). lanes 10 finrough 12: as in lane 9, reacted wifin 8.4 ng of H144 for 0, 30, and 60 min respectively. Lane 13: pCP234 Nhe1-Pvu2 restriction digest, 0.2 pg. lanes 14 finrough 16: as in lane 13, reacted with 8.4 ng of H144 for 0, 30, and 60 min respectively. Reactions in lanes 2 through 4, 6 through 8, 10 finrough 12, and 14 finrougln 16 were heated before loading. Key: The name of each plasmid, phase, and state of cleavage at fine K-DNA bend is indicated. Ofiner symbols as on figure 12. - 4' (.4 <4414‘QAlil...“..“'----wq-—_.z . .14444444...4.4.-1_.-.-' .4 .4 .4 - 11.4--_.<.r.a_..... .J 104 ‘ M246810121416 2200 1 000 600 '— [650] 1 00 <— 85 pCP phase cleavage yes no yes no Figure 14. Effect of helical phasing on cleavage at fine K-DNA bend. 6% polyacrylarrnide gels shined wifin efinidium bromide. lane M: 100 bp ladder, 0.1 pg. lane 1: pCP230 Nhe1-Pvu2 restriction digest, 0.2 pg (0.1 pmole). lanes 2 finrough 4: as in lane 1, reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 30, and 60 min respectively. Lane 5: pCP232 Nhe1-Pvu2 restriction digest, 0.2 pg. lanes 6 through 8: as in lane 5, reacted with 8.4 ng of H144 for 0, 30, and 60 rrnin respectively. lane 9: pCP235 Nhe1-Pvu2 restriction digest, 0.2 pg (0.1 pmole). Lanes 10 finrough 12: as in lane 9, reacted wifin 8.4 ng of H144 for 0, 30, and 60 rrnin respectively. lane 13: pCP234 Nhe1-Pvu2 restriction digest, 0.2 pg. Lanes 14 finrough 16: as in lane 13, reacted wifin 8.4 ng of H144 for 0, 30, and 60 min respectively. Reactions in lanes 2 through 4, 6 finrough 8, 10 finrough 12, and 14 finrough 16 were heated before loading. Key: The name of each plasmid, phase, and shte of cleavage at tlne K-DNA bend is irndicated. Other symbols as on figure 12. 105 to helical phasing. Changing fine helical phasing of fine K-DNA bend with respect to lacO by 1540ne-half helix turns, alterrnately prorrnotes or greafiy reduces cleavage. In order to make cerhin finat fine observed lack of cleavage was not due to fine presence of a fortuitous inhibitor in fine reactions wifin pCP232, pCP234, or two ofiner plasmids - pCP233 and pCP236 - employed in later expedments (see below), a-35S-labeled Nhel-PvuZ restriction fragnents of pCPZ30 wee added to fine pCP232 reaction mixture. Cleavage at fine K-DNA bend was inhibited by approximately 50%, which is the extent expected due to non-productive birnding of H144 at lacO in fine pCP232 reaction (fig. 15). There was no cleavage of fine K—DNA bend on pCPZBZ DNA irn fine mixed reaction, since such cleavage would give products finat could be distinguished on 6% polyacrylarrnide native gels. Moreover, when finis experiment was performed wifin radiolabeled pCP232 DNA and urnlabeled pCP230 DNA, no cleavage at fine K- DNA bend was detected in pCP232 DNA (data not shown). Identical results were obhirned when pCP234 (or pCP233, or pCP236) were used instead of pCP232. Thus, the apparent inability of H144 to cleave at fine K-DNA bend in pCP232, pCP234, and pCP236 is not due to fine presence of a fortuitous inhibitor. Cleavage mediated by DNA looping is affected by fine structure of fine looping segment The lacO/K-DNA restriction fragments of fine plasmids in which fine phasing of K- DNA wifin respect to lacO has been changed by sequential additions or deletions of one-half turn next to K-DNA, exhibit a striking oscillation in apparent size (fig. 14, lanes 1, 5, 9, and 13). The apparent size of fine lacO-conhirning Nhe1-P0142 fragnent of pCPZ30 or pCP235 is 790 bp, wheeas finat of fine corresponding fragnent in pCPZBZ or pCPZB4 is 850 bp. This diffeenoe in apparent size on polyacrylamide gels must be due to a phasing effect between fine K-DNA bend and a small native bend in fine sequence near fine operator. This phenomenon has been obseved wifin ofine pairs of bends (Salvo and Grindley, 1987; Zinkel and Crofiners, 1987). In agreement wifin fine published literature (Burkhoff and Tullius, 1987), finis small native bend was mapped between coordinates 140 and 170 in fine sequence of pCPZBO (Clnapter 2, fig. 7), corresporndirng to 106 '- b. 242468100 242468100 V V —————————— *—85-—' - - \ 72 Figure 15. Cleavage at fine K-DNA bend in reactions conhining bofin pCP230 and pCP232 DNA. Panel a: 6% polyacrylamide gel stained with efinidium bromide. Lane M: 100 bp ladder, 0.1 pg. lane 1: pCP230 Nhe1-P0112 restriction digest, labeled at fine Nhe1 site by filling-in wifin Klenow in the presence of a-355 dCTP. 0.2 pg (0.1 pmole) DNA. lanes 2 finrough 5: as in lane 1, but reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 rrnin respectively. lane 6: as in lane 1 plus an equal amount of unlabeled pCP232 Nhe1-Pvu2 restriction digest. Lanes 6 finrough 10: as in lane 6, but reacted wifin 8.4 ng (0.16 pmole) of H144 for 0, 15, 30, and 60 rrnin respectively. Lane 4’: size marker ( . 275—' ‘ ’ m8 Figure19 .. .W- W , '4 ' *9'""-"f.r‘.- - . WWW “mM.-fi‘CQHus—IUJJ . x « ~ec—u-u-aw \ltfis'fimwufi 0“."tt‘-‘-““‘.O“~"C.-4r"’-~."..’---““4.‘--.-.fl_" 136 Figure 19. CHEF of DNA samples prepared during induction of T7.3 synfiesis in W3110 recA laclq F'/pCP69. Hybddization wifin selected E. coli K12 gees. Panel a: DNA prepared in agarose blocks from irduced and unirduced cells, separated on a 1% agarose gel, and shined wifin efinidium bromide. lares 1 ard 16: 2 ladders (FMC). Lare 2: EMG2 genomic DNA (uninduced). Lane 3: W3110 recA lach F' genomic DNA (unirduced). lane 4: as in lane 2, but restricted wifin Natl. lane 5: S. cerevisiae YNN295 chromosomal DNA (Biorad). lanes 6 finrough 13: DNA from induction presented on figure 19. Samples prepared at 0, 20, 75, 120, 180, 240, 300, ard 360 nnin respectively. lare 14: S. pambe chromosomal DNA (FMC). Lane 15: W3110 recA lach F' genomic DNA (uninduced) restricted wifin Natl. Conditions for CHEF: The gel was run at 2.9 V/cm applied volhge. Initial pulse time was for 25 sec, and was linearly ramped to 40 min ove a period of 48 hours. Subsequenfiy a 40 min conshnt pulse was applied for an addifional 140 hours. Tie temperature of fine electrophoresis buffe was mainhined at 12°C finrouglnout fine run. Panel b: Autoradiogram of in situ hybddization wifin a ptsM probe. Panel c: Autoradiogam of in situ hybddization wifin an rpaB probe. Panel d: Autoradiogram of in situ hybddization wifin a secA probe. Key: The positions of Band I, ard of fine bands finat hybddize to each probe are marked. Their size is also indicated. 110 mi awasfor 340!“in IphOl‘Sls 137 13579111315 13579111315 2200 240 .. c d. 1 15 1 3 5 7 9 1 1 13 15 v ‘ - ' .— I _. {5 240 —> ‘ 275 ——o ‘ . \ ’ 4703 sea . Figure 19 4‘“ 136 Figure 19. CHEF of DNA samples prepared during induction of T73 synfiesis in W3110 recA lach F'lpCP69. Hybridization wifin selected E. coli K12 genes. Panel a: DNA prepared in agarose blocks from induced ard unirduced cells, separated on a 1% agarose gel, and shined wifin efinidium bromide. lares 1 and 16: 2 ladders (FMC). Lare 2: EMG2 genorrnic DNA (uninduced). lane 3: W3110 recA lach F' genomic DNA (uninduced). Lane 4: as in lane 2, but restricted wifin Natl. lane 5: S. cerevisiae YNN295 chromosomal DNA (Biorad). Lanes 6 finrough 13: DNA from induction presented on figure 19. Samples prepared at 0, 20, 75, 120, 180, 240, 300, and 360 min respectively. lane 14: S. pambe chromosomal DNA (FMC). Lane 15: W3110 recA lacl‘l F' genomic DNA (uninduced) restricted wifin Natl. Conditions for CHEF: The gel was run at 2.9 V/ cm applied volhge. Initial pulse time was for 25 sec, and was linearly ramped to 40 min over a pedod of 48 hours. Subsequenfiy a 40 min conshnt pulse was applied for an additional 140 hours. The temperature of fine electrophoresis buffer was mainhined at 12°C finroughout fine run. Panel b: Autoradiogam of in situ hybridization wifin a ptsM probe. Panel c: Autoradiogram of in situ hybddization wifin an rpaB probe. Panel d: Autoradiogram of in situ hybddization wifin a secA probe. Key: The positions of Barnd I, ard of fine bands finat hybridize to each probe are marked. Tleir size is also indicated. 137 a. b. 13579111315 13579111315 2200 . l 240 ‘ g. c. d. 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15 ' ' V ._ I —- i‘ t 24o—> 275—. ‘ \ P 4703 . 59M . ~ Figure 19 138 Table 4 Results of hybddization of E. coli genes wifin Band I probe rrnin khan Band I Not 1 Not 1 Not 1 recA' EMG2 EMGZ W3110 secA 2.45 108.6 + 275 (D) 240 480 lpr 4.5 210 - 275 (D) ND. 480 mutD 5.5 250 - 275 (D) 240 480 dnaX 10.87 501 - 360 (C) 270 480 galK 17.0 799.6 - 205 (K) 157 157 ptsM 40.1 N.R. - 130 (N) 130 130 31115 69.5 N.R. - 1000 (A) 415 1(XJO rle 70.2 N .R. - 1000 (A) ND. 1000 ariC 84 4003 - 203 (L) 203 2(13 asnA 84 4003.5 - 203 (L) 2(B 203 rbs 84.3 4010 - ND. ND. N .D il v ORFI 84.6 4028 + 203 (L) 2(13 200 rmB terrrn. 89.7 4243.8 + many many many rpaB 90.0 4256.7 + 240 (H) 240 240 lexA 91.6 4325 + 240 (H) 240 240 Key: (N .R.) not reported in Rudd et al. (1990); (ND) not determined; (+) hybddizes; (') does not hybddize. Letters in parenfinesis denote fine name given to each band on fine physical map by Smifin et al. (1987). Note: The 11718 termirnator (rrnB term.) probe lights up many bands due to fie high degree of homology that it has wifin ofiner rRNA terminator sequences (Ellwood and Nomura, 1982; Liebke and Hatfull, 1985). 139 E. coli K12 ~ 4 720 000 bp slyS Figure 20. Aligned physical/ genetic map of fine E. coli K12 genome. Circular map based on data compiled by Rudd et al. (1990). The positions of fine probes utilized, fine Natl sites (0), and fine region encompassed by Band I (--) are shown. Note: a deletion of fine lac - praAB region is present between mutD and dnaX in W3110 recA lach F'. 140 cells finat did not harbor fine plasmid (lane 2) or irn EMG2 cells (lane 3). The Natl restriction fragments finat carry rpaB or secA, also hybddized selectively to fie respective probes (lanes 4 and 15, panels c and d, respectively; hble 4). In contrast, Band I did not hybridize to fine ptsM probe (fig. 19, panel b, lanes 6 finrough 13). However, ptsM hybddized to fine expected size band in fie Natl digest (ibid, lanes 4 and 15), suggesting finat its inability to hybddize to Band I was not artifactual. Other probes were utilized in similar expedments to furfiner map fie boundades of Band I, as sunnmadzed in hble 4. Of fiese probes, rbs did not hybddize to Band I, but ilvORFl did. As before, fine expected size Natl restriction fragments were equally well detected wifin bofin probes. Thus, one end of Band I lies between fine rbs and ilvORFI loci, which are located at 84.3 and 84.6 min on fine aligned physical / genetic rrnap respectively. This interval defines a region of 20 kb, within which one putative cleavage may be located. Likewise, Band I hybddized to secA (fig. 19, panel d) at 2.45 min, but did not hybddize to lpr, located at 4.5 min on fine map (hble 4). This interval defines a region of 100 kb wifinin which anofiner cleavage site may be located. As expected, DNA probes for genes located between 84.6 (ilvORFl) ard 2.5 min (secA) hybddized to Band I, wheeas probes for genes located between 4.5 rrnin (lpr) and 84.3 min (rbs) did not hybddize to Band I. In all cases, fine expected size EMG2 DNA Natl restdction fragnent hybddized wifin each probe. An apparent difference between fine physical maps of EMG2 and W3110 recA lach F' in fine region located between mutD and dnaX (see hble 4) adses from a 150 kb chromosomal deletion encompassing fine lac — praAB region (I. Fandl and N. Parnayohtos, personal communication). This deletion removes fie Natl site at fie boundary of fie 275 and 360 kb bands in fine EMG2 Natl restriction rrnap (hble 4, Nail EMG2, Bands D and C, respecfively), but does not affect fie sequences between 0 and 4.5 min (dah not shown). Thus, by screening fie genomic DNA wifin fine probes listed in hble 4, fine approximate boundaries of Band I were identified at 84.45 :l: 0.3 min and at 3.48 :l: 1 min. This distarnce correspords to an 800 kb to 920 kb fragnent on fie aligned physical/genetic map. Howeve, fine 141 apparent electrophoretic mobility of Band I is greater than 2 Mb on the CHEF system. Moreove, when Band I is resolved under PHOGE conditions it migrates as a 1200 kb fragnent (data not shown). In bofin cases fine apparent size is larger from finat determined above by mapping. The reasons accounting for the aberrant electrophoretic mobility of Band I unde different PFGE systems by comparison to its genetically determined size, are unknown. Several possible explanations are presented in fine Discussion. The F' factor in W3110 recA lacI‘l F'lpCP69 is not fragmented during induction of T73 endonuclease synfinesis In order to prove finat Band II corresponds to fine F' factor in W3110 recA lach F cells, DNA from induced W3110 recA lacI‘l F/pCP69 cells was separated by PHOGE, under conditions finat would best resolve DNA fragments ranging from 40 to 2000 kb in size. Band II, migrating at 240 kb by comparison to S. cerevisiae chromosomal DNA and l ladders, was visible in fine efinidium bromide-stained gel (fig. 21, panel a, lanes 4 finrough 11). A second band, migrating above 1100 kb by comparison to fine size markers, was also present in lares 4 and 5 (Band 111) corresponding to 0 and l min of induction, but not in lanes 6 finrough ll, i.e. after 60 min of induction. As noted previously, fine relative amount of Band 11 increased as induction progressed. The DNA was prepared for in situ hybridization, and was probed wifin pifA, an F factor-specific gene (Manis and Kline, 1978). Autoradiography showed finat pifA hybridized bofin to Band II (240 kb; fig. 21, panel b, lanes 4 through 11) and Band III (lanes 4 and 5), as well as to an "unresolved high molecular size complex" above Band III (lanes 4 finrough 11). Tie pifA probe also hybridized to an 100 kb band in lanes containing a Natl digest of W3110 recA lach F‘ or EMGZ (fig. 21, lanes 14 and 16 respectively). Tle size of finis band was identical to fine size reported for F factor when cleaved at its single Natl site (Smifin, Econome et al., 1987). Moreover, the 1" factor of W3110 recA laclq P' carries a copy of the lac operon. The chromosomal allele of fie lac operon is deleted in finis strain (J. Fandl and N. Panayotatos, 142 Figure 21. PHOGE of DNA samples prepared during induction of T73 synfiesis in W3110 recA lach F'/pCP69. Hybridization wifin an F factor probe (péfA). Panel a: DNA prepared in agarose blocks from induced and unirnduced cells, separated on a 1% agarose gel, and stained wifin efinidium bromide. [ares 1 and 15: ¢I>T4 and ¢I>T7 DNA (145 arnd 40 kb respectively). Lanes 2 and 12: 3. ladders (FMC). Lanes 3 and 13: S. cerevisiae YNN295 chromosomal DNA (Biorad). Lanes 4 finrough 11: DNA from induction samples; 0, 1, 60, 120, 190, 360, 420, and 480 min respectively. Lane 14: W3110 recA laclq F' genomic DNA restricted with Natl. Lane 16: EMG2 genomic DNA restricted with Natl. Conditions for PHOGE: The gel was run at 6 V/ cm applied voltage. Longitudinal pulse time was for 5 sec, and was linearly ramped to 100 sec over a period of 2 hours. The run was repeated wifin fine same parameters, except finat ramping was over a period of 64 hours. Tle duration of transverse pulses was double that of longitudinal pulses. The temperature of the electrophoresis buffer was maintained at 15°C finroughout fine run. Panel b: Autoradiogram of in situ hybridization wifin a pifA probe. Key: The positions of fine F' factor, form II, III, and 1" linear are marked. 143 13579111315 b‘ 1 3 5 7 9 11 1315 s—Ill 240— . o..... ' ‘_" 100 — . . s—F' linear ' ‘I Figure 21 142 Figure 21. PHOGE of DNA samples prepared during induction of 17.3 synfiesis in W3110 recA lach F'/pCP69. Hybridization wifin an F factor probe (pifA). Panel a: DNA prepared in agarose blocks from induced and uninduced cells, separated on a 1% agarose gel, and stained wifin efinidium bromide. Lanes 1 and 15: T4 and T7 DNA (145 and 40 kb respectively). Lanes 2 and 12: A ladders (FMC). Lanes 3 and 13: S. cerevisiae YNN295 chromosomal DNA (Biorad). Lanes 4 finrough 11: DNA from induction samples; 0, 1, 60, 120, 190, 360, 420, and 480 min respectively. Lane 14: W3110 recA laclq F' genomic DNA restricted with Natl. Lane 16: EMGZ genomic DNA restricted with Natl. Conditions for PHOGE: The gel was run at 6 V/ cm applied voltage. Longitudinal pulse time was for 5 sec, and was linearly ramped to 100 sec over a period of 2 hours. The run was repeated with fine same parameters, except finat ramping was over a period of 64 hours. The duration of transverse pulses was double that of longitudinal pulses. The temperature of the electrophoresis buffer was maintained at 15°C finrouglnout fine run. Panel b: Autoradiogram of in situ hybridization wifin a pifA probe. Key: The positions of fie F' factor, form 11, III, and F' linear are marked. :inW3110rtl paratrdmnl‘. )NA(145an140 "visit: YNN295 es; 0, 1, 60, 11L DNA retried iinalpulsetime unW'illel’aml maduiitifll‘0f erature 0‘ a! .. F. n .. 143 a. y 13579111315 fi 4“”Q .1 ..a ._. A ..v—— ‘—|I| -..seie- _‘ L- 'V‘““""““‘-‘V‘Wfi“'“""w“ tr“‘ I‘fi-WIT‘W‘V‘V‘W'V‘ *—III 240— ' .'.'.o 9. ‘—II 100 — . ‘ e—F‘ linear ‘ -| Figure21 1 I val 144 personal communication). When radiolabeled lacOP was used to probe W3110 recA lach F' DNA, fine 240 kb band was detected on uncut DNA, whereas a 100 kb band was detected on Natl-restricted DNA (data not shown). Thus, fine prominent 240 kb band (Band II) is a non- linear (supercoiled?) form of the F' factor in W3110 recA lacl‘l F'. The abnormal mobility exhibited by a non-linear F factor is consistent wifin fine migration of supecoiled molecules under PFGE (Beverley, 1988; Simske arnd Scherer, 1989). Band III and fine unresolved high molecular size complex may represent a different form F', or alternatively, F' associated wifin bacterial chromosomal DNA (Miller and Kline, 1979). Some non-specific hybridization to fine high excess of DNA associated wifin the unresolved complex may also contribute to fine intensity of fine signal. DISCUSSION Evidence has been presented finat during induction of T73 endonuclease synfinesis in a recA host, fie genomic DNA is fragnented and degraded into progressively smaller fragments, ranging in size from 4.7 Mb (fine size of fine linearized E. coli chromosome) to 240 kb during fine early stages of induction. The size of fine fragnents decreases as induction progresses, but it renains above 200 kb. Among finese fragments, two specific bands (Band I and Band II) were observed and characterized. Band I is a genomic DNA fragment finat is released as a result of 17.3 synfinesis Band I encompassed fine region between fine ilvORFl and secA loci, at 84.3 arnd 2.5 min on fine aligned physical / geetic map respectively (fig. 20), defining a region of 800 kb. One end of Band I is located between rbs and ilaORFI (at 84.3 and 84.6 min, respectively). This interval defines a region of approximately 20 kb. Tie ofier end has been mapped wifin less resolution. It lies between secA and lpr (at 2.45 and 4.5 min, respectively), which define a region of approximately 100 kb. This characterization established finat fine size of Band I is at least 800 but smaller finan 920 kb. Interestingly, Band I migrated abnormally slowly on bofin CHEF and PHOGE gels, exhibiting an apparent size greater finan 2 Mb on fine former arnd approximately 1200 kb on fine latter system. Since aberrant mobilities have been observed before for circular molecules when resolved by PFGE (Beverley, 1988; Hightower et al., 1987; Sirnske and Scherer, 1989), it is possible finat Band I is a large non-linear DNA molecule. Predicting what exacfiy may be causing fine abnormal rrnobility of Band I is complicated by fine fact that fine migration of 145 146 circular molecules greater finan 100 kb, catenated circles, knotted forms, large loops, or ofiner complex tertiary conformations on pulsed field gels has not been studied. In fie absence of sequence information of fine precise cleavage sites it is not possible to make predictions on fine structures finat may be localized at fine regions which give rise to fine ends of Band 1. Direct cloning of fine ends of Band I was not attempted because it displayed a large size, it was present in small amounts, and it could not be sufficienfiy resolved from ofiner fragnented or degraded DNA molecules finat migrated at fine same position on the gels. However, the fact finat induction of 17.3 synfinesis led to fine release of a distinct genomic fragment, indicates fine presence of at least two highly preferred targets in viva, on the bacterial chromosorre. Band II is a non-linear form of an F' factor Band 11 corresponds to a non-linear form of fine F' factor present in W3110 recA lacl‘l F' cells. Anofiner form of fie F‘ factor was seen at low concentration during fine early stages of induction (Band III). Whereas finis form could not be detected after 120 mirn of induction, fine intensity of Band 11 increased as induction progressed. 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H144 was constructed to direct T73 at lacO DNA fragnents via its LacI domairn. H144 was shown to be a unique enzymatic probe of intrinsically bent DNA and potentially ofier unusual conformations, which may exhibit single-strandedness. Cleavage by H144 at the physical center of intrinsic bends revealed the presence of a previously unrecognized unusual conformtion at finat site. Effective recognition of target structures depended on fine presence of lacO in cis, on fie helical phasing of targets wifin respect to lacO, as well as the conformation of fine sequence between a target and lacO. These results demonstrated finat lacO-bound H144 cleaves at a target wlen an appropriate DNA loop forms to bring fine target in contact wifin fine nucleolytic domain of H144. This molecular mechanism is functionally equivalent to the mechanism by which enhancers are finought to mediate transcriptional activation by binding at distal sites. More importantly, it provides fine first example of DNA looping between non-cognate (functionally different) sites by a single protein. Since fine site contacted by fine T73 domain is not tighfiy bound (no gel retardation can be seen wifin T73 and fine DNA fragments used), looping nediated by H144 between lacO and a target, should not strain fine helix. Thus, fine degree to which H144 cleaves a specific target on supercoiled or linear DNA may perhaps be used as a measure of how close two DNA segments are to one anofine along fine lnelix. Moreover, H144 may be used as a probe of intrinsic bends in viva, by employing an approach similar to finat utilized for probing cruciforms wifin T73 152 153 T7.3 was used to probe for unusual DNA structures on fine E. coli chromosome in viva, by extending fine approach utilized for probing for cruciforms on supercoiled plasmids. Synfinesis of T7.3 in a recA host, resulted in progressive fragmentation of fine genomic DNA into many different large fragnents, as visualized on pulsed-field gels. In addition, one distinct genomic fragment was resolved, indicating finat at least two highly preferred target sites are present on fine chromosome. Its ends were mapped by hybridization to known E. coli genes, and were localized on fie aligned physical / genetic map. It is postulated finat an unusual structure(s) is recognized and cleaved at finese sites. Howeve, fine cloning and sequencing of fine region encompassing finose sites remains to be completed. This task will be facilitated when the sequence of fine corresponding regions of fine E. coli genome becorres available.