IHIIHUIJIHII'HMHUIHIHHIIHIl'llliWIHIIHIIHUI 31293 01053 5148 . LIBRARY Michigan State University PLACE It RETURN BOXtomnovothbctnckthnywmd. TO AVOID FINES mum on Of baton data duo. DATE DUE DATE DUE DATE DUE MSU to An Afflrmutivo MOVE“ Opponmlty lmtttuion Wm: EFFECTS OF ISOLATED MISSENSE MUTATIONS FROM THE DNA/15 AND DNAA46 ALLELES IN INITIATION OF ESCHERICHIA COLI ORIC REPLICATION By Kevin Michael Carr A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1994 ABSTRACT EFFECTS OF ISOLATED MISSENSE MUTATIONS FROM THE DNAA5 AND DNAA46 ALLELES IN INITIATION OF ESCHERICHIA COLI ORIC REPLICATION By Kevin Michael Carr The temperature sensitive alleles dim/l5 and dnaA46 each contain two missense mutations. These have been separated by molecular genetic techniques and the resulting mutant proteins studied in initiation of DNA replication in vitro. An alanine—to—valine substitution at amino acid 184 is common to both of the alleles and is responsible for the thermolabile defect and prolonged lag in DNA synthesis of DnaAS and DnaA46. This protein was also defective in binding ATP as are DnaAS and DnaA46. Aggregated, wild type DnaA was also severely impaired in ATP binding, suggesting protein aggregation and not the amino acid substitution, per se, is the primary cause of defective ATP binding. The dnaA5 allele also contains a mutation resulting in substitution of glycine at residue 426 with serine. The single mutant is partially thermolabile in activities of DNA replication as well as binding to oriC DNA. DnaA46 and the A184V mutant product bind oriC DNA more efficiently at 30 °C than DnaA+ and both retained affinity for oriC at elevated temperature. The effects on out binding of the A184V and G426S mutations balance each other when they are both present in DnaAS. All of the mutant forms of DnaA were able to form complexes with oriC but a reduced abundance of certain complexes was observed for proteins harboring the A184V mutation. By contrast, the histidine 252 to tyrosine alteration of the dnaA46 allele, when present alone, did not affect the biochemical activities of DnaA protein. To Mom Dad Iohn Karen Chris Acknowledgements While my path here might be considered nontraditional, I will stick to tradition by acknowledging those people who have helped me in this endeavor. First, of course, is my thesis advisor, Dr. Jon Kaguni, who found me wandering in the lobby of the biochem building one night and decided to offer me a job. This work would not have been possible without his guidance and patience. He provided me a place to develop as a scientist and I provided him with several beers by betting on Michigan - Notre Dame football games. I must also thank my second thesis advisor, Dr. Laurie Kaguni, free spirit, latin scholar, keen wit, for many helpful insights and suggestions along the way. My appreciation also to Dr. Zach Burton, ad hoc member of my thesis committee. To my many colleagues over the years, I owe many thanks. Ted Hupp and.Deog Su Hwang, who laid the foundation for my work and provided excellent examples of how science is done. Dave (if my speakers were just a little bit bigger) Lewis, friend, golf partner, TOH enthusiast and educator in the ways of liberalism. Remember, the GOP is the Big Tent party. Mark (Ditka) Sutton, for many heated discussions on the relative merits of genetics, the Chicago Bears, and for stories about Danny. Cindy (Steffi) Petersen for jokes, M&Ms, kindness and friendship always. To all my other friends and comrades over the years, Qingping Wang, Carluchi Margulies, Iarek Marszalek, Wenge Zhang, Joe Lipar, Andrea Williams, Angie Kohloff, Matthew Olson, Carol Farr, J. J. Wang, Richie Halberg, Eveline Yao, Chuck, Mary and Paul Tyler Campbell, Diane Cox, Julie Osterle, who shared many things including the occasional excursion to the Dairy Store or the Peanut Barrel. Finally, my deepest appreciation and thanks are for my parents, whose support, encouragement and faith, while tested at times, were constant. Table of Contents List of Tables ....................................................... ix List of Figures ....................................................... x List of Abbreviations ................................................. xi Chapter I. Literature Review ..................................... 1 Introduction ..................................................... 2 Principles of Regulation ........................................... 3 The origin of replication .......................................... 7 Structural features ............................................. 7 Transcription around oriC ..................................... 11 Membrane attachment and dam methylation ................... 12 Replication from oriC in vitro .................................... l3 oriC plasmids/minichromosomes ............................. 13 In vitro replication of ant plasmids ............................ 14 vi DnaA Protein ................................................... 18 Physical and biochemical properties ............................ 18 DnaA function in transcription ................................ 20 Regulation of chromosome replication by DnaA ................. 21 Mutant DnaA proteins ........................................ 22 Chapter 11. Effects of Isolated Missense Mutations from the dnaAS and dnaA46 Alleles in Initiation of Escherichia coli oriC Replication .................................................... 29 Introduction .................................................... 30 Experimental Procedures ......................................... 34 Materials .................................................... 34 Enzymes and Proteins ......................................... 34 Buffers ...................................................... 35 Bacterial strains and DN As .................................... 35 DNA Manipulations .......................................... 38 Subcloning of dnaA into a pET expression vector ............. 38 Separation of the individual mutations in dnaAS and dnaA46. 42 Purification of DnaA protein .................................. 42 DNA replication assays ........................................ 45 ATP binding assays ........................................... 46 DNA binding assays .......................................... 46 Protein Determinations ....................................... 48 vii Results ......................................................... 49 Confirmation of mutations in the various dnaA alleles .......... 49 The thermolabile defect of dnaAS and dnaA46 is due to the mutation at amino acid 184 ................................. 57 Complementation of dnaA204(Ts) by the mutant proteins ........ 60 The prolonged lag associated with DnaA5 and DnaA46 proteins is due to the mutation at amino acid 184 ....................... 62 The ATP binding defect appears to be a function of the A184V mutation ................................................. 65 The mutant proteins retain affinity for oriC DNA ............... 68 Discussion ...................................................... 75 Chapter 111. Summary and Perspectives .......................... 82 Bibliography ........................................................ 87 viii List of Tables Chapter II Table 1. Bacterial strains and plasmids .............................. 36-37 Table 2. In vivo complementation of dnaA204 by the alleles of dnaA ..... 61 ix List of Figures Chapter I Figure 1. Schematic representation of the relationship between cell division and chromosome replication cycle ...................... 5 Figure 2. Consensus sequence of the minimal origin of the bacterial chromosome .................................................. 9 Figure 3. Hypothetical model for the initiation of chromosome replication in E. coli ........................................... 16 Figure 4. Map of mutations and correlated phenotypes in dnaA ........ 24 Chapter II Figure 1. Physical map of pKC plasmids carrying alleles of dnaA ........ 41 Figure 2. Digestion of dnaA plasmids with Sph I ...................... 51 Figure 3. Heteroduplex analysis of dnaA mutants ..................... 54 Figure 4. DNA sequence of dnaA—HZSZY mutant ...................... 56 Figure 5. Temperature sensitivity of mutant DnaA proteins in in vitro replication ................................................... 59 Figure 6. Time course for DNA synthesis by mutant DnaA proteins ..... 64 Figure 7. ATP binding by mutant DnaA proteins ...................... 67 Figure 8. Fragment retention assay for oriC binding by mutant DnaA proteins ..................................................... 70 Figure 9. Gel shift assay of MC binding by mutant DnaA proteins ...... 74 bp BSA DnaA box dNTP DTT EDTA HEPES IPTG kDa PVA SDS SSB Tris List of Abbreviations base pair Bovine serum albumin the 9 bp consensus sequence recognized by DnaA deoxyribonucleotide dithiothreitol ethylenedinitrilo tetraacetic acid 4-(2-hydroxyethyl)-1-piperidineethane sulfonic acid isopropyl B—D—thiogalactopyranoside kilodalton polyvinyl alcohol sodium dodecyl sulfate single stranded DNA binding protein Tris(hydroxymethy1)aminomethane xi Chapter I Literature Review Study of the mechanisms of DNA replication is of significance because of the fundamental nature of this process to all life. With the initial description of the structure of DNA, a possible mode by which the genetic material can direct its own duplication through a semiconservative process became apparent (Watson and Crick, 1953). The semiconservative nature of DNA replication was soon demonstrated (Messelson and Stahl, 1958). Intense study of the mechanism and regulation of DNA replication has been continuous ever since. The Escherichia coli chromosome is a circular, duplex DNA molecule of 4,700 kilobase pairs (reviewed in McMacken, et al., 1987; von Meyenburg and Hansen, 1987). Replication of the genome of E. coli begins at a unique site on the chromosome and replication forks proceed bidirectionally around the circular DNA to the site where replication terminates. Daughter molecules segregate to complete the cycle of chromosome duplication. Principles Qf Regulation Early in the study of DNA replication in E. coli, it was recognized that replication is a very regular event (Messelson and Stahl, 1958). Soon after it was understood that the regulated step in synthesis of DNA is at the initiation step (Maaloe and Kjeldgaard, 1966). Jacob and coworkers proposed a model for the control of replication, called the replicon model, which has two components (Jacob, et al., 1963). The first is the replicator which is the site of regulation and where replication begins, now known to be oriC. The second is the initiator, which acts on the replicator to initiate replication in a regulated manner. The DnaA protein is the best candidate for the initiator in this model. The concept of initiation mass (Donachie, 1968) is central to all models attempting to describe regulation of initiation. Initiation mass is defined as the ratio of cell mass to the number of origins present in the cell at the time of initiation. If initiation of replication is tightly regulated and coordinated to the cell cycle then it is predicted the initiation mass remains constant. Anything which disrupts the regulation of initiation will alter the initiation mass. Examination of different periods in the cell cycle (Cooper and Helmstetter, 1968; Helmstetter, 1968) revealed that the period between initiation and termination, the C time which represents the complete duplication of the chromosome, is constant at all growth rates and is 40 minutes long (Figure 1). More recent analysis has shown that at very slow Figure 1. Schematic representation of the relationship between cell division and chromosome replication cycle (von Meyenburg and Hansen, 1987). C, the time between initiation (ini) and termination (ter) of replication, may be extended at very slow growth rates but is constant and equal to 40 min at more rapid growth rates. D, the time between termination and cell division (div), is 20 min at all growth rates. The time between initiations varies according to the doubling time (to) of the cell (a tD=90 min; b tD=60 min; c tD=35 min). If to is less than C + D (eg. in c) then the cells must initiate a new round of initiation before previous rounds are completed. lnitiations are synchronous on all origins present in the cell. E C~———) CE CZ—D’—— 63 VVV div div 1 I ~ 1 l ini ter ini C E ) 6—1—6 C22: C>—3 07—9 VV div div i . i . ini ter in! ter (é; cg/ cg/ ca? 2/ 29/ " iv div div . I I I ter ter ter 6 growth rates this time can be extended (Churchward and Bremer, 1977; Kubitschek, 1974; Kubitschek and Newman, 1978), but it is never shorter than 40 minutes. The time between termination and subsequent division, D time, was also a constant. This time is 20 minutes, resulting in a minimum of 60 minutes between the time replication is initiated and the appearance of two new daughter cells from a particular round of replication. However,E. coli is capable of doubling time as short as 15—20 minutes, which means it must also initiate a new round of replication at this same time interval. In rapidly growing cultures, new round of replication are started before ongoing rounds are completed, resulting in chromosome structures in varying stages of duplication (Figure 1). Flow cytometry has been used to determine the number of genome equivalents of DNA in a cell in which new initiation of DNA replication and cell division have been inhibited (Boye, et al., 1988; Skarstad, et al., 1986; Skarstad, et al., 1983; Skarstad, et al., 1985). It indirectly measures the number of origins which were present when the drugs were administered to the cell. In samples of normal cultures, the vast majority of the cells are found to have 2" (e.g. 2, 4 or 8) genome equivalents. This finding indicates that in rapidly growing cells with multiple origins present, initiation occurs simultaneously at all origins. This property is referred to as synchronous initiation and it is further evidence of the high level of regulation of chromosomal replication. Mutants which affect the synchronous initiation of replication have been identified in a subclass of dnaA mutants (Skarstad, et al., 1988) that have a 7 mutation near the ATP binding site. Other cellular factors potentially involved in this process have been identified by mutants which result in an asynchronous phenotype. These include dam (Dam methylase) (Boye and Lobner-Olesen, 1990), squ (sequestration) (Lu, et al., 1994), him (II-IF), fis (FIS protein) (Boye, et al., 1993) and gyrB (gyrase B subunit) (von Freiesleben and Rasmussen, 1991). The roles these proteins may play in regulation of oriC function are discussed below. I] "tl’l' Structural features The origin of replication, oriC, was identified and localized to 83.5 minutes on the E. coli chromosome based on examination of gene dosage in replicating cells (Bird, et al., 1972; Masters and Broda, 1971). Later, the origin region was cloned by its ability to confer autonomous replication in E. coli to a DNA fragment carrying a drug resistance gene but no functional origin (Hiraga, 1976; Yasuda and Hirota, 1977). Progressive deletions identified the minimal functional origin as 245 bp (Oka, et al., 1980). Sequence information from the E. coli origin and the origins of other enteric bacteria was obtained (Cleary, et al., 1982; Zyskind, et al., 1983; Zyskind, et al., 1981). The aligned sequences showed a high degree of homology with several clusters of identity distributed throughout, suggesting sites of functional importance (Figure 2). These clusters are separated by regions of nonconserved sequence but of fixed Figure 2. Consensus sequence of the minimal origin of the bacterial chromosome (Zyskind, et al., 1983). The consensus sequence is derived from six bacterial origins indicated. The alignment of the sequences is such that the least number of changes are introduced into the consensus sequence. In the consensus sequence, a large capital letter means that the same nucleotide is found in all six origins; a small capital letter means the nucleotide is present in five of the six sequences; a lowercase letter is used when that nucleotide is present in three or four of the six origins but only two different nucleotides are found at that site; and where three or four of the four possible nucleotides, or two different nucleotides plus a deletion, are found at a site, the letter n is used. The minimal origin of E. coli is enclosed within the box and the numbering of the nucleotide positions is the used for E. coli. Bold large capital letters located at positions 149. 242, and 267, indicate where single- base substitutions produce an on’C 'phenotype in E. coli. GATC (dam methylation) sites are underlined in the consensus sequence. The four dnaA boxes are indicated as R1, R2, R3 and R4; and their orientations indicated by arrows. The three 13mer repeats are indicated by arrows in the upper left. .3223 2.5.; 3 33333333333 3.333.333.3333 .333. <3 . 3. 3 333333.333. 893388 3.3.32.3 3 33333333333 3.3. 3.3333333. 33323 3. 3 3. 3 333323333. 3.3.65.3... 3:33:33 3 23333.33 3.3. 3333.33.33 .3... 3. . . 33 3 333.333: 25323 #333235 3 23333333133 333. 33.33.33.333 .3... 3. . 3. 3 3333.333. 5.3.3.3333: 3:38.333 3 233333.333 3.3. 3333.33.33 .3... 3. . .3. 3 33332333. :8 3.3332333 3 133333.333 3.3. 3333.33.33 .3... 3. 2 . 3. 3 3332333. 3 E 335833 33.333333 3 33.22232355335333 8335.3333c.635.333?33333332835333.35ch333.. 5.353 . . illr \ z. 33 2:353 3.3 3 333 3 3.3333. 3. 3 333 3 3333333 ..3 33 . 333333 .33333 333333 . 3333 . 333 3 3.333.. 33 . ... . <.333.3 ... .. .33333 333333 33333. . 3.33 e (U0 ( o.°((oo ‘0 o 0.. O ”okouou 0.. .. .hhhuu O(<°‘< “hog“. a hope a hug U 000((00 U. o 0.. o Uehkoeu co. .0 ouk“0‘ o“°‘< ”060‘. o hoh< . 333 3 33333.3 3. . ... . 3.333.3 ... .. .33333 .33333 33333. . 3.33 o hU( < 0300(30 U. o .00 . U.(O°o U 0.. 00 r“hh( o‘<‘°° UU(33 2.93 3 339.3 333on 3339.3 52 and unlabeled fragment from the other alleles of dnaA. Mispaired bases are chemically modified by base specific chemicals and the strand subsequently cleaved by piperidine. Hydroxylamine hydrochloride chemically modifies mispaired cytidine residues (Fraenkel-Conrat and Singer, 1972). All of the missense mutations in dnaA5 and dnaA46 are CCDT transitions, two on the sense strand (A184V and H252Y) and one (G426S) on the antisense strand (Hansen, et al., 1992). In samples with the sense strand of dnaA+ labeled, two cleavage products of the expected size were seen for the wild type/dnaA46 heteroduplex, corresponding to the mutations at amino acids 184 and 252 (Figure 3A). The wild type/dnaA—A184V heteroduplex resulted in a single cleavage product. Its size was similar to the product generated by the corresponding mutation in dnaA46. Heteroduplex mapping with the antisense strand of d naA+ as the labeled strand resulted in a cleavage product expected from the missense mutation that gives rise to the G426S substitution in dnaA5 (Figure 38). By comparison to similarly treated homoduplexes, no other significant bands were observed in the heteroduplexes. Treatment of heteroduplexes with osmium tetroxide, which modifies mispaired thymine residues, did not detect mispairing (data not shown). DNA sequencing confirmed the expected CCDT base change of the mutation in dnaA-H252Y (Figure 4). 53 Figure 3. Heteroduplex analysis of dnaA mutants. Heteroduplexes were formed with an end-labeled dnaA" restriction fragment and unlabeled fragments of the various dnaA alleles as indicated. (A) Heteroduplex reactions with the sense strand of dnaA*as the labeled strand. (B) Heteroduplex reactions with the antisense strand al the labeled strand. The lanes marked Untreated are dnaA" duplex samples not treated with hydroxylamine or piperidine and indicate the position of uncleaved fragment. 54 e e 6 33.233.33.31 .3. $3.33: +\+ 332332335 +\+ . 383.93%: 3.33%-...351 33333.35: . 33.3.35: 33.3%: +\+ 332303335 +\+ 55 Figure 4. DNA sequence of dnaA-H252Y mutant. Plasmids pK0596 (dnaA*) or pKC-H252Y were used as templates for sequencing reactions by the Sanger dideoxy method (Sambrook, et al., 1989). Primer JK—21 (15—mer, 5’-ATAACCCGTTGTTCC—3’) corresponds to nucleotides 494-508 of the coding sequence of dnaA. The sequence in the region surrounding the substitution is shown on the left with the expected Cd>T change at nucleotide 754 of the coding sequence. The nucleotide substitution in the sequence of dnaA-H252Yis indicated on the right (arrow). DnaA“ DnaA-H252Y G A T C T C >>CD>CD-i-i-i—i-iOO>O>OO 57 The thermolabile defect of dnaA5 and dnaA46 is due to the mutation at amino acid 184 DnaA protein is active in initiation of DNA replication in vitro. Replication activity can be measured in either a crude enzyme preparation from a dnaA204 (Ts) strain or reconstituted with purified enzymes (Fuller, et al., 1981; Kaguni, et al., 1985). Both replication systems require supercoiled plasmid DNA containing either the complete (463 bp) or minimal (245 bp) replication origin from E. coli. DnaA5 and DnaA46 proteins were active in the crude enzyme replication assay at a temperature permissive for activity in vivo. At 40 °C, the non—permissive temperature, they were inactive in the initiation of DNA synthesis, consistent with the phenotype of these mutants. The isolated single mutants were also examined in this assay (Figure 5). The mutant A184V was thermolabile for DNA replication. By contrast, DnaA—H252Y displayed no thermolability. Its activity was indistinguishable from that of wild type DnaA. The mutant DnaA—G426S showed partially temperature sensitive activity. Its activity was reduced approximately 50% at 40 °C relative to its activity at 30 °C. This defect is probably masked by the more severe temperature-sensitivity of the A184V mutation, and probably does not contribute significantly to the thermolabile phenotype of dnaA5. Effects of the individual mutations on the activity of DnaA at the permissive temperature were subtle. DnaA—H252Y did not differ significantly in replication activity from wild type DnaA protein. For DnaA—G426S, there 58 Figure 5. Temperature sensitivity of mutant DnaA proteins In In vitro replication. The various DnaA proteins were titrated in DNA replication assays at 30 °C (0) or 40 °C (O). Times of incubation were: DnaA” and DnaA—H252Y, 20 min; DnaA-(34268, 30 min; DnaA5, DnaA46 and DnaA-A184V, 40 min. DNA Synthesis (pmol) 800 600 400 200 800 600 400 200 800 600 400 200 ’9 DnaA+ DnaA-A184V o I I '- I l l l l 0 50 100 150 200 0 50 100 150 200 DnaA46 DnaA5 e - - . 3. 0 50 100 150 200 0 50 100 150 200 DnaA-H252Y DnaA-(34268 0 50 100 150 200 0 50 DnaA Protein (ng) 100 150 200 60 was a more severe inhibition of activity at higher concentrations compared to DnaA+. By contrast, DnaA-A184V protein was more active at this higher concentration. Complementation of dnaA204(Ts) by the mutant proteins Activity of the mutants in vivo was assayed by their ability to complement a strain which harbors the dnaA204 allele (Table 2). The dnaA204 allele confers a temperature—sensitive phenotype. dnaA5, dnaA46 and dnaA-A184V were unable to complement the temperature—sensitive phenotype of this strain, consistent with the thermolability observed in in vitro replication assays. There was a slight increase in colony forming ability observed with dnaA5 but it is not clear from this assay if this represents a significant difference in its phenotype relative to dnaA46 or dnaA—A184V. The dnaA-HZSZY allele supported growth at 42 °C almost as efficiently as dnaA", consistent with the observation that its replication activity was not thermolabile in vitro. Interestingly, dnaA—64268, when supplied on a plasmid, resulted in an intermediate efficiency of colony formation. In vitro, this protein showed partially temperature—sensitive replication activity. It should be noted that the in vivo activity of these proteins at 30 °C can not be assessed with this assay since the chromosomal dnaA204 allele is active at this temperature. Also, the level of plasmid—encoded protein was not determined and may have varied between the different mutants. None of the 61 Table 2. in vivo complementation of dnaA204 by the alleles of dnaA dnaA* 0.94 dnaA46 0.7 x 10'5 dnaA5 1.3 x 10“ dnaA-H252Y 0.79 dnaA-64268 1.5 x 10'2 dnaA-A184V 2.5 x 10'5 Strain BL21(DEs)dnaA204(pLysS) was transformed with plasmids harboring the various alleles of dnaA as well as the vector pET11a. Transformants were selected on LB plates containing 50 ug/ml ampicillin and 25 ug/ml chloramphenicol at 30°C. Plasmid minipreps were performed to verify the presence of the appropriate plasmid. Overnight cultures grown in LB media containing ampicillin and chloramphenicol were diluted in LB media and spread in duplicate on LB plates with the same antibiotics. Relative plating efficiency at 42 °C versus 30 °C was determined after overnight incubation. (My appreciation to Joe Lipar for assistance in collecting this data.) 62 plasmid-encoded proteins had an observable effect on growth at the permissive temperature. The prolonged lag associated with DnaA5 and DnaA46 proteins is due to the mutation at amino acid 184 In vitro replication assays dependent on DnaA are characterized by lag period of 2—5 minutes prior to incorporation of deoxynucleotides (Fuller, et al., 1981). Previous biochemical characterizations of DnaA5 and DnaA46 protein have shown that the lag period associated with these proteins is much longer (12—18 minutes). The extended lag period is the result of a required activation step in this assay for DnaA5 and DnaA46 which involves the heat shock proteins DnaK and GrpE. This interaction takes place prior to interaction with oriC (Hupp and Kaguni, 1993b; Hwang and Kaguni, 1988a). A time course of DNA synthesis was performed with the various mutant proteins (Figure 6). Reactions containing 50—65 ng of the DnaA proteins were incubated at 30 °C, and stopped at times indicated. As previously observed, wild type DnaA had a short lag period of approximately 2 minutes before incorporation of dNTPs. DnaA5 and DnaA46 had much longer lag periods of 10—15 minutes. The lag period of DnaA-A184V was similar to that of DnaA5 and DnaA46. DnaA—H252Y had a lag period similar to that of DnaA”. The lag period for DnaA—G426S, while longer than that of wild type was shorter than that of DnaA—A184V. The A184V and G426S mutations did not appear to have an additive effect on the lag period of 63 Figure 6. Time course for DNA synthesis by mutant DnaA proteins. DNA replication assays were assembled as described in Experimental Procedures with constant amounts of the specified protein (50—65 ng). The reactions were incubated at 30 °C, stopped at the indicated times and DNA synthesis was quantitated (Experimental Procedures). 64 250 - 200 L- DNA Synthesis (pmol) 01 O I 0 0 150 3 100 - 10 a i i I 20 30 40 Time (min) til-i DnaA+ —)(— DnaA-A184V DnaAagg —I— DnaA-H252Y DnaA46 -B— DnaA-64268 DnaA5 65 DnaA5. The extended lag of DnaA5 was similar to the lag period of the single mutant A184V. This may indicate that the lag periods represent independent processes and the shorter lag period resulting from mutation at amino acid 426 is masked by the longer process associated with the mutation at amino acid 184. Aggregated DnaA+ was also examined and the lag period of 5—10 minutes seen was longer than for monomeric DnaA protein. The ATP binding defect appears to be a function of the A184V mutation DnaA protein binds ATP with high affinity (KD=0.03uM), and bound ATP is required for replication activity (Sekimizu, et al., 1987). DnaA5 and DnaA46 proteins fail to bind ATP. This defect had previously been attributed to the amino acid substitution at amino acid 184 (Hupp and Kaguni, 1993c; Hwang and Kaguni, 1988a), common to both, since it is very close to a consensus ATP binding motif (P—loop). Using a filter binding assay, DnaA+ was observed to bind with high affinity (Figure 7). The calculated ratio of ATP bound per monomer of DnaA was 0.42. Less than stoichiometric binding of ATP by DnaA+ has consistently been reported in the literature (Hwang and Kaguni, 1988a; Sekimizu, et al., 1987) with values ranging from 0.1—0.3 ATP bound per DnaA monomer. DnaA-H252Y bound ATP with an affinity similar to DnaA+and with a ratio of 0.38 ATP bound per monomer. DnaA5, DnaA46 and DnaA-A184V failed to bind ATP at any appreciable level. This would tend to confirm that the ATP binding defect is attributable to the 66 Figure 7. ATP binding by mutant DnaA proteins. increasing amounts of [a—32P] ATP were incubated with a fixed amount of DnaA protein (2 pmol). ATP binding was determined by retention on nitrocellulose filters (Experimental Procedures). Curves were drawn, the dissociation constant (KD) and stoichiometry of binding (n) were determined by fitting the data to the binding equation :[ATP]bound = n x K,aq x [E.] x [ATP],,ee/(1 + K9q x [ATP],,99); [Ed = concentration of DnaA protein (0.08uM as monomer), KD = 1/K9q. KD and n values calculated are DnaA“, 0.01 (M, 0.42; DnaA—H252Y, 0.025 uM, 0.38; DnaA—64268, 0.014 uM, 0.055; DnaAagg, 0.04 (M, 0.02. [ATPJbound (uM) 0.04 0.03 0.02 0.01 67 [ATPJfree (uM) DnaA+ DnaAagg DnaA5 DnaA46 DnaA-A184V DnaA.H252Y DnaA-G4268 68 mutation at amino acid 184. However, assay of DnaA“lgg showed it to have an extremely low, but detectable ATP binding activity. ATP binding by DnaA—G426S was only slightly better than the aggregate. The DnaA+ (monomer) used in this study was derived directly from the DnaAagg sample by guanidine hydrochloride treatment followed by gel filtration chromatography (Experimental Procedures). This method did not result in active protein except for H252Y. The very low extent of ATP binding by the aggregated DnaA protein suggests that the physical state of the protein has a significant impact on its ability to bind ATP. In addition to DnaA+, DnaA-H252Y was subjected to guanidine treatment to obtain monomer. High affinity ATP binding by DnaA—H252Y was observed before or after guanidine treatment, and an increase in the ratio of ATP bound after treatment was observed (data not shown). Extended incubation of DnaAagg at 0 °C or 25 °C did not result in increased binding of ATP by DnaA“‘gg (data not shown). The mutant proteins retain affinity for oriC DNA DnaA protein has been shown to bind cooperatively to oriC at the four DnaA boxes present (Fuller, et al., 1984). In vitro, binding to a supercoiled oriC plasmid has been shown to result in local unwinding of the DNA (Bramhill and Kornberg, 1988a). This unwinding is postulated to be required for binding of other prepriming components. The ability of the various mutant proteins to recognize and bind to oriC was tested using a linear DNA 69 Figure 8. Fragment retention assay for oriC binding by mutant DnaA proteins. 32P—labeled, 461 bp Sma l—Xho I fragment from pBSoriC was incubated with the indicated amounts of DnaA proteins at 30 °C or 40 °C. The amount of fragment retained on filters was quantitated by liquid scintillation counting. Binding is expressed relative to the amount of fragment bound by the highest level of DnaA+ at 30 °C (=1.0). (A). Binding by DnaA46 and corresponding single mutants (A184V & H252Y) relative to wild-type DnaA. (B). Binding by DnaA5 and its corresponding single mutants (A184V 8. G4268). Binding by DnaA+ and DnaA—A184V from (A) are shown again for comparison. 70 1.2 1.0 0.8 0.6 0.4 oriC Fragment Bound 0.2 0.0 ' I I I I I 0.0 I I I I I 0 50 100 150 200 250 0 50 100 150 200 250 DnaA Protein (ng) 1.2 40 °C 1.0 0.8 0.6 0.4 oriC Fragment Bound 0.2 0.0 l l l l l 00 ' " I l l l l 0 50 100 150 200 250 0 50 100 150 200 250 DnaA Protein (ng) O DnaA+ x DnaA-A184V O DnaA46 I DnaA-H252Y A DnaA5 El DnaA-64263 71 fragment containing oriC by a filter binding assay. All of the forms of DnaA were able to bind to the origin at either 30 °C or 40 °C (Figure 8). The cooperative nature of binding by DnaA+ is suggested by the sigmoidal shape of the curve. DnaA46 and DnaA—A184V, while retaining affinity for oriC DNA, appeared altered in their binding (Figure 8A). Binding of oriC did not appear to be cooperative and was more efficient than DnaA+ at lower concentrations of protein. DnaA—H252Y protein bound to oriC in a sigmoidal manner similar to wild type. Apparently, this mutation does not influence the DNA binding activity of DnaA46 since its binding was similar to that of DnaA-A184V. Binding to oriC by DnaA46 and DnaA—H252Y was unaffected at 40 °C relative to 30 °C, whereas DnaA—A184V showed a slight reduction. It was still able to bind to oriC at least as well as the wild type at the elevated temperature. That the DNA binding activity of DnaA46 appears similar to DnaA-A184V suggests that this common mutation affects DNA binding. The DnaA—H252Y mutant protein appears similar to wild type in DNA binding suggesting that this residue is not important in this activity. DnaA5 and the corresponding single mutation, DnaA—G426S, were also measured for oriC binding activity (Figure 88). Binding by DnaA5 to oriC was slightly temperature sensitive as was previously reported (Hupp and Kaguni, 1993c). DnaA—G426S protein was reduced in its ability to bind to oriC at 40 °C compared to 30 °C. Relative to wild type DnaA, the binding to oriC was somewhat reduced at 30 °C. Interestingly oriC binding by DnaA5 protein was intermediate between the activity observed for the two single mutant 72 proteins suggesting that their effects counteract each other when both are present. Whether the activity of DnaA5 results from an interaction between the two mutations or represents an average of independent effects cannot be determined from this experiment. Fragment retention or filter binding assays only measure the total amount of DNA bound by protein but reveal little about the nature of the protein—DNA complexes. DNA gel shift assays were performed to examine the complexes formed between the mutant proteins and a restriction fragment containing oriC (Figure 9). Addition of increasing amounts of DnaA+ protein resulted in the appearance of a number of complexes which are resolved by native gel electrophoresis. These complexes represent the ordered binding of DnaA to the four DnaA boxes present in oriC (C. E. Margulies and J. M. Kaguni, unpublished results). Examination of the mutant proteins revealed that DnaA—H252Y and DnaA-G426S formed complexes nearly identical to DnaA+. The other three proteins, DnaA5, DnaA46 and DnaA—A184V also formed at least some of the same complexes with oriC as wild type DnaA. However, there was a reduction in the abundance of intermediate complexes formed. This could be the result of the A184V mutation present in these proteins. Alternatively, it may be related to the physical state of the purified protein. No new or anomalous complexes were observed between the mutant DnaA proteins and oriC suggesting that gross alterations in the association of these proteins with oriC were not occurring. 73 Figure 9. Gel shift assay of oriC binding by mutant DnaA proteins. The indicated amounts of DnaA protein were incubated with DNA fragment (described in the legend for Fig. 8) containing oriC. Separated complexes were visualized by autoradiography. The uppermost band corresponding to the position of the well are of complexes which did not enter the gel. 74 DnaA‘ DnaA-H252Y DnaA-G426S D ifi ii i .o 6 254050 6122550 6122550ng DnaA-A184V DnaA46 DnaA5 I ”7 ii I 0 122550 61225506122550ng . 2! 1.. Discussion In order to understand better the contribution of the individual missense mutations in dnaA5 and dnaA46 to their observed biochemical defects, the missense mutations were separated from each other and the resulting single mutant proteins were studied. The mutation at amino acid 184 (alanine ED valine) is common to both of these alleles and was found to be responsible for the thermolabile defect and the prolonged lag in initiation of chromosomal replication. Correlation of these two biochemical defects with a single mutation would be predicted because both of the defects were previously shown to be related. The prolonged lag period is due to a required activation of the mutant proteins by the heat shock proteins DnaK and GrpE (Hupp and Kaguni, 1993a; Hwang and Kaguni, 1991) and it is this interaction which was thermolabile (Hupp and Kaguni, 1993b; Hwang and Kaguni, 1988a). Aggregated DnaA+ protein also showed a lag in in vitro DNA synthesis that was shorter than that for these proteins with the A184V mutation. Since there was only a very short lag for monomeric DnaA+, these results suggest that the activation process involves disaggregation of the mutant proteins. This is supported by reports that DnaAagg, like DnaA5 and DnaA46 is unable to function in in vitro replication systems using purified proteins without prior activation by DnaK protein (Hwang, et al., 1990). The DnaK activation does result in conversion of DnaA to a monomeric form. 75 76 The mutation at amino acid 184 results in a less efficient and thermolabile interaction with heat shock proteins. The short lag period observed for DnaA-G426S is probably also the result of protein aggregation like that of DnaAagg and not related to its defect in binding to oriC, since it has been shown previously that the activation of DnaA5 protein, which harbors the 426 mutation, can occur prior to its interaction with oriC (Hupp and Kaguni, 1993b). The ATP binding defect previously reported for DnaA5 and DnaA46 (Hupp and Kaguni, 1993c; Hwang and Kaguni, 1988a) had been attributed to the A184V mutation since it is very close to a consensus ATP binding (P—loop) motif. No ATP binding was observed with DnaA-A184V, whereas detectable binding by the other two single mutant proteins was observed. This supports the hypothesis that the mutation at amino acid 184 is directly responsible for the inability of DnaA5 and DnaA46 to bind ATP. Comparison of the ATP binding activities of DnaA+ and DnaAagg, however, suggests that physical state of the purified protein greatly influences the ability of DnaA to bind ATP. Aggregated DnaA+ protein is known to be associated with phospholipids, among them cardiolipin (Hwang, et al., 1990). Acidic phospholipids have been implicated in regulating the activity of DnaA and cardiolipin has been shown to be able to prevent ATP binding by DnaA under certain conditions (Sekimizu and Kornberg, 1988) or alternatively to allow the exchange of ADP and ATP on DnaA. Presumably the guanidine hydrochloride treatment in the purification of DnaA+ and DnaA-H252Y 77 removes bound phospholipids but this has not been examined directly. DnaA-G426S, which like DnaAagg, has not been treated with guanidine, binds ATP only slightly better than DnaAagg and significantly less well than DnaA+. In those proteins where binding can be detected, the KD values determined are similar to that previously reported for DnaA of 0.03 uM (Sekimizu, et al., 1987) but they vary tremendously in the calculated n value. It is a measure of the number of ATP molecules bound per molecule of DnaA protein, but the n value may also be interpreted as a representing the fraction of DnaA molecules in the sample capable of binding ATP. This fraction may vary as a function of aggregation, the presence of bound phospholipids, or the contribution of both of these factors. These results further suggest that the processes of activation, disaggregation and ATP binding are related. A number of reports in the literature would tend to support this view. Nucleotide binding by DnaA stabilizes the monomeric form of the protein (Yung, et al., 1990). Cardiolipin can, under varying conditions, inhibit nucleotide binding by DnaA or facilitate the turnover from the inactive, ADP-bound DnaA to active ATP—bound DnaA (Sekimizu and Kornberg, 1988). Aggregated DnaA protein, which is inactive in in vitro replication using purified proteins, is complexed with phospholipids. The aggregated protein can be activated for initiation of DNA replication by DnaK protein or by phospholipase A2, an enzyme which degrades phospholipids (Hwang, et al., 1990). 78 It is still a possibility that the mutation at amino acid 184 does result in a defect in ATP binding not dependent on aggregation. DnaA5 was unable to form the characteristic open complex at oriC (Hupp and Kaguni, 1993c). Formation of this complex is dependent on the ATP—bound form of DnaA (Sekimizu, et al., 1987) so the deficiency of DnaA5 was attributed to its lack of ATP binding. DnaA5 which had been activated for replication by DnaK and GrpE was still unable to form this complex (cited in Hupp and Kaguni, 1993a), which may indicate it is still unable to bind ATP. Disaggregation of DnaAagg resulting in ability to bind ATP may not be an appropriate model for activation of DnaA5 and DnaA46. DnaAalgg is activated for replication in reconstituted systems by DnaK alone (Hwang, et al., 1990), whereas DnaA5 and DnaA46 absolutely require GrpE in addition to DnaK (Hupp and Kaguni, 1993a). Binding of DnaA protein to a DNA fragment containing the oriC sequence, measured by nitrocellulose filter binding assay, was altered by two of the missense mutations, A184V and G426S. DnaA-A184V and DnaA46 bound oriC more efficiently at low protein concentrations but the cooperative nature of the DNA binding appeared reduced. A previous report that DnaA46 failed to bind in vitro to DNA containing the consensus DnaA binding sequence was apparently erroneous (Hwang and Kaguni, 1988a). The activity of these mutant proteins is very labile when purified (numerous personal observations) and this may have been a factor in the previous observations. The ability of DnaA46 to bind to oriC does however contradict 79 numerous other reports that DnaA46 cannot repress transcription from the dnaA promoter at the nonpermissive temperature (Kucherer, et al., 1986). Inability to repress transcription has been taken to indicate that the mutant protein does not bind to DNA at elevated temperature. A number of factors may explain this apparent discrepancy, most obvious is that the binding to two different DNA fragments is being measured; oriC contains four DnaA boxes whereas the dnaA promoter contains only one. Another difference is the assay used. In the fragment retention assay the mutant protein is exposed to the elevated temperature for only a short time. In in vivo transcriptional repression assays, the protein is held at the nonpermissive temperature for a longer period. The presence of other cellular factors in the in vivo assay may also negatively effect the ability of the protein to bind at elevated temperatures. DnaA-H252Y was not altered in binding to oriC relative to DnaA+ and the mutation did not influence the DNA binding activity of the DnaA46 protein. The altered nature of the oriC binding activity of DnaA46 was apparently due to the A184V mutation alone. The mutation G426S gave rise to a partially temperature sensitive defect in binding to DNA and it is likely the cause of the observed partially thermolabile replication activity. Although no direct evidence has been reported in the literature, there are suggestions that the C—terminal domain of DnaA is involved in DNA binding (cited in Messer and Weigel, 1994; Skarstad and Boye, 1994). The clear defect in DNA binding by DnaA—G426S is the first biochemical evidence documenting the involvement of this region of DnaA in DNA binding. 80 Further investigation of the interaction of the mutant DnaA proteins with oriC was done using a gel shift assay. The similarity of complex formation observed for DnaA+ and DnaA—G426S indicates that the defect resulting in the temperature sensitive binding of DnaA-G426S did not apparently alter the structure of the DnaAzoriC complex, at least at 25 °C, the assay temperature for the gel shift. The reduced abundance of the intermediate complexes seen with mutants harboring A184V may be due directly to the mutation, or secondarily to some altered conformation of the protein resulting from the mutation. DnaA5 protein appeared to be the most affected in formation of these intermediate complexes. Previous examination of the interaction of DnaA5 with oriC by DNase I footprinting (Hupp and Kaguni, 1993c) revealed that the protein was altered in its association with oriC, protecting broader and less well defined regions within oriC. Altered patterns in the gel shift assay support the previous report that DnaA5 is somehow altered in its association with oriC. Since both of the amino acid substitutions in DnaA5 were shown to affect interaction with oriC, it is not clear if one of them or interaction of the two is responsible for the altered footprint. An alanine—to—valine substitution at amino acid 184 has been identified in two other mutants of dnaA besides dnaA5 and dnaA46. Like them, these other mutants also have a second missense mutation (Hansen, et al., 1992). Though no direct evidence exists, this curious finding has been used to suggest that a mutation at this amino acid is so deleterious it can only 81 be maintained if there is a secondary mutation to compensate. The oriC binding activity of DnaA5, measured in the fragment retention assay, was affected by both of the mutations present. Increased binding to oriC caused by the A184V mutation was balanced by a decrease in binding due to the G426S mutation. The net result was activity more closely approximating wild type DnaA. The oriC binding activity of DnaA46 was unaltered relative to DnaA—A184V however, which would suggest that this altered activity does not represent a lethal effect. Other than the DNA binding activity, the only other difference between the DnaA—A184V and the double mutants observed in this study was in the DNA replication. The increased activity at higher levels of protein observed for DnaA-A184V and the more pronounced inhibition seen with DnaA—G426S appeared to offset each other when present jointly in DnaA5. The same reduction in activity of DnaA46 relative to DnaA-A184V was seen, and was the only difference observed between DnaA—A184V and DnaA46. In all of the biochemical activities examined thus far, DnaA-H252Y was indistinguishable from DnaA+, so there is no apparent explanation for having this effect in DnaA46. Since amino acid 252 is so highly conserved (identical in 14 out 15 species, reviewed in Messer and Weigel, 1994) as are 184 and 426, it is surprising that a mutation at this position did not alter the activity of the protein in at least some small way. A phenotype resulting from this mutation is of course still possible since the entire range of DnaA protein function has not been examined, or an effect may only result through interaction with the A184V mutation. Chapter III Summary and Perspectives 82 Three missense mutations that are found in the dnaA5 and dnaA46 alleles were separated in order to correlate biochemical defects to the individual mutations. Recombinant DNA techniques were used to separate the mutations and the T7 RNA polymerase expression system (Novagen) used to overproduce the various forms of DnaA for study. The alanine—to—valine substitution at amino acid 184 that is common to both of the mutants was responsible for the thermolabile defect and prolonged lag prior to initiation of DNA replication in vitro. A partial temperature sensitive defect was also observed when glycine 426 is substituted with serine, the mutation unique to DnaA5. The ability of the various mutant or wild type d naA alleles to complement the temperature sensitive phenotype of dnaA204 in vivo correlated with the results obtained in vitro. Examination of the mutant alleles in this genetic background however precludes assessing their function in vivo at the permissive temperature since the dnaA204 strain is viable at 30 °C. Reintroducing the alleles of dnaA with the isolated mutations onto the chromosome in an otherwise normal genetic background will allow for a more careful examination of their phenotypes in vivo. Physiological studies of the single mutants would also provide more insight into their effects on DnaA protein function in initiation of replication. Of particular interest would be flow 83 84 cytometry experiments to examine the synchrony of initiation with each of the alleles. It will also be possible to address directly whether or not the A184V mutation is lethal when present alone and if the second mutations compensate for this. ATP binding by mutants carrying A184V was not detected. This is expected since the mutation is very close to the putative ATP binding site. Aggregated, wild type DnaA, however, was also unable to bind ATP at any significant level, suggesting an alternate explanation for the inability of these mutants to bind ATP. Determination of the ability of these mutants to bind ATP is of central importance to understanding the role ATP binding plays in regulating the activity of DnaA. The effects of DnaK/GrpE activation of DnaA5, DnaA46 and DnaA—A184V on the physical state of these proteins can be examined by centrifugation or gel filtration studies. Whether activation of the mutant proteins for replication also confers the ability to bind ATP may be addressed by crosslinking and SDS—PAGE analysis to separate the crosslinked DnaA protein from DnaK. DnaK also binds ATP. DNA binding activity to oriC DNA was found to be altered by two of the mutations examined, A184V and G426S. DnaA-A184V and DnaA46 bound to oriC more efficiently at lower protein concentrations and the binding was not thermolabile. DnaA-G426S was partially temperature sensitive for binding to oriC and likely explains the thermolability observed in the DNA replication assay. This defect did not result in detectable alterations of the DnaAzoriC interaction in a gel shift assay. Alteration of 85 oriC:DnaA interaction with these mutants had already been suggested by genetic and biochemical studies. Deletion of topA, encoding topoisomerase I can suppress the temperature sensitive phenotype of dnaA46 (Louarn, et al., 1984). Loss of topoisomerase I activity results in a greater degree of negative superhelicity in the chromosome. Altered supercoiling may compensate for a defect in the proteinzDNA interaction. DNase I protection patterns at oriC were altered with DnaA5 relative to DnaA+. Further investigation of the defects observed in DNA binding with these mutants by footprinting, determination of affinity on supercoiled versus relaxed templates and ability to promote strand unwinding will better clarify the nature of the defect(s) in DNA binding. The histidine—to-tyrosine change at amino acid 252 in DnaA46 did not alter the activity of the protein relative DnaA+ in any of the assays used in this study. This is quite surprising given that this amino acid is conserved in 14 out of 15 homologues of dnaA which have been sequenced thus far (reviewed in Messer and Weigel, 1994). It also did not have a significant influence on the activity of DnaA46. Except for a subtle difference in activity in the DNA replication assay, no other difference was observed between DnaA46 or the single mutant DnaA—A184V. An alternate function of this region is suggested by the observation the codon for amino acid 252 is part of a DnaA box present in the reading frame of dnaA. A transcriptional termination event has been shown to occur in this region of dnaA but it is 86 unclear if it is dependent on DnaA protein or this binding sequence (Wende, etaL,1991) Identification of specific biochemical defects associated with a mutation and correlation to physiological alterations are common and useful methods to understand of the normal functioning of proteins. Genetic studies strongly suggest that the mutation at amino acid 184 is responsible for the asynchronous initiation phenotype observed in dnaA5 and dnaA46 strains (reviewed in (Hansen, et al., 1992). There are multiple biochemical defects observed for DnaA—A184V, including an increased tendency for self aggregation, a possible defect in ATP binding and an alteration in oriC interaction. All of these have been proposed as being involved in the regulatory function of DnaA protein. Biochemical characterization of other dnaA mutants will complement knowledge gained from the study of DnaA5, DnaA46 and the corresponding single mutant proteins. Such studies will be easier due to improved expression of dnaA in the pET system allowing more rapid purification of new mutant proteins. Bibliography Abe, M. and Tomizawa, J. (1971) Chromosome replication in Escherichia coli K12 mutant affected in the process of DNA initiation. Genet. 69:1-15. Asada, K., Sugimoto, K., Oka, A., Takanami, M. and Hirota, Y. (1982) Structure of replication origin of the Escherichia coli K-12 chromosome: the presence of spacer sequences in the ori region carrying information for autonomous replication. N ucl Acids Res. 10:3745-3754. Atlung, T. (1984) Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli. Mol Gen Genet. 197:125-128. Atlung, T., Clausen, E. S. and Hansen, F. G. (1985) Autoregulation of the dnaA gene of Escherichia coli K12. Mol Gen Genet. 200:442-450. Atlung, T. and Hansen, F. G. (1993) Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli. J Bacteriol. 175:6537-6545. Augustin, L. B., Jacobson, B. A. and Fuchs, J. A. (1994) Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion. J Bacteriol. 176:378-387. Bird, R. 15., Chandler, M. and Caro, L. (1976) Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures. J Bacteriol. 126:1215-1223. Bird, R. E., Louarn, J., Martuscelli, J. and Caro, L. (1972) Origin and sequence of chromosome replication in Escherichia coli. J Mol Biol. 70:549-566. Boye, E. and Lobner-Olesen, A. (1990) The role of Dam methyltransferase in the control of DNA replication in E. coli. Cell. 62:981-989. Boye, E., Lobner-Olesen, A. and Skarstad, K. (1988) Timing of chromosomal replication in Escherichia coli. Biochim BiOphys Acta. 951:359-364. Boye, E., Lyngstadaas, A., Lobner-Olesen, A., Skarstad, K. and Wold, S. (1993) Regulation of DNA replication in Escherichia coli. DNA replication and the cell cycle 43:15-26. Bradford, M. (1976) Anal Biochem. 72:248-254. 87 88 Bramhill, D. and Kornberg, A. (1988a) Duplex opening by DnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell. 52:743-755. Bramhill, D. and Kornberg, A. (1988b) A model for initiation at origins of DNA replication. Cell. 54:915-918. Bremer, H. and Churchward, G. (1985) Initiation of chromosome replication in Escherichia coli after induction of dnaA gene expression from a lac promoter. J Bacteriol. 164:922-924. Bukau, B. and Walker, G. C. (1989) Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J Bacteriol. 171:2337-2346. Burgers, P. M., Kornberg, A. and Sakakibara, Y. (1981) The dnaN gene codes for the beta subunit of DNA polymerase HI holoenzyme of Escherichia coli. Proc Natl Acad Sci, USA. 78:5391-5395. Campbell, J. L. and Kleckner, N. (1990) E. coli oriC and the dnaA gene promoter are sequestered from Dam methyltransferase following the passage of the chromosomal replication fork. Cell. 62:967-979. Chiaramello, A. E. and Zyskind, J. W. (1989) Expression of Escherichia coli dnaA and mioC genes as a function of growth rate. J Bacteriol. 171:4272- 4280. Churchward, G. and Bremer, H. (1977) Determination of deoxyribonucleic acid replication time in exponentially growing Escherichia coli B/r. J Bacteriol. 130:1206-1213. Cleary, J. M., Smith, D. W., Harding, N. E. and Zyskind, J. W. (1982) Primary structure of the chromosomal origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae: comparisons and evolutionary relationships. J Bacteriol. 150:1467-1471. Cooper, S. and Helmstetter, C. E. (1968) Chromosome replication and the division cycle of Escherichia coli B/ r. J Mol Biol. 1:519-540. de Massy, B., Fayet, O. and Kogoma, T. (1984) Multiple origin usage for DNA replication in sdrA(rnh) mutants of Escherichia coli K-12. Initiation in the absence of oriC. J Mol Biol. 178:227—236. Donachie, W. D. (1968) Relationship between cell size and time of initiation of DNA replication. Nature. 219:1077-1079. 89 Eberle, H. and Forrest, N. (1982) Regulation of DNA synthesis and capacity for initiation in DNA temperature sensitive mutants of Escherichia coli. 11. Requirements for acquisition and expression of initiation capacity. Mol Gen Genet. 186:66-70. Evans, 1. M. and Eberle, H. (1975) Accumulation of the capacity of initiation of deoxyribonucleic acid replication in Escherichia coli. J Bacteriol. 121:883- 891. Fayet, 0., Louarn, J. M. and Georgopoulos, C. (1986) Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. Mol Gen Genet. 202:435-445. Felton, J. and Wright, A. (1979) Plasmid pSC101 replication in integratively suppressed cells requires dnaA function. Mol Gen Genet. 175:231-233. Filutowicz, M., Ross, W., Wild, J. and Course, R. L. (1992) Involvement of Fis protein in replication of the Escherichia coli chromosome. J Bacteriol. 174:398-407. Fraenkel-Conrat, H. and Singer, B. (1972) The chemical basis for the mutagenicity of hydroxylamine and methoxyamine. Biochim Biophys Acta. 262:264-8. Frey, J., Chandler, M. and Caro, L. (1981) The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures. Mol Gen Genet. 182:364-366. Fuller, R. S., Funnell, B. E. and Kornberg, A. (1984) The DnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell. 38:889-900. Fuller, R 5., Kaguni, J. M. and Kornberg, A. (1981) Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci, USA. 78:7370-7374. Fuller, R. S. and Kornberg, A. (1983) Purified DnaA protein in initiation of replication at the Escherichia coli chromosomal origin of replication. Proc Natl Acad Sci, USA. 80:5817-5821. Funnell, B. E., Baker, T. A. and Kornberg, A. (1987) In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome. J Biol Chem. 262:10327-10334. 9O Gielow, A., Kucherer, C., Kolling, R. and Messer, W. (1988) Transcription in the region of the replication origin, oriC, of Escherichia coli: termination of aan transcripts. Mol Gen Genet. 214:474-481. Gille, H., Egan, J. B., Roth, A. and Messer, W. (1991) The Fis protein binds and bends the origin of chromosomal DNA replication, oriC, of Escherichia coli. Nucl Acids Res. 19:4167-4172. Gille, H. and Messer, W. (1991) Localized DNA melting and structural perturbations in the origin of replication, oriC, of Escherichia coli in vitro and in vivo. Euro Mol Biol Org J. 10:1579-1584. Hansen, E. B., Atlung, T., Hansen, F. G., Skovgaard, O. and von Meyenburg, K. (1984) Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli. Mol Gen Genet. 196:387- 396. Hansen, E. B., Hansen, F. G. and von Meyenburg, K. (1982) The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K-12. Nucl Acids Res. 10:7373-7385. Hansen, F. G., Koefoed, S. and Atlung, T. (1992) Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli. Mol Gen Genet. 234:14-21. Hansen, F. G., Koefoed, S., Sorensen, L. and Atlung, T. (1987) Titration of DnaA protein by oriC DnaA-boxes increases d naA gene expression in Escherichia coli. Euro Mol Biol Org J. 6:255-258. Hansen, F. G. and Rasmussen, K. V. (1977) Regulation of the dnaA product in Escherichia coli. Mol Gen Genet. 155:219-225. Helmstetter, C. E. (1968) DNA synthesis during the division cycle of rapidly growing Escherichia coli B/ r. J Mol Biol. 1:507-518. Hiraga, S. (1976) Novel F’ factors able to replicate in Escherichia coli Hfr strains. Proc Natl Acad Sci, USA. 73:198-202. Hirota, Y., Atsuhiro, O., Kazunori, S., Kiyozo, A., Hitoshi, S. and Takanami, M. (1981) Escherichia coli origin of replication: structural organization of the region essential for autonomous replication and recognition frame model. p. 1-12 in Ray, D. S. (eds.),The Initiation of DNA Replication. Academic Press, Los Angeles. 91 Hirota, Y., Mordoh, J. and Jacob, F. (1970) On the process of cellular division in Escherichia coli. III. Thermosensitive mutants of Escherichia coli altered in the process of DNA initiation. J Mol Biol. 53:369-387. Hirota, Y., Ryter, A. and Jacob, F. (1968) Thermosensitive mutants of E. coli affected in the processes of DNA synthesis and cellular division. Cold Spring Harb Symp Quant Biol. 677-693. Holz, A., Schaefer, C., Gille, H., Jueterbock, W. R and Messer, W. (1992) Mutations in the DnaA binding sites of the replication origin of Escherichia coli. Mol Gen Genet. 233:81-88. Hupp, T. R. and Kaguni, J. M. (1988) Suppression of the Escherichia coli dnaA46 mutation by a mutation in trxA, the gene for thioredoxin. Mol Gen Genet. 213:471-478. Hupp, T. R. and Kaguni, J. M. (1993a) Activation of DnaA5 protein by GrpE and DnaK heat shock proteins in initiation of DNA replication in Escherichia coli. J Biol Chem. 268:13137-13142. Hupp, T. R and Kaguni, J. M. (1993b) Activation of mutant forms of DnaA protein of Escherichia coli by DnaK and GrpE proteins occurs prior to DNA replication. J Biol Chem. 268:13143-13150. Hupp, T. R. and Kaguni, J. M. (1993c) DnaA5 protein is thermolabile in initiation of replication from the chromosomal origin of Escherichia coli. J Biol Chem. 268:13128-13136. Hwang, D. 5., Crooke, E. and Kornberg, A. (1990) Aggregated DnaA protein is dissociated and activated for DNA replication by phospholipase or DnaK protein. J Biol Chem. 265:19244-19248. Hwang, D. S. and Kaguni, J. M. (1988a) Interaction of DnaA46 protein with a stimulatory protein in replication from the Escherichia coli chromosomal origin. J Biol Chem. 263:10633-10640. Hwang, D. S. and Kaguni, J. M. (1988b) Purification and characterization of the dnaA46 gene product. J Biol Chem. 263:10625-10632. Hwang, D. S. and Kaguni, J. M. (1991) DnaK protein stimulates a mutant form of DnaA protein in Escherichia coli DNA replication. J Biol Chem. 266:7537-7541. 92 Hwang, D. S. and Kornberg, A. (1992a) Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF. J Biol Chem. 267:23083-23086. Hwang, D. S. and Kornberg, A. (1992b) Opposed actions of regulatory proteins, DnaA and IciA, in opening the replication origin of Escherichia coli. J Biol Chem. 267:23087-23091. Hwang, D. S., Thony, B. and Kornberg, A. (1992) IciA protein, a specific inhibitor of initiation of Escherichia coli chromosomal replication. J Biol Chem. 267:2209-2213. Jacob, F., Brenner, S. and Cusin, F. (1963) On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp Quant Biol. 28:329-348. Junker, D. E., Jr., Rokeach, L. A., Ganea, D., Chiaramello, A. and Zyskind, J. W. (1986) Transcription termination within the Escherichia coli origin of DNA replication, oriC. Mol Gen Genet. 203:101-109. Kaguni, J. M., Bertsch, L. L., Bramhill, D., Flynn, J. E., Fuller, R S., Funnell, B., Maki, S., Ogawa, T., Ogawa, K., van der Ende, A., et a1. (1985) Initiation of replication of the Escherichia coli chromosomal origin reconstituted with purified enzymes. Basic Life Sci. 141-150. Kaguni, J. M., Fuller, R. S. and Kornberg, A. (1982) Enzymatic replication of E. coli chromosomal origin is bidirectional. Nature. 296:623-627. Kaguni, J. M. and Kornberg, A. (1984) Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes. Cell. 38:183-190. Kano, Y. and Imamoto, F. (1990) Requirement of integration host factor (H-IF) for growth of Escherichia coli deficient in HU protein. Gene. 89:133-137. Kellenberger-Gujer, G., Podhajska, A. J. and Caro, L. (1978) A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature. Mol Gen Genet. 162:9-16. Koch, C. and Kahmann, R. (1986) Purification and properties of the Escherichia coli host factor required for inversion of the G segment in bacteriophage Mu. J Biol Chem. 261:15673-15678. Kogoma, T. and Kline, B. C. (1987) Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process. Mol Gen Genet. 210:262-269. 93 Kogoma, T. and Lark, K. G. (1975) Characterization of the replication of Escherichia coli DNA in the absence of protein synthesis: stable DNA replication. J Mol Biol. 94:243-256. Kohiyama, M. (1968) DNA synthesis in temperature sensitive mutants of Escherichia coli. Cold Spring Harb Symp Quant Biol. 317-324. Koonin, E. V. (1993) A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucl Acids Res. 21:2541-2547. Koppes, L. J. and von Meyenburg, K. (1987) Nonrandom minichromosome replication in Escherichia coli K-12. J Bacteriol. 169:430-433. Kubitschek, H. E. (1974) Estimation of the D period from residual division after exposure of exponential phase bacteria to chloramphenicol. Mol Gen Genet. 135:123-130. Kubitschek, H. E. and Newman, C. N. (1978) Chromosome replication during the division cycle in slowly growing, steady-state cultures of three Escherichia coli B/r strains. J Bacteriol. 136:179-190. Kucherer, C., Lother, H., Kolling, R, Schauzu, M. A. and Messer, W. (1986) Regulation of transcription of the chromosomal dnaA gene of Escherichia coli. Mol Gen Genet. 205:115-121. Lark, K. G. (1972) Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15T. J Mol Biol. 64:47-60. Liu, L. F. and Wang, J. C. (1987) Supercoiling of the DNA template during transcription. Proc Natl Acad Sci, USA. 84:7024-7027. Lother, H., Kolling, R, Kucherer, C. and Schauzu, M. (1985) DnaA protein- regulated transcription: effects on the in vitro replication of Escherichia coli minichromosomes. Euro Mol Biol Org J. 4:555-560. Louarn, J., Bouche, J. P., Patte, J. and Louarn, J. M. (1984) Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli. Mol Gen Genet. 195:170-174. Lu, M., Campbell, J. L., Boye, E. and Kleckner, N. (1994) Squ: a negative regulator of replication in E. coli. Cell. in press. Maaloe, O. and Kjeldgaard, N. O. (1966) Control of Macromolecular Synthesis. 94 Malki, A., Hughes, P. and Kohiyama, M. (1991) In vitro roles of Escherichia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids. Mol Gen Genet. 225:420-426. Marszalek, J. and Kaguni, J. M. (1994) DnaA protein directs the binding of DnaB protein in initiation of DNA replication in Escherichia coli. J Biol Chem. 269:4883-4890. Masters, M. and Broda, P. (1971) Evidence for the bidirectional replications of the Escherichia coli chromosome. Nature New Biol. 232:137-140. Masters, M., Paterson, T., Popplewell, A. G., Owen-Hughes, T., Pringle, J. H. and Begg, K. J. (1989) The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the fist) and ftsA genes: in vivo experiments. Mol Gen Genet. 216:475-483. McHenry, C. S. (1985) DNA polymerase HI holoenzyme of Escherichia coli: components and function of a true replicative complex. Mol Cell Biochem. 66:71-85. McMacken, R, Silver, L. and Georgopoulos, C. (1987) DNA Replication. p. 564-612 in Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M. and Umbarger, H. E. (eds.),Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C. Meijer, M. and Messer, W. (1980) Functional analysis of minichromosome replication: bidirectional and unidirectional replication from the Escherichia coli replication origin, oriC. J Bacteriol. 143:1049-1053. Messelson, M. and Stahl, F. (1958) The replication of DNA in E. coli. Proc Natl Acad Sci, USA. 44:671-682. Messer, W. (1972) Initiation of deoxyribonucleic acid replication in Escherichia coli B/ r: chronology of events and transcriptional control of initiation. J Bacteriol. 112:7—12. Messer, W., Egan, B., Gille, H., Holz, A., Schaefer, C. and Woelker, B. (1991) The complex of oriC DNA with the DnaA initiator protein. Res Microbiol. 142:119-125. Messer, W., Hartmann-Kuhlein, H., Langer, U., Mahlow, E., Roth, A., Schaper, S., Urmoneit, B. and Woelker, B. (1992) The complex for replication initiation of Escherichia coli. Chromosoma. 102:81-6. 95 Messer, W. and Noyer-Weidner, M. (1988) Timing and targeting: the biological functions of Dam methylation in E. coli. Cell. 54:735-737. Messer, W. and Weigel, C. (1994) Initiation of chromosome replication. in press in Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M. and Umbarger, H. E. (eds.),Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C. Nishimura, Y., Caro, L., Berg, C. M. and Hirota, Y. (1971) Chromosome replication in Escherichia coli. IV. Control of chromosome replication and cell division by an integrated episome. J Mol Biol. 55:441-456. Norris, V. (1990) DNA replication in Escherichia coli is initiated by membrane detachment of oriC. A model. J Mol Biol. 215:67-71. Ogawa, T. and Okazaki, T. (1991) Concurrent transcription from the gid and mioC promoters activates replication of an Escherichia coli minichromosome. Mol Gen Genet. 230:193-200. Ogden, G. B., Pratt, M. J. and Schaechter, M. (1988) The replicative origin of the E. coli chromosome binds to cell membranes only when hemimethylated. Cell. 54:127-135. Ohki, M. and Smith, C. L. (1989) Tracking bacterial DNA replication forks in vivo by pulsed field gel electrophoresis. Nucleic Acids Res. 17:3479-90. Oka, A., Sugimoto, K., Takanami, M. and Hirota, Y. (1980) Replication origin of the Escherichia coli K-12 chromosome: the size and structure of the minimum DNA segment carrying the information for autonomous replication. Mol Gen Genet. 178:9-20. Parada, C. A. and Marians, K. J. (1991) Mechanism of DnaA protein- —dependent pBR322 DNA replication. DnaA protein-mediated trans-strand loading of the DnaB protein at the origin of pBR322 DNA. J Biol Chem. 266:18895-18906. Polaczek, P. (1990) Bending of the origin of replication of E. coli by binding of H-IF at a specific site. New Biol. 2:265-271. Polaczek, P. and Wright, A. (1990) Regulation of expression of the dnaA gene in Escherichia coli: role of the two promoters and the DnaA box. New Biol. 2:574-582. 96 Rokeach, L. A. and Zyskind, J. W. (1986) RNA terminating within the E. coli origin of replication: stringent regulation and control by DnaA protein. Cell. 46:763-771. Sakakibara, Y. (1988) The dnaK gene of Escherichia coli functions in initiation of chromosome replication. J Bacteriol. 170:972-9. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning; A Laboratory Manual. Saraste, M., Sibbald, P. R. and Wittinghofer, A. (1990) The P-loop—a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 15:430-434. Schaefer, C. and Messer, W. (1988) Termination of the Escherichia coli aan transcript. The DnaA protein/ DnaA box complex blocks transcribing RNA polymerase. Gene. 73:347-354. Schaus, N., O'Day, K., Peters, W. and Wright, A. (1981) Isolation and characterization of amber mutations in gene dnaA of Escherichia coli K-12. J Bacteriol. 145:904—913. Schauzu, M. A., Kucherer, C., Kolling, R, Messer, W. and Lother, H. (1987) Transcripts within the replication origin, oriC, of Escherichia coli. Nucl Acids Res. 15:2479-2497. Sekimizu, K., Bramhill, D. and Kornberg, A. (1987) ATP activates DnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell. 50:259-265. Sekimizu, K. and Kornberg, A. (1988) Cardiolipin activation of DnaA protein, the initiation protein of replication in Escherichia coli. J Biol Chem. 263:7131-5. Sekimizu, K., Yung, B. Y. and Kornberg, A. (1988) The DnaA protein of Escherichia coli: abundance, improved purification, and membrane binding. J Biol Chem. 263:7136-7140. Skarstad, K., Baker, T. A. and Kornberg, A. (1990) Strand separation required for initiation of replication at the chromosomal origin of E.coli is facilitated by a distant RNA—DNA hybrid. Euro Mol Biol Org J. 9:2341-2348. Skarstad, K. and Boye, E. (1994) The initiator protein DnaA: evolution, properties and function. Biochim Biophys Acta. 1217:111-130. 97 Skarstad, K., Boye, E. and Steen, H. B. (1986) Tinting of initiation of chromosome replication in individual Escherichia coli cells [published erratum appears in EMBO J 1986 Nov;5(11):3074]. Euro Mol Biol Org J. 5:1711-1717. Skarstad, K., Lobner-Olesen, A., Atlung, T., von Meyenburg, K. and Boye, E. (1989) Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein. Mol Gen Genet. 218:50-56. Skarstad, K., Steen, H. B. and Boye, E. (1983) Cell cycle parameters of slowly growing Escherichia coli B / r studied by flow cytometry. J Bacteriol. 154:656- 662. Skarstad, K., Steen, H. B. and Boye, E. (1985) Escherichia coli DNA distributions measured by flow cytometry and compared with theoretical computer simulations. J Bacteriol. 163:661-668. Skarstad, K., von Meyenburg, K., Hansen, F. G. and Boye, E. (1988) Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles. J Bacteriol. 170:852-858. Stuitje, A. R, de Wind, N., van der Spek, J. C., Pors, T. H. and Meijer, M. (1986) Dissection of promoter sequences involved in transcriptional activation of the Escherichia coli replication origin. Nucl Acids Res. 14:2333-2344. Szyf, M., Gruenbaum, Y., Urieli-Shoval, S. and Razin, A. (1982) Studies on the biological role of DNA methylation: V. The pattern of E.coli DNA methylation. Nucl Acids Res. 10:7247-7259. Tesfa-Selase, F. and Drabble, W. T. (1992) Regulation of the gua operon of Escherichia coli by the DnaA protein. Mol Gen Genet. 231:256-264. Theisen, P. W., Grimwade, J. E., Leonard, A. C., Began, J. A. and Helmstetter, C. E. (1993) Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli. Mol Micro. 10:575-584. Torrey, T. A., Atlung, T. and Kogoma, T. (1984) dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants. Mol Gen Genet. 196:350-355. von Freiesleben, U. and Rasmussen, K. V. (1991) DNA replication in Escherichia coli gyrB(Ts) mutants analyzed by flow cytometry. Res Microbiol. 142:223-227. 98 von Meyenburg, K. and Hansen, F. G. (1987) Regulation of chromosome replication. p. 1555-1577 in Neidhardt, F. C., Ingraham, J. L., Low, K. B., Magasanik, B., Schaechter, M. and Umbarger, H. E. (eds.), Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C. von Meyenburg, K., Hansen, F. G., Riise, E., Bergmans, H. E., Meijer, M. and Messer, W. (1979) Origin of replication, oriC, of the Escherichia coli K12 chromosome: genetic mapping and minichromosome replication. Cold Spring Harb Symp Quant Biol. 121-128. Wahle, E., Lasken, R S. and Kornberg, A. (1989a) The DnaB-DnaC replication protein complex of Escherichia coli. I. Formation and properties. J Biol Chem. 264:2463-2468. Wahle, E., Lasken, R. S. and Kornberg, A. (1989b) The DnaB-DnaC replication protein complex of Escherichia coli. H. Role of the complex in mobilizing DnaB functions. J Biol Chem. 264:2469-2475. Wang, Q. P. and Kaguni, J. M. (1987) Transcriptional repression of the dnaA gene of Escherichia coli by DnaA protein. Mol Gen Genet. 209:518-525. Wang, Q. P. and Kaguni, J. M. (1989) DnaA protein regulates transcriptions of the rpoH gene of Escherichia coli. J Biol Chem. 264:7338-7344. Watson, J. D. and Crick, F. H. C. (1953) Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature. 171:737-738. Wende, M., Quinones, A., Diederich, L., Jueterbock, W. R. and Messer, W. (1991) Transcription termination in the dnaA gene. Mol Gen Genet. 230:486-490. Woelker, B. and Messer, W. (1993) The structure of the initiation complex at the replication origin, oriC, of Escherichia coli. Nucl Acids Res. 21:5025- 5033. Yamamori, T. and Yura, T. (1982) Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12. Proc Natl Acad Sci, USA. 79:860-864. Yang, C. C. and Nash, H. A. (1989) The interaction of E. coli IHF protein with its specific binding sites. Cell. 57:869-880. Yasuda, S. and Hirota, Y. (1977) Cloning and mapping of the replication origin of Escherichia coli. Proc Natl Acad Sci, USA. 74:5458-5462. 99 Yuasa, S. and Sakakibara, Y. (1980) Identification of the dnaA and dnaN gene products of Escherichia coli. Mol Gen Genet. 180:267-273. Yung, B. Y., Crooke, E. and Kornberg, A. (1990) Fate of the DnaA initiator protein in replication at the origin of the Escherichia coli chromosome in vitro. J Biol Chem. 265:1282-1285. Yung, B. Y. and Kornberg, A. (1989) The DnaA initiator protein binds separate domains in the replication origin of Escherichia coli. J Biol Chem. 264:6146-6150. Zyskind, J. W., Cleary, J. M., Brusilow, W. S., Harding, N. E. and Smith, D. W. (1983) Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: oriC consensus sequence. Proc Natl Acad Sci, USA. 80:1164-1168. Zyskind, J. W., Harding, N. B., Takeda, Y., Cleary, J. M. and Smith, D. W. (1981) The DNA replication origin region of the enterobacteriaceae. p. 13- 28 in Ray, D. S. (eds.),The Initiation of DNA Replication. Academic Press, Los Angeles. "lllllllllllllllllllllllll