... I... . 3! but: .15» E117 J! :uu1JIseit Imus?! . s. . . 'nF-nv’ c .2 ta «1} 1:3,. UL 1336’: .1 It}; 37... a: If. . .1 L. 4 . .p L A . W .s ((21 v 2,... L? 3r hi q r. utvo}-.r3~!§!.v! .91. .. 3:5, 1.1:... I... ; 1.21.9...12‘1i: s. IteJEs 1.355.. ‘ 3A .3}. up.“ 9 I 5.. 5.3.3; 1:]?! ‘ . . r: 11.1.! .......I :13}?- ! '1’3E-ul ‘ {bill's- 3.35.193... ,1! 32.55 ‘»llv .. 1.1. ‘5. A. . i I nv~ Vl-uvKOk... a .~7.r.f.7... » ‘ 3:55.15: . 9.133.1AP. FA 55.. g. ff! 4.); z... .. , $1) o .331... i -. {I .. f 0.1.. K?! >,! . lo 3;. OX :13... .ov ‘ )‘L- flu»! .rar. ICHIGAN STAYE UNIVE SI L 85‘! um I ”le m Llflfllflllfil!llllifil 3 1 93 00899 7722 This is to certify that the dissertation entitled The Interaction of Escherichia coli Gene Products with Mutant Forms of dnaA Protein presented by Theodore Robert Hupp has been accepted towards fulfillment of the requirements for Ph-D- degreein_BinchemisLLy Wt. ' 95! mm“ Date M299— MS U i: an Afflrmau‘w Action/Equal Opportunity Institution 0- 12771 rw LIBRARY Illehluen State University K 1 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution emu-diam”.- THE INTERACTION OF Escherichia coli GENE PRODUCTS WITH MUTANT FORMS OF dnaA PROTEIN by Theodore Robert Hupp A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1990 (flyw‘. .3 cf“ 7" ABSTRACT THE INTERACTION OF Escherichia coli GENE PRODUCTS WITH MUTANT FORMS OF dnaA PROTEIN by Theodore R. Hupp The initiation of DNA replication depends upon the activity of the dnaA initiator protein. Although the mechanism of dnaA protein function has been elucidated, the efficiency and regulation of dnaA protein dependent initiation of replication is not understood. Mutant forms of dnaA protein are altered in some aspect of initiation of replication. Characterization of the alterations associated with these mutant initiator proteins would enlarge our understanding of initiation processes. Two approaches were used to identify proteins in E. coli that interact with mutant forms of dnaA protein. The first relied on the genetic characterization of an extragenic suppressor of the temperature sensitive phenotype of cells which harbor the dnaA46 mutation. This suppressor, named dasC, was mapped to the thioredoxin gene suggesting that thioredoxin may interact with dnaA46 protein to allow initiation of replication to occur at elevated temperatures. The second approach involved the purification and biochemical characterization of a mutant form of dnaA protein, called dnaAS protein. This mutant initiator protein is inactive in the replication of oriC plasmids. Two heat shock proteins, dnaK and grpE, are required for activating dnaAS protein in replication. Although it is not known if these gene products - thioredoxin, dnaK protein, and grpE protein - interact with the wild type dnaA protein, these studies have provided a direction for the further understanding of previously unknown aspects of initiation of DNA replication. this song is dedicated to my mother my father Lisa Laura papa gramma mamo grampy and old bean with thanks, honor, and love iii ACKNOWLEDGEMENTS An incomplete expression of joyful gratitude for the many, worthy people who were all, in every way, the catalyst of this document, will be sonorated in the lines below. Jon M. Kaguni, my thesis advisor and the owner of a sagacious scientific phiIOSOphy, for teaching the ways of Science, encouragement during the times when Science became a matter of Faith, instilling in myself the extreme love of genetics, an unforgettable canoe trip, and occasional golf outings. Members of my Ph.D. committee, including John Wilson, Arnie Revzin, Larry Snyder, Greg Zeikus, and alternate member, Zach Burton, for their helpful discussions and ideas. Members of the Kaguni lab past and present including: Deog Su Hwang, my best friend of the lab and foundational author of the bulk of my Ph.D. project, for many discussions about philosophy, women, and sex (and, of course, science), many meals cooked by his wife and during such meals, again, many discussions about philosophy, women, and sex; David Siemieniak for peanut barrel excursions and exchanging knowledge of fundamental particle physics; Richie Halberg for his enthusiastic presence, coffee breaks, photographic excursions and living evidence that being manied is good; Kevin Mundella Carr, the bastion of eclectic Conservative and Liberal philosophy and co-discoverer of the balloon shift, for being the roots of stimulating dialogue; Qing Ping Wang, living proof that women are better at doing the art of science than men, for her always friendly presence; Modori Harris, more living proof that women are better at doing the art of science than men, for her endlessly enthusiastic demeanor; Scott Cherry, ex-undergraduate extraordinaire, the source of perpetual amusement and source of most knowledge; Diana Hart, Wisdom personified, for many things; Marintha Heil, a Heglian and sister, for many THINGS THAT REALLY MATTER including smiles, encouragement, funny jokes, conversations, and other iv important things like that; Jen'y Marshall for being a spark plug of humor in the lab, life- inspiring dialogue, and dealings with the law; and the new lab member, Cindy Peterson for funny things. Members of the other Kaguni lab including, Laurie Kaguni; Phyllis Y. Cashman; Mikey Fairbanks, co—discoverer of the balloon shift; Matthew Olsen, one of the ugliest men on campus; Dave Lewis, bob marley incarnate and arch enemy of William F. Buckley, Jr.; Rhoderick Elder, and Mike Conway, for many unforgettable collaborations. Catherine M. Wemette, my other best friend of the lab and founder of the Society of Disembodied Biochemists, for sharing her family and witticism over the years, encouragement and reprimands, showing the art of wine drinking, appreciation of poetry readings at the weekly meeting of the Society of Disembodied biochemists, and forming our primitive philosophy Be Free, Love, and Be Kind. Kathryn Ball, a song of the amazon wilderness and soul mate, for many things, known and unknown, seen and unseen, past and future; my parents for strong support and love, Lisa for always being around and caring; Steven C. Steele, the twin brother that I never had, for endless excursions to an infinite number of eternities and dimensions, including Binky’s, Fireball, University Circle, do-nut shops, and other planets, for sharing in the beauties of our Mother, and for being there during the growing years; Julie Beckert, the sister-in-law that I never had, for being the sensitive voice of the oppressed, inspiring myself with her vision of time, sharing her life as Miss Bottle Queen from 1985-1988, and for being the leader of rain forest excursions; Timothy J. Cothrel, the other twin brother that I never had, for being himself. Jack Preiss for being available to talk about things and for the use of his virus-laden computer and Betty Brazier for exchanging things of life. TABLE OF CONTENTS Page List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xiii List of Abbreviations ............. , ...................................................................................................... xv Chapter I Literature Review ............................................................................................................ 1 I. The physiology and genetics of DNA replication A. Structure of the chromosomal origin of replication ................................................. 2 B. Gene products that function in the initiation of replication ................................... 10 C. Regulation of the initiation of replication .............................................................. 12 II. The biochemistry of DNA replication A. The mechanism of DNA polymerase function ...................................................... 15 B. A biochemical model describing the enzymatic requirements at a replication fork ................................................................................................ 17 C. The initiation of DNA synthesis from oriC plasmids catalyzed by purified enzymes ............................................................................... 22 III. The heat shock response A. The heat shock proteins of E. coli ....................................................................... 27 B. The biochemical role of 70 Rd heat shock proteins ............................................. 28 References .............................................................................................................. 36 Chapter II Suppression of the Escherichia coli dnaA46 mutation by a mutation in trxA, the gene for thioredoxin ............................................................................... 47 Abstract ..................................................................................................................... 48 Introduction .............................................................................................................. 49 vi Chapter III Experimental Procedures .................................................................................. 51 Results Mapping of dasC by P1 transduction ........................................................... 54 Transduction of a mat null mutant into TC382 ........................................... 54 Transduction of dnaA46 into TRHl through 'I‘RH18 ................................. 57 Mapping of dasC with recombinant plasmids ............................................. 63 The dust” mutation supports T7 and M13 growth ...................................... 66 Discussion ........................................................................................................ 69 References ....................................................................................................... 72 The Escherichia coli dnaA5 protein is inactive in the replication of oriC plasmids in reconstituted enzyme systems .............................................. 75 Abstract ........................................................................................................... 76 Introduction .................................................................................................... 77 Experimental Procedures ................................................................................. 79 Results Overproduction of dnaA5 protein ............................................................... 85 Purification of dnaA5 protein ..................................................................... 85 dnaA5 protein is temperature sensitive in the replication of oriC plasmids in a system containing a crude enzyme fraction .......................... 89 dnaA5 protein is active in the replication of P1 phage origin containing plasmids .......................................................................... 92 dnaA5 protein retains affinity for DNA containing oriC at permissive temperatures ............................................................................ 100 dnaA5 protein retains affinity for DNA containing oriC at the nonpermissive temperature ....................................................................... 101 dnaA5 protein retains its affinity for DNA containing the dnaA promoter .......................................................................................... 102 dnaA5 protein retains specificity in binding to its consensus DNA sequence .......................................................................................... 109 vii grpE protein is the second activating protein ........................................... 155 The amounts of dnaK protein and grpE protein required for the activation of dnaA5 protein .................................................................... 155 Discussion ...................................................................................................... 180 References ..................................................................................................... 184 Chapter v ’ The Escherichia coli dnaA5 protein is activated by the grpE and dnaK heat shock proteins prior to its interaction with oriC ................................... 187 Abstracts“: ................................................................................................... 188 Introduction ................................................................................................. 189 Experimental Procedures ............................................................................. 192 Results The time course of DNA synthesis in activation reactions .................. 194 Staging the activation of dnaA5 protein ............................................. 194 The time course of the staged activation of dnaA5 protein ................ 195 dnaK protein exhibits a concentration dependence in staged activation reactions ............................................................................ 196 grpE protein is required in stoichiometric levels during the staged activation of dnaA5 protein .................................................... 197 The temperature dependence of the activation reaction .................... 197 The influence of solvents on the activation reaction ......................... 198 ATP is required for the activation of dnaA5 protein ........................ 199 The ATPase activity of dnaK protein is not stimulated by dnaA5 protein .................................................................................... 199 dnaA5 protein and dnaA protein react differentially with monoclonal antibodies ...................................................................... 200 The activation of dnaA5 protein produces a conformational change in the protein ......................................................................... 201 viii The interaction of dnaA5 protein with DNA containing oriC is altered ...................................................................................................... 109 dnaA5 protein is defective in ATP binding ................................................. 110 dnaA5 protein is inactive in the replication of oriC plasmids in systems containing purified enzymes ....................................................................... 111 An extended lag precedes dnaA5 protein activity in a replication system employing a crude enzyme fraction ................................................ 124 dnaA5 protein inhibits dnaA protein activity in a replication system containing purified enzymes ....................................................................... 124 dnaA5 protein inhibits dnaA protein dependent unwinding at oriC ........... 127 dnaA5 protein interferes with the formation of a structure characteristic of an active dnaA protein-oriC complex .............................. 128 Discussion ......................................................................................................... 135 References ........................................................................................................ 139 Chapter IV The Escherichia coli dnaA5 protein is activated in oriC plasmid replication by grpE and dnaK heat shock proteins ............................................................. 141 Abstract ............................................................................................................ 142 Introduction ..................................................................................................... 143 Experimental Procedures ................................................................................. 146 Results The activation of dnaA5 protein in a DNA replication system containing purified enzymes ..................................................................... 149 The activating factor is heat sensitive ....................................................... 150 The activating factor is a protein ............................................................... 151 dnaK protein is one of the activating proteins ........................................... 151 The second activating factor is a protein ................................................... 152 The second activating protein is heat sensitive .......................................... 153 The partial purification of the second activating protein .......................... 153 The purification of grpE protein ............................................................... 154 ix Discussion ................................................................................................ 221 References ............................................................................................... 226 Chapter VI Summary and Perspectives ...................................................................... 229 List of Tables Page Chapter II 1. Bacterial strains .......................................................................................................... 53 2. Transduction of dnaA46 into TRH 1 through TRH 18 .............................................. 58 3. Interference of dasC function by multicopy plasmids containing wild type chromosomal DNA ............................................................................................ 64 4. Growth of phages T7 and M13 on dasC and trxA mutant strains .............................. 67 Chapter III 1. A summary of dnaA5 protein purification ................................................................ 87 Chapter IV 1. The activation of dnaA5 protein by WM433 Fraction II ........................................ 173 2. The activating factor is heat sensitive ..................................................................... 174 3. dnaA5 protein is required to observe Fraction III dependent replication activity..l75 4. The activating factor is trypsin sensitive ................................................................ 176 5. The second activating protein is heat sensitive ...................................................... 177 6. The partial purification of the second activating protein ....................................... 178 Chapter V 1. A. Staged activation of dnaA5 protein B. grpE and dnaK proteins are required during Stage I reactions .......................... 218 2. Stage I solvent requirements .................................................................................... 219 3. Solvent replacements in the staged activation reactions .......................................... 220 xi List of Figures Page Chapter I 1. A scheme describing the C and D periods in the E. coli cell cycle .............................. 5 2. The evolutionary sequence conservation of oriC DNA ................................................ 8 3. A model describing the enzymatic requirements at a replication fork ........................ 21 4. A. A linear sequence of the origin region B. A model describing the stages in dnaA protein dependent initiation ..................... 26 5. A model describing the stages and enzymatic requirements of initiation of DNA replication from the lambda phage origin of replication ............................... 33 6. A. A model describing heat shock protein function in eucaryotic cells B. Hsp 70, in the presence of ATP, binds to protein-protein complexes to catalyze the dissociation of specific proteins from the complex ....................... 35 Chapter II 1. Physical map of the trxA region .................................................................................. 56 2. Southern analysis of chromosomal DNA from dasC transductants ............................ 61 Chapter III 1. SDS polyacrylamide gel electrophoresis of dnaA5 protein fractions .......................... 91 2. The temperature dependent replication activity of dnaA5 and dnaA proteins ............ 95 3. The requirements for the replication of Plori plasmids .............................................. 97 4. The temperature dependent replication activity of dnaA5 and dnaA proteins in a Plori plasmid replication system ............................................................................... 99 5. The binding of dnaA5 and dnaA proteins to oriC ..................................................... 104 6. dnaA5 protein and dnaA protein affinity to oriC ...................................................... 106 7. dnaA5 and dnaA protein binding to the dnaA promoter fragment ............................ 108 8. The DNase I protection pattern of the dnaA promoter when bound by dnaA and dnaA5 proteins ................................................................................................... 113 xii 9. The DNase I protection pattem of the moH promoter when bound by dnaA and dnaA5 proteins ......................................................................................... 115 10. DNase I protection pattern of oriC when bound by dnaA and dnaA5 proteins ........ 117 ll. dnaA5 protein and dnaA protein binding affinity to ATP ....................................... 119 12. dnaA5 protein is inactive in the reconstituted enzyme system containing primase as the sole priming enzyme ....................................................................... 121 13. dnaA5 protein is inactive in the RNA polymerase dependent reconstituted enzyme system...t .................................................................................................... 123 14. Time course of dnaA5 protein and dnaA protein activity in the replication system containing a crude enzyme fraction ............................................................. 126 15. dnaA5 protein inhibition of dnaA protein replication activity ................................. 130 16. dnaA5 protein and dnaA protein influence on Fl“ formation ................................. 132 17. dnaA5 protein inhibition of dnaA protein binding to oriC ...................................... 134 Chapter IV 1. The influence of dnaK protein on Fraction III dependent activation of dnaA5 protein ......................................................................................................... 158 2. Titration of dnaK protein in activation reactions ..................................................... 160 3. The second activating factor is trypsin sensitive ..................................................... 162 4. Mono Q chromatography of the second activating protein ..................................... 164 5. A. Superose 12 chromatography of Fraction IV B. ELISA of Superose 12 fractions ........................................................................ 166 6. SDS polyacrylamide gel of grpE protein containing fractions ................................ 168 7. grpE protein replaces a Superose 12 fraction in the activation of dnaA5 protein .......................................................................................................... 170 8. A. dnaK protein titration in activation reactions B. grpE protein titration in activation reactions ..................................................... 172 Chapter V 1. A. Time course of activation of dnaA5 protein B. Time course of DNA synthesis dependent upon dnaA protein .......................... 203 xiii 2. Time course of staged activation ............................................................................. 205 3. A. The amounts of dnaK protein required during Stage I B. The amounts of grpE protein required during Stage I. ....................................... 207 4. A. Thermolability of activation B. Thermoresistance of the activated form of dnaA5 protein .................................. 209 5. The concentration of glycerol in Stage I required for the activation of dnaA5 protein .......................................................................................................... 211 6. The concentration of ATP in Stage I required for the activation of dnaA5 protein ......................................................................................................... 213 7. The influence of dnaA5 protein on the ATPase activity of dnaK protein .............. 215 8. A. The affinity of monoclonal antibodies to dnaA and dnaA5 proteins B. The affinity of monocloanl antibody A4 to dnaA5 protein after Stage I reactions in the presence and absence of ATP ................................................. 217 xiv List of Abbreviations D'IT BSA SSB SDS ' TLC PVA kd ng ug ul hsp dithiothreitol bovine serum albumin single-stranded binding protein sodium dodecylsulfate thin layer chromatography polyvinylalcohol kilodalton nanograms micrograms microliters heat shock protein XV Chapter I LITERATURE REVIEW I. The physiology and genetics of DNA replication A. Structure of the chromosomal origin of replication. The chromosome of E. coli is a closed circular duplex molecule (1) with a molecular weight of 4,0(X) kilobase pairs (2). The knowledge that DNA is a double helix composed of complementary strands (3) resulted in the prediction and subsequent confirmation that DNA is replicated semi-conservatively (4). The importance of chromosomal replication in the life of a cell has resulted in emphasis being placed on understanding the mechanisms of DNA replication and its regulation. Understanding the mechanisms of macromolecular synthesis in relation to both growth rate and age was important in demonstrating the elegant regulatory mechanisms which a cell utilizes to control its life. The amounts of some macromolecules, including ribosomes and DNA, change exponentially as a function of the growth rate (5). Analyzing the relative concentrations of macromolecules as a function of the cell size has led to the formation of mathematical relationships which describe global cell composition in terms of DNA, RNA, and protein content (6). The amount of DNA within a cell is subjected to a stringent regulatory system which allows for the initiation of chromosomal replication at a specific time during the cell cycle to ensure that each daughter cell obtains a copy of the chromosome. DNA synthesis is cell cycle regulated in that a certain cell mass or volume must be achieved in order for DNA synthesis to be initiated (7). The initiation of DNA synthesis requires de novo protein synthesis (8,9) and RNA synthesis (10) conoborating the idea that initiation factors must be synthesized and accumulate to allow for the initiation of replication to be triggered during the cell cycle. 3 Cooper and Helmstetter determined that, although the concentration of DNA can change as a function of the growth rate, there are biological constants that describe the relationship between the frequency of initiation of replication and cell division (11). The constants in Operation were termed C and D, where C represents the time required for the chromosome to be replicated and D describes the time elapsed between termination of DNA replication and cell division. The C and D periods are 40 and 22 minutes, respectively, at a given 81' owth rate. If the C and D periods are true constants, then how can the total time required for the C and D periods to elapse be longer than the life cycle of a rapidly growing cell? To address this question, they hypothesized that more than one initiation event occurs during the cell cycle in response to a rapid growth rate to account for the fact that bacteria can divide in a time shorter than that elapsing during the C and D periods (Figure l). The values for C and D were recently determined in slowly growing cells using flow cytometric analysis (13) and were in agreement with the data obtained 15 years earlier (11). Although the initiation of DNA replication could be measured experimentally, it was not known if the initiation site was unique or if initiation occurred at a random site on the chromosome during successive generations. Jacob (14) proposed a mechanism of initiation of DNA synthesis based on a mechanism known to operate during the regulation of protein synthesis (15). In the latter case, a negative regulator influences the expression of a gene product by binding to an operator element in the DNA to inhibit its synthesis. By analogy and subsequent experimentation, it was proposed that a positive control circuit affects the initiation of DNA replication. In this model, the chromosome is assumed to be a circular structure with two genetic determinants. One being a structural gene giving rise to a diffusible element, the initiator, and the second being a cis-acting operator-like DNA Figure 1. A scheme describing the C and D periods in the E. coli cell cycle (13, 1). The time required for chromosomal DNA replication (C period) and the time elapsin g be- tween the termination of chromosomal DNA replication and cell division (D period) is depicted as a function of differing growth rates; (A) 90 minute doubling time, (B) 60 minute doubling time and (C) 35 minute doubling time. ‘ini’ is the initiation of DNA replication, ‘ter’ is the termi- nation of DNA replication, and ‘div’ is cell division. a mac c-ss o-zo E25 C—E- /E-)l' --— CE, fig-é“ - ' VVV v div div it'll tgr ini l T F ' 6-3 Cari—D Cr: 6:) C_:>_-__-‘ Cij—‘D div div ihi ter ihi tér C 5x35 C=lo0 0:20 / / /_ C2 C23 div div div div ini I tni ' il‘ti'\ 6 element that can be acted upon by the initiator possibly to unwind the duplex DNA and lead to DNA synthesis after the formation of a primer. The existence of this cis—acting locus, the chromosomal origin of replication, oriC (16,17), was confirmed, indicating that replication always initiates at a specific site during the cell cycle. More precise experimentation further localized oriC to the ilv region of the Chromosome (18). By the quantification of the relative abundance of marker genes after the initiation of DNA synthesis in a synchronized cell culture, the bi—directional mode of initiation was proven (19-22). The cloning of oriC onto plasmids allowed for restriction site analysis and provided for a more precise physical map of oriC (23). Recombinant DNA techniques were used to clone oriC based on its ability to confer autonomous replicating properties to recombinant DNAs (24) and based on its linkage to the asn locus (25). The minimal functional segment required for confenin g replication activity to vector DNAs is 245 base pairs (26). Subsequent DNA sequencing of the minimal origin fragment (27, 28) identified the presence of extensive structural motifs, including A-T rich regions, GATC methylation sites, and inverted and repeated sequences (29). Some mutations within oriC that perturb the primary sequence, including base substitutions, insertions and deletions, alter the origin activity (30). In addition, the evolutionary conservation of the sequence motifs among enterobactericeae (Figure 2) (31) and the ability of oriC from a marine bacterium to function in E. coli (32) provided evidence that the structure of the origin is important in directing enzymes and proteins to initiate replication. Each structural motif is involved in origin activity through its interaction with various cellular enzymes. Methylation at the sequence GATC, which is present 11 times within the minimal origin, by the dam methylase gene product (33) is thought to be important in maintaining origin integrity and function. The dam gene product is not essential for the growth of E. coli, as dam mutants are viable (34). This implies that methylation of the GATC motifs is not an essential step in oriC directed DNA replication, but this idea Figure 2. The evolutionary sequence conservation of oriC DNA (32). The sequences from the origin of DNA replication from six bacteria are aligned to maximize visual sequence identity. The minimal origin required to confer activity is enclosed within the box and bold capitol letters indicate where base substitutions inactivate origin function. Conserved motifs are identified as follows: GATC repeats are underlined; contiguous left to right arrows are beneath the A-T rich 13-mer sequences; Rl-R2-R3-R4 mark the location of the dnaA consensus sequence; a large capital letter indicates conservation of the base in all six origins; a small capital letter indicates the base is present in five of the six origins examined; a lower case letter indicates the base is present in three or four origins; and ‘n’ is inserted where there seems to be no sequence homology, or there are insertions or deletions within the region. 0<<X174, and G4, were used as model systems for the identification of E .cali enzymes involved in DNA replication and the priming of DNA synthesis (Figure 3, (145)). 18 The viral DNA of M13 exists in the phage coat as a single-stranded molecule (146) and in the first stage of its replication cycle, it is converted to the duplex form by E. coli enzymes. The requirement for RNA polymerase in the initial stages of viral DNA replication in viva was infened from the fact that the reaction is sensitive to rifampicin (147), a known inhibitor of RNA polymerase. The rifampicin sensitive RNA polymerizing event could be duplicated in vitra, demonstrating for the first time (148), a biochemical role for RNA polymerase in the priming of DNA replication. An RNA primer is synthesized near a hairpin loop in the DNA at the origin of replication and, in the presence of SSB, DNA polymerase III holoenzyme synthesizes an entire DNA strand complementary to the original parental viral DNA (149). Another viral DNA, G4, exists in a single stranded form in the phage coat (146), and the mechanism responsible for its replication is differentiated from M13 by the fact that the RNA priming of its replication is not dependent upon E. coli RNA polymerase, but by another RNA priming enzyme called primase (150), encoded by dnaG . This rifampicin resistant RNA polymerase forms an RNA primer at the G4 origin of replication (151), and in the presence of SSB, DNA polymerase III holoenzyme replicates the parental DNA strand. The two bacteriophages described above utilize two different enzymes to prime DNA replication at the origin of replication. The mechanism of RNA priming utilized by the single-stranded bacteriophage Xl74 rolling circle mode of replication (158). The hydrogen bonds between bases at a replication fork must be broken to allow for the propagation of DNA chains. Therefore, the inclusion of a helicase in this model is required to augment authentic replication fork movement. The rep gene product is not essential for E. coli viability (159), indicating that it is not the only helicase functioning at the replication fork. However, mutations in rep decrease the rate of replication fork movement and increase the number of replication forks per cell (160), indicating it is involved in some aspect of DNA replication in viva. 20 Figure 3. A model describing the enzymatic requirements at a replication fork (145). 21 5' 3' fife rep PROTEIN: $5.33 PREPRIMING (HELICASE) «at. PROTEINS (n "on u c) fie ° ,3. \ N/(a dnaB PROTEIN quaint”0 \I/ DNA POLYMERASE I ® ”pppALL\ ’ " l/ 50 ’9 .2, \ a I’ ..3. \x 3 -— § PRIMASE ”WA PRIMER e‘ /’*a \\ l \\ ’ pppA \\ DNA 4’) / é Q POLYMERASEIII é \\\ HOLOENZYME / \\\ rNMP \\\ dNMP e“ e“ 5' LIGASE — r ’ 3. LEADING LAGGING STRAND STRAND 22 Other helicases discovered since then (161) may replace the function of rep helicase in this model describing the propagation of a replication fork. uer or Helicase II (162) can unwind DNA with flanking 3' single-stranded regions in a manner similar to rep helicase. Mutations in rep or uer do not result in cell death, but double mutations in rep and uer are lethal to the cell (163) indicating that at least one of them is required for replication fork propagation and cell viability. The unwinding of the duplex DNA into single strands at a replication fork relays a positive torsional strain into the double helix in the vicinity of the growing replication fork. DNA gyrase can introduce negative supercoils into covalently closed DNA (164) thus relieving the torsional strain which would have hindered fork movement and the rate of DNA replication. The immediate cessation of DNA synthesis in viva after inhibiting the activity of DNA gyrase by antibiotics (165) or after a temperature upshift in cells containing a temperature sensitive gyr allele (166) supports the idea that DNA gyrase is required for progressive fork movement in DNA replication (167). C. The initiation of DNA synthesis from oriC plasmids catalyzed by purified enzymes. Understanding the mechanisms of viral DNA replication led to the identification of the many cellular enzymes involved in chromosomal replication. These studies gave information concerning the propagation of a replication fork, but information was not obtained on the mechanism of initiation of replication from oriC or of its regulation. The cloning of the chromosomal origin, oriC, into plasmid vectors (24, 25) provided the substrate needed to identify enzymes required for the initiation of replication in vitra. A technological breakthrough (90) led to the isolation of a crude enzyme fraction that could authentically initiate DNA synthesis from oriC . The requirements of initiation in this in vitra system mimic those in viva. These include 1) rifampicin sensitivity in initiation 23 demonstrating the requirement of RNA polymerase, 2) sensitivity to inhibitors of DNA gyrase and 3) a bidirectional mode of DNA synthesis (168). The essential and unique requirement for the dnaA gene product also exists in the in vitra oriC replication system. A crude enzyme fraction from a dnaA mutant strain could not support the replication of oriC plasmids unless supplied with extracts or fractions containing elevated levels of dnaA protein. This complementation assay was used to purify dnaA protein to near homogeneity (169). Further refinement of the replication system employing a crude enzyme fraction led to the formation of a reconstituted enzyme system composed of purified enzymes and proteins that could initiate DNA synthesis from oriC (132). The minimal complement of enzymes required for the initiation and propagation of DNA chains was divided into three sets; initiation factors (which include the proteins dnaA, HU, dnaB, dnaC, DNA gyrase, and RNA polymerase), elongation factors (which include the proteins dnaB, dnaC, primase, SSB, DNA gyrase, and DNA polymerase III holoenzyme) and specificity factors (topoisomerase I and RNase H) which maintain oriC dependent initiation of replication. The reductionist approach was used to dissect this complex system into more simple components in an effort to understand the role of dnaA protein in initiation. dnaA protein binds cooperatively to oriC DNA and other DNAs containing the consensus sequence T'I‘ATCCACA (40). The ability of dnaA protein to bind to oriC DNA implied that it was one of the first participants in the cascade leading to DNA synthesis. dnaA protein binds nucleotides with a very high affinity (170). This form of dnaA protein binds to oriC DNA with an affinity equivalent to the non-nucleotide form of dnaA protein, indicating that its high affinity binding to ATP does not affect its affinity for DNA and may influence other properties of the protein. Subsequent studies (171) demonstrated that, in the presence of ATP, dnaA protein locally unwinds an A-T rich region at one end of the chromosomal origin to allow for the formation of single-stranded regions that are utilized as substrates by the dnaB-dnaC complex (Figure 4) (42). dnaB and dnaC prOteins 24 form a complex in the presence of ATP (172) that results in the dnaC protein dependent delivery of dnaB helicase to single-stranded DNA (173). The binding of dnaB protein results in the release of dnaC protein and this activates dnaB helicase (174). Further unwinding by the helicase leads to the formation of a small bubble capable of being utilized as a substrate by primase which can subsequently lead to DNA synthesis (175). The regulation of this event may occur at the site of dnaA protein action, in that the ADP form of dnaA protein cannot initiate DNA replication because it is unable to unwind oriC DNA (107). Acidic phospholipids, like cardiolipin, reverse the ADP inhibition of dnaA protein, suggesting that a role exists for phospholipids in modulating dnaA protein activity in the cell (176). This oriC unwinding model system is incomplete due to the fact that it excludes other proteins, like RNA polymerase, t0poisomerase I, and RNase H, all known to be present in the cell and involved in oriC-dnaA protein dependent replication in vitra (177, 178). RNase H degrades RNA in DNA-RNA hybrids and it is believed that RNase H removes RNA synthesized by RNA polymerase at sites other than ariC (85) and thus maintains oriC specific initiation of DNA synthesis. This idea is confirmed by the observation that RN ase H mutant cells lose oriC dependent initiation of replication and initiation occurs at other sites on the chromosome (84,179). The requirement for RNA polymerase was thought to be in forming a RNA molecule in concert with dnaA protein to prime DNA synthesis initiating at oriC (79). This was supported by the results describing the existence of promoters within oriC , RNA transcripts terminating within oriC , and the existence of RNA-DNA transition sites homologous to origin DNA (4447), A recent study demonstrated that a RNA polymerase dependent RNA transcript does not prime DNA synthesis at oriC, but transcriptional activation by RNA polymerase aids dnaA protein in melting the helix in the vicinity of the A-T rich region near oriC (180). However, a more complex role for RNA polymerase cannot be eliminated. 25 Figure 4. (A) A linear sequence of the origin region (180). The bold line represents DNA contiguous to the origin region (boxed). Promoters within and proximal to the origin region are labeled according to the direction of transcription and locus. Consensus sequences required for origin function are represented as filled rectangles within the origin box. (B) A model describing the stages in dnaA protein dependent initiation (180). The diagram depicts four stages in the dnaA protein dependent initiation process; 1) bind- ing of dnaA protein in the presence of ATP and HU protein to oriC, 2) formation of an open complex at elevated temperatures, 3) entry of the dnaC-dnaB helicase and 4) RNA priming and DNA synthesis. 26 xmqaioo 02.5Emmmn 20.231595 IIIII' 02¢ 02.2.": whjnsfi... omioomwnom “Gums—00 vane—200 $0 zmao ..(Ez. ah< Once once .3 ‘IJII 66 e a (25.9: 3: ..x8 <2? ace 9 use 92. a eta \. . .x . a T i T . s: can. mmwa 4mm... alas. III. The heat shock response A. The heat shock proteins of E. coli. The heat shock response is thought to function as a protective mechanism preventing cellular damage in response to enviromental stress (181). However, growing evidence indicates that heat shock proteins play a role in normal cell physiology (182, 183). In E. coli, the expression of heat shock proteins is under the control of the htpR (rpaH) gene product (184), a sigma factor that associates with RNA polymerase. When growing cells are transferred from 30°C to 42°C the expression of the htpR gene product is induced and it directs the synthesis of at least seventeen heat shock proteins (185, 186). The rate of synthesis of the heat shock proteins can increase from 5 to 20 times and, 5 to 10 minutes after the heat shock is induced, a new steady state level of expression is achieved. Seven of the seventeen heat shock proteins from E. coli correspond to known gene products and are named Ian, rpaD, graEL, graES, dnaK, dual, and grpE. The Ian gene product is an ATP dependent protease (187, 188) that is not required for cell viability, but it is required in maintaining a healthy SOS response by proteolytically cleaving a cell division inhibitor (189). The rpoD gene encodes sigma 70, the RNA polymerase subunit that directs transcription from most E. coli promoters (190). It is thought that an increase in the levels of sigma 70 after heat shock ensures that the cell will be able to rapidly return to normal steady state gene expression and growth. The other five heat shock gene products described above, were first identified as mutants unable to support lambda phage propagation. graEL and groES form an operon and are required for lambda phage body assembly (191, 192) and phage Mu development (193). When graE is present at an elevated copy number the tlrermolability of a dnaA 2 7 28 temperature sensitive mutant is suppressed (88,89). The role of graE in E. coli metabolism is not well understood, but some mutants are defective in RNA and DNA synthesis at elevated temperatures (194). Recently, graE was shown to be essential for E. coli viability (195). Mutations in dnaK (196, 197), dual (198), and grpE (199) restrict lambda DNA synthesis and affect the global synthesis of E .cali RNA, DNA and protein. These three gene products function in a diverse series of reactions in combination or singly. dnaK is required for mini-F DNA replication (203), while dnaK, dual and grpE are required for phage P1 DNA replication (200) and for the initiation of lambda DNA synthesis (201, 202). A novel dnaK mutant is specifically defective in initiation of DNA replication indicating a possible role for dnaK in that process (66). dnaK (204) and grpE (205) are required for normal cell growth and they both were shown to interact in viva and in vitra (206). The diverse associations amongst different heat shock proteins reflects the cells ability to respond to a wide variety of stimuli. B. The biochemical role of the 70 kd heat shock proteins. Bacteria, yeast, insects, plants and mammals all respond to heat shock by synthesizing a set of proteins highly conserved within a cell and among other organisms (207). Antibodies to a 70 kd heat shock protein from chicken cross react with proteins in yeast and humans (208, 209). The 50% homology in the amino acid sequence between a 70 kd heat shock protein from yeast and Drosophila (210) confirmed the high homology that exists between heat shock proteins from different organisms. Another common feature of 70 kd heat shock proteins, which may be required for biochemical activity (see below), is the high affinity for ATP (211). The biochemical role of the E. coli 70 kd heat shock protein, dnaK protein, is not known; however, its biochemical function in lambda phage DNA replication is known 29 (Figure 5) and its role in that process may provide clues concerning its function in normal E. coli growth. Lambda phage DNA replication in viva (212) and in vitra (213-215) requires phage encoded and host encoded gene products to authentically initiate bi- directional DNA synthesis (216) from the phage origin of replication, ariL. Lambda encodes two of its own initiation proteins, lambda O and P proteins (217-218), that form an initiation complex at ariL with the dnaB helicase (219). This initiation complex containing dnaB helicase is similar to the initiation complex at oriC containing dnaB protein (173), except that in lambda replication, the activity of dnaB helicase is inhibited when bound by lambda P protein. dnaK protein functions in the presence of dnaJ protein to dissociate lambda P protein from the initiation complex which results in the activation of the dnaB helicase activity (220). This leads to further duplex strand unwinding and subsequent DNA synthesis. grpE protein is absolutely required for lambda DNA replication in viva, but it is dispensible in vitra (221). Although dnaK protein alone can dissociate lambda P protein from the ariL initiation complex, grpE protein reduces by ten fold the level of dnaK protein required to dissociate lambda P protein. The mechanism of grpE protein action is unknown, but it was postulated that grpE protein could bind to the dissociated form of lambda P protein. This would reduce the level of dnaK protein required for the lambda P dissociation reaction by preventing non-specific binding of free lambda P protein to dnaK protein. grpE and dnaK proteins can form a complex that is stable in the presence of high levels of salt, but it is unlikely that the two proteins from a complex in initiation, as the reaction contains high levels of ATP and the grpE-dnaK protein complex is dissociated by ATP (202). The role of dnaK in dissociating protein-protein complexes mimics the biochemical role of heat shock proteins in eucaryotic cells. In Saccharamyces cerevisiae a family of 70 kd heat shock proteins is involved in post- translational import of proteins into the membranes of organelles (Figure 6A). A series of heat shock protein mutants accumulates unprocessed precursors of proteins destined for the 30 mitochondria or the endoplasmic reticulum (222). The heat shock proteins are thought to affect the conformation of proteins, thus the transport of the precursors into the mitochondria requires that the transported protein maintain a conformation suitable for translocation (223). Proposals for the mechanisms of heat shock protein function include 1) the disassembly of aggregates formed from untranslocated precursor proteins, 2) the alteration of the tertiary structure of proteins prior to insertion into membranes and 3) binding to unfolded domains within proteins to prevent the acquisition of conformations incompatible with translocation. A S. cerevisiae cytosolic protein, shown to be a member of the 70 kd heat shock protein family, stimulates the translocation of prepro-alpha factor across microsomal membranes (224). The rate of in vitra protein import, independent of the heat shock protein, could be increased upon urea denaturation of the precursor protein. This denaturation stimulated translocation of prepro-alpha factor mimics the role of the heat shock protein, suggesting that the mechanism of action is through a denaturation of the precursor protein prior to insertion into the membrane. Another well documented biochemical function of heat shock proteins was observed in mammalian systems (Figure 6B). A 70 kd polypeptide from bovine brain, subsequently shown to be a heat shock protein (225), dissociates clathrin from coated vesicles in an ATP dependent manner (226, 227). This heat shock protein, called uncoating ATPase, hydrolyzes ATP in the process of dissociating clathrin from coated vesicles and it is bound in molar amounts to the dissociated form of clathrin. The uncoating ATPase recognizes a specific conformation in clathrin, as only the form of clathrin bound to coated vesicles is able to elicit its ATPase activity (228, 229). During heat stress, proteins are thought to becorrre partially denatured resulting in the exposure of hydrophobic domains that interact to form aggregates. Heat shock proteins may bind to such exposed domains to linrit aggregation and to promote refolding of the protein to restore its native structure (Figure 6B). During normal cellular metabolism, 31 protein complexes that are naturally occurring, yet exhibit faces similar to heat denatured proteins, may be acted upon by the heat shock proteins to facilitate the acquisition of a specific conformation (230). 32 Figure 5. A model describing the stages and enzymatic requirements of initiation of DNA repli- cation from the lambda phage origin of replication (221). Stage 1, binding of lambda 0 protein to the origin; Stage 2, binding of the lambda P protein- dnaB helicase to the initiation complex; Stage 3, binding of dual protein to the initiation com- plex; Stage 4, binding of dnaK protein to the initiation complex; Stage 5, dissociation of lambda P protein from the initiation complex to activate dnaB helicase activity; Stage 6, RNA priming and DNA synthesis. 33 ori DNA Synthesis DnaG.dNTP DNA Pol III DNA gyrase 34 Figure 6. A model describing heat shock protein function in eucaryotic cells. (A) Hsp 70, in the presence of ATP, binds to unprocessed proteins destined for the endoplasmic reticulum or the mitochondria. Hsp 7O alters the conformation of the protein to catalyze its in- sertion into membranes (223). (B) Hsp 70, in the presence of ATP, binds to protein-protein complexes to catalyze the dissocia- tion of specific proteins fiom the complex (230). 35 5"???) 99°F? 11. 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. REFERENCES Cairns, J. (1963) J. 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Chapter II SUPPRESSION OF THE Escherichia coli dnaA46 MUTATION BY A MUTATION IN trxA, THE GENE FOR THIOREDOXIN 47 ABSTRACT The dasC mutation was mapped by P1 transduction near the rep, rho and trxA region of the E. coli chromosome. The dasC mutation could not be separated from trxA by P1 transduction indicating that the dasC mutation maps within trxA. Multic0py plasmids containing an intact ch gene were able to reverse the suppressive effect of the dasC mutation on the dnaA46 phenotype. Introduction of a frameshift mutation into the cloned trxA coding region abolished the ability of the recombinant plasmids to reverse the suppressive effect. These results indicate that dasC and trxA are allelic. 48 INTRODUCTION Initiation of DNA replication from the Escherichia coli chromosomal origin, oriC, depends on numerous gene products identified by genetic and biochemical analyses. Physiological and genetic studies have indicated that the gene products of dnaA, dnaB, dnaC, dnaG, dnaK, and gyrB are required during the initiation process (1-4). An RNA polymerase mediated event is also required as infened from the inhibition of initiation of DNA synthesis by rifampicin, a specific inhibitor of RNA polymerase (5, 6). Biochemical studies of dnaA protein indicate that it acts early in the replication of recombinant plasmids containing the oriC sequence and that it binds to DNA sequences within oriC (7-9). Other studies have revealed different mechanisms of priming of DNA replication by primase, the product of the dnaG locus, by RNA polymerase, or by both enzymes (10, 11). Formation of a pre-priming complex has been observed in replication primed by primase (12). The requirement for proteins, dnaA, dnaB, dnaC, and DNA gyrase suggests their interaction in complex formation and substantiates observations from in vivo studies. A determination of additional properties of dnaA protein and proteins it may interact with will further the understanding of initiation of DNA replication. A genetic approach to analysis of complex processes has been to select and to characterize extragenic suppressors of mutations in genes whose products are known to be involved in that process (13). This approach has also been used in the isolation of mutants which have not been obtained by conventional genetic approaches (14, 15). Such extragenic suppressors may either interact functionally with the gene product of the primary mutation, alter the levels of the gene product of the prirmry mutation, or by-pass the requirement of the primary gene product. This approach has been applied to the analysis of the initiation of DNA replication. Temperature resistant derivatives of thermosensitive alleles of the dnaA gene have been isolated (14, 16, 17). One of the first characterized extragenic suppressors of dnaA mutants 4 9 50 was mapped in the rpoB gene which encodes the B subunit of RNA polymerase (14, 17). Allele specific suppression has been observed in that only some rpoB alleles are capable of suppressing particular dnaA alleles. Based on this evidence, it has been proposed that RNA polymerase and the dnaA gene product interact in a transcriptional event required for the initiation of DNA replication. Six additional dnaA suppressors have been isolated and mapped (14). One of these extragenic suppressors, dasF, is allelic with the gene encoding RNaseH (18). This suppression appears to by-pass the normal requirement for dnaA mediated initiation of DNA replication at oriC, by allowing dnaA independent initiations to occur at other sites on the E. coli chromosome (19). A second suppressor, dasC, has been mapped by the use of specialized transducing phages to the ilv region of the chromosome (14). This region is well characterized genetically and includes the rep gene which encodes a helicase required fro the replication of bacteriophages, the rho gene which encodes a transcriptional termination factor, and m ,tlre gene encoding thioredoxin (20-22) . This study extends the characterization of dasC by implicating the involvement of thioredoxin in suppression. EXPERIMENTAL PROCEDURES Bacterial strains, phages, and plasmids. The bacterial strains used or constructed for this study are listed in Table 1. Phages Plvir, M13Gari l (23), and T7, and plasmid pBR322 were laboratory stocks. pJG31 (24) was a gift from D. Calhoun (Mount Sinai School of Medicine). Strain constructions. The dnaA46 allele was transduced from K0778 into AB1157 by linkage to tnaA::Tn10 (25). A tetracycline-resistant, temperature-sensitive transductant named ABA46 was obtained and used in the indicated experiments. The trxAxkan allele was transduced from A179 into ABA46. A kanamycin-resistant, temperature-sensitive transductant named TRA46 was obtained. The trxAxkan allele was transduced from TRA46 into TC382. A temperature-sensitive isolate (TRH202) was obtained. A temperature-resistant isolate was obtained which resulted from the reversion of dnaA46 to dnaA+. This temperature resistant isolate, TRH204, is isogenic with TRHZ through 7 and TRH9 through 18 (Table 2). F1::Tn10 from K603 was transferred into TC187 and TC382 with selection by plating the mating mixture onto LB plates containing tetracycline (30 ug/ml) and streptomycin (25 g/ml). LB media and minimal A media supplemented with 0.5% glucose and the required L-amino acids were prepared as described. Where indicated ampicillin (30 ug/ml) and tetracycline (12 ug/ml) were added to the above media. Plasmid constructions. Plasmid pJGAKpn was constructed by digestion of pJG3l DNA (Figure 1) with KpnI followed by recircularization of the larger fragment with T4 DNA ligase (New England Biolabs) to delete coding sequences for rho and trxA. Plasmid pRl-1023 was constructed by digestion of pJG31 with Pqu and HindIII, followed by isolation of the 3.2 kilobase (kb) fragment containing the rho and trxA genes. Fragment purification from agarose gels was performed by electrophoresis onto Whatrnan DE81 paper, elution from the paper by incubation in 1.7 M N aCl, 10 mM Tris-HCl (pH 8), and 1 mM EDTA at 37°C for 2 hr and concentration by ethanol precipitation. This fragment was 5 1 52 ligated to the 2.3 kb PvuII-HindIII fragment of pBR322 containing the replication origin and B-lactamase gene. Self-ligation of the vector was minimized by treatment with calf intestinal phosphatase (Boehringer Mannheim). Plasmid pRH0117 was constructed by linearization of pRHOQS at the single BclI site in the rho gene with Bell restriction enzyme. The cohesive termini were end-filled with the large fragment of DNA polymerase I (New England Biolabs) and religated to produce a frameshift mutation in the rho coding region. To form pRH0206, a 12 base pair synthetic BamHl-EcoRl linker (Worthington) was inserted into pRH023 DNA at the Bcll site. pTXA2 was constructed by restriction of pRH0206 with BamHl and recircularization of the larger fragment containing the pBR322 origin of replication, the B-lactamase gene, and the m gene. pTXA114 and pTXAl 15 were constructed by cleavage of pTXA2 with ClaI. The cohesive termini were end-filled with the large fragment of DNA polymerase I and religated to introduce a frameshift mutation in the trxA coding region. In the above constructions, transformants were initially screened for plasmids with the appropriate structure by restriction analysis of DNA prepared by the method of Davis et al. (26). Recombinant DNAs with the appropriate structure were purified by the cleared-lysate procedure (27) in CsClz-ethidium bromide gradients and the structure was reconfirmed by restriction analysis. Gradient purified DNAs were used for the above constructions. Restriction enzyme digestions, treatment with calf intestinal phosphatase, end-filling with the large fragment of DNA polymerase I, and transformation of CaClz-treated competent cells were performed as described (28). 53 Table 1. Bacterial Strains Construction. source, Sin'm W mm TC187 M46thiargI-ImetBllistrppyrE (14) lacxyltarpsLtna ileuhp + 1082 da€382a (l4) TC383 da€383a (l4) BW6159 ilv.-:Tn10 thil spaTI relAl Hfi CGSCd K0778 fllac bglR praA trp39700 trp::Tn9 his tlu' .mpF 81 rpsL tsx dnaA46 tnaA::Tn10 CGSC AB1157 thr-l era-14 leuB6 fl(gpt-proA)62 lach tax-33 supE44gaIK2 - hisG4 rpsL31 xyl-5 mti-I argE? dri-l ABA46 dnaA46 DIanTnIOb A179 HfiC supD Mskm TRA46 dnaA46 tnaA::Tn10 trxAr. kart TRH202 dnaA46 trankan TRH204 trxAchan (isogenic with TRH2) TRHZ-TRI-U, trxAukan TRH9-TRH18 'I'RHl. TRH8 dnaA46 dasC tranlran K603 Fl::Tn10 thr-l leuB6 trpE63 thi-l arc-14 lacYI galKZ galI‘ZZ xyl-5 mtl-l supE44 RK4349 pro-3 entA403 his-218 iva7 metE163::Tn10 fllac6 xyl-S or xyl-7 rpsL109 supE4 hst rpsL metBI C600 thi-l thr-I 1048610ch tonAZI supE44 4|st JM103 F'traD36fl(lacpro) thi strA supE endA sbc hst proAB laleZflMl5 W a isogenic at other loci with TC187 b isogenic at other loci with AB1157 c isogenic at other loci with TC382 d E. coli Genetic Stock Center CGSC This study (31) This study AsinTablerroman malogousexperiment. As in Table 2 from an analogous experiment. Table 2 Table 2 CGSC CGSC Lab collection Lab collection (42) J RESULTS Mapping of dasC by P1 transduction. The dasC mutations in TC382 and TC383 which suppress the temperature sensitive phenotype of the dnaA46 mutant were reported to be 20-50% linked to the ilv locus near 84 minutes on the E. coli genetic map ((14); Figure 1). To confirm the map position of the dasC mutation, P1 transductions were performed. P1 lysates from BW6159 (relevant genotype, ilv::Tn10) were used to transduce the dasC, dnaA46 strains TC382 and TC383 to tetracycline resistance at 30°C. Drug-resistant transductants containing ilv::Tn10 should also obtain the wild type dasC gene with a frequency proportional to the distance between the two loci. Of the 96 tetracycline-resistant transductants of TC382 examined, 68 became temperature sensitive at 40°C presumably due to introduction of the wild type dasC gene (71% cotransduction frequency). Transduction of metE::Tn10 from RK4349 (metE::Tn10) into TC382 (dasC, dnaA46) resulted in 14 temperature-sensitive colony isolates out of 96 drug-resistant transductants (15% cotransduction frequency). The cotransduction frequencies between dasC and ilv::Tn10, dasC and dnaA46 (data not shown), and dasC and metE::Tn10 correlates well with the physical map of Figure 1A indicating that the dasC mutation maps near the ilv operon. It was not possible to demonstrate linkage of dasC to ilv::Tn10 with the strain TC383. Work with TC383 was not continued. Transduction of a trxA null mutant into TC382. Experiments performed concurrently (described below) indicated that the dasC mutation was located in the trxA gene which encodes thioredoxin. The insertion mutation in A179 (trxA::kan) was used to test whether m and dasC were allelic. Because of the proximity of the dnaA gene to trxA, the trxA::kan allele was transduced into ABA46 (dnaA46) followed by selection for kanamycin-resistant transductants. A temperature- 54 55 Figure 1. Physical map of the trxA region (31, 32, 35). (A). Relevant genes near mm. (B). Genes contained in the 7 kb Hind III fragment include the C-tenninal coding region of rep, the trxA and rho gene (open boxes), and the region encoding a 38 kilodalton (kd) protein of unknown function (crosshatched box). DNA fragments contained in the different recombinant plasmids (solid line) as well as regions which have been deleted are indicated. Mutations introduced by end-filling of cohesive tennini by DNA polymerase I (large fragment) and ligation of blunt-ends (NW ), or by insertion of a BamHI -EcoRI linkers ( m ) are indicated. Restriction enzyme sites : H, HindIIl; P, Pvull; K, Kpnl; C, ClaI; B, Bcll. 56 A A v "n A V 1 WV ‘77 uvrqr V [K tnA nmfli a’ac rho «a d rep nxA r—-1 ..Pl 1K I'll"- ‘ H'|'-‘|'H"'l 1K it pJGSl tMGAKmnei pRH023 .P L I pRHOIIT . pRHOZOég AM ' pTXA2 pTXAIM am pTXAllS T 500bp 57 sensitive, kanamycin-resistant transductant, TRA46 (dnaA46, trxA::kan), was obtained and used to transduce the insertion mutation trxAzzkan into the dasC, dnaA46 strain TC382. Of 576 kanamycin-resistant transductants, 558 were temperature sensitive for growth due to the replacement of dasC by the tncA::kan mutation. This suggests that a trxA null mutant cannot suppress the temperature-sensitive phenotype of dnaA46 and that dasC and HM might be closely linked. However, further analysis of the remaining 18 temperature- resistant transductants designated TRI-Il through TRH18 indicated that they arose either by reversion of dnaA46 to dnaA‘l', or by an unusual event of gene duplication. The inability to separate dasC from m indicates that they are allelic. Transduction of dnaA46 into T RHI through TRH18. Each of the temperature-resistant transductants, TRHl through TRH18, was used as a recipient in transduction of tnaA::Tn10 and the linked dnaA46 mutation from ABA46. Tetracycline-resistant isolates from this experiment were screened for temperature sensitivity. Sixteen of the eighteen transductants named TRHl through TRH18 appeared to have acquired the dnaA46 mutation in this experiment near the expected cotransduction frequency (90-95%) with tnaA::Tn10 (Table 2A). These results indicate that the temperature-resistant transductants TRH2 to TRH7, and TRH9 to TRH18, arose due to reversion of the dnaA46 mutation to wild type. As a control, transduction of tnaA::Tn10 and the linked dnaA46 mutation into the parental strain TC382 (dasC, dnaA46) did not result in a high proportion of temperature-sensitive transductants (Table 2B). This reversion rate (16 temperature-resistant isolates of 576 kanamycin-resistant transductants obtained by transduction of trxA::kan into TC382) is abnormally high relative to the reversion of dnaA46 to temperature resistance in other strains. This may be due to a deleterious effect of the trxAxkan allele on growth when introduced into TC382. Such strains grow poorly with a generation time of 65 min in comparison to 35 min for TC382. 58 Table 2 Transduction of dnaA46 into TRHl through TRH18 tetracycline-resistant temperature-resistant . . l . l . A. TRHl 24 22 TRHZ 24 0 TRH3 24 3 TRH4 24 0 TRH5 24 1 TRH6 24 1 TRH7 24 O TRH8 24 21 TRH9 24 0 TRHIO 24 3 TRHll 24 0 TRI-112 24 2 TRH13 24 0 TRH14 24 0 TRH15 24 0 TRH16 24 0 TRHl7 24 0 TRH18 24 1 B. TC382 48 47 The dnaA46 allele was cotransduced with maA::Tn10 from ABA46 into the indicated recipient strains.Tetracycline-resistant transductants were obtained and screened for temperature resistance by growth on LB plates at 40°C compared to 30°C. 59 In contrast, transduction of tnaA::Tn10 and the linked dnaA46 mutation from ABA46 into TRHl or TRH8 resulted in most tetracycline-resistant isolates remaining temperature resistant (Table 2). This may suggest that dasC is located close to but not within MA with a linkage of 99.6% (558 temperatme-sensitive isolates of 560 kanamycin-resistant transductants). Using the formula derived by Wu (49), this cotransduction frequency may suggest a physical distance of 60 bp separating the two markers . By further examination, TRHl and TRH8 appeared to have arisen by an unusual tandem duplication to retain both the intact tncA gene, presumably dasC, and trxAxkan based on three lines of evidence. First, if trxA::kan and dasC are linked with a cotransduction frequency of 99.6%, P1 transduction of ilv::Tn10 from BW6159 into TRHl or TRH8 should result in a cotransduction frequency of about 70% between ilv::Tn10 and dasC+. Of 153 tetracycline-resistant transductants of TRHl obtained by this cross, only 7 were temperature-sensitive for growth with a linkage of 4.5%. Of 63 tetracycline-resistant isolates of TRH8 examined, 11 were temperature sensitive for growth indicating a linkage of 17.5%. These cotransduction frequencies suggest that dasC and ilv::Tn10 are not as closely linked as expected. Second, Southern analysis (47) of chromosomal DNA from TRHl through TRH8 digested with Kpnl restriction enzyme indicated the presence of both the trxAxkan allele and the trxA gene, presumably containing the dasC mutation (Figure 2). In this analysis, pTXA2, a pBR322 derivative containing the MA gene in a 1.3 kb chromosomal DNA fragment (Figure 113), was nick-translated with DNA polymerase I and [ot- 32p] (1ch and used as a probe. Plasmid pJG31 contains a 7 kb I-IindIII fragment of chromosomal DNA in pBR322 (24). Kpnl digestion of this plasmid produced fragments of 2 kb containing trxA, and 11 kb containing the vector and remaining chromosomal DNA (lane 1). Both fragments are homologous to the probe. Two fragments of about 2, and 8.5 kb were detected in Kpnl-digested chromosomal DNA from AB1157, TC187 (dnaA46), and TC382 (dasC, dnaA46) (lanes 2-4). The 2 kb fragment conrigrates with the smaller Kpnl 60 Figure 2. Plasmid (30 ng) and chromosomal DNA (1 ug unless indicated) purified (48) from the strains listed were restricted with Kpn I, electrophoresed on a 0.7% agarose gel and transferred to nitrocellulose by the method of Southern (47). Southern analysis was performed using 32P-labeled pTXA2. Lane 1, pJG31; 2, AB1157; 3, TC187; 4, TC382; 5, TRHl (3 ug); 6, TRH8; 7, TRA46; 8, TRH202; 9, TRHZO4. Hind III-digested DNA was visualized by ethidium bromide staining prior to transfer and used as a molecular weight marker (sizes indicated in kb) 61 El 2 3:4 56789 1,. 62 fragment from p163] and contains the m gene. The larger fragment of about 8.5 kb corresponds in size with the 8.5 kb Kpnl fragment located to the left of the chromosomal Kpnl fragment containing tncA (Figure 1, (29)) and homologous to the left portion of the inserted fragment in pTXA2. This fragment appears less intense presumably due to the short region of homology. Southern analysis was performed with chromosomal DNA from TRA46 (dnaA46, trxAzzkan), TRH202 (dnaA46, chxkan), and TRH204 (tnA::kan) (lanes 7-9). TRH202 and TRH204 are isogenic at other loci with TC382 (Table 2). In addition to the 8.5 kb Kpnl fragment from the left of trxA, a 3.8 kb fragment was observed corresponding in size to that expected (4 kb) by rcplacement of mat in the chromosome with the mtAxkan allele (31). The 2 kb fragment containing trrA was not observed. In contrast, Southern analysis of Kpnl-digested chromosomal DNA from TRHl and TRI-IS indicated the presence of the 2 kb fragment presumably containing the dasC mutation in m, the 3.8 kb fragment containing mrAxkau, and the 8.5 kb fragment located to the left of 0704 (lanes 5, 6). Gene duplications such as this involving mat, and trxA::kan have been observed following general transduction and are unstable (30). A similar analysis of HindIII digested chromosomal DNA from these strains confirmed that both n'xA::kan and trxA are present in TRHl and TRH8 (data not shown). A 7 kb chromosomal I-IindIII fragment from pJG31, AB1157, TC187 (dnaA46), or TC382 (dasC, dnaA46) was observed using 32P-labeled pTXA2 as a probe. The kanamycin- resistance gene in trxA::kan contains a single HindIII site (31). Chromosomal fragments of about 3.5 kb and 5.2 kb were observed from TRA46 (dnaA46, trxA::kan), TRH202 (dnaA46, axA::kan), and TRH204 (trxA::kan). These fragment sizes correspond approximately to the fragment sizes expected (5.5 and 3.5 kb) in which 0704ka has replaced mm. The presence of the 7, 3.5, and 5.2 kb fragments in chromosomal DNA from TRHl and TRH8 not only indicates the presence of trxA and trxAxkan but also that the event of gene duplication does not involve insertion into the 7 kb HindIII fragment. In addition, other genes in this region also have been duplicated. 63 The third line of evidence that trxA and trxA..kan are present in TRHl and TRH8 is that both kanamycin—resistant isolates support T7 phage growth which requires an intact trxA gene (data not shown, see below). From the above experiments, we were unable to separate dasC from mrAxkan by P1 transduction unless a gene duplication event occurred. These results indicate that m and dasC are allelic. Mapping of dasC with recombinant plasmids. The dasC mutation was originally mapped by use of ilv transducing phages to the right of the ilv operon of the E. coli chromosome (14). Introduction of particular ilv transducing phages into the dasC, dnaA46 strain TC382 reversed the suppressive effect of the dasC mutation and conferred temperature sensitivity. This region of the chromosome has been well characterized and includes rep, rho, trxA, and a gene encoding a 38 kilodalton protein of unknown function (Figure 1B, (21, 22, 32, 33)). Experiments were performed to correlate the dasC gene to one of the known genes in the region by an independent method. This approach relied on introduction of a multicopy number plasmid containing wild type chromosomal DNA from this region into TC382, the dnaA46 strain suppressed by dasC. By a gene dosage effect, the wild type gene product encoded by the plasmid may interfere with the suppressor function of the dasC gene product to make the transformed strain become temperature sensitive. The recombinant plasmid, pJG31 (24), containing 7 kb of wild type chromosomal DNA from the dasC region inserted into pBR322 (Figure 13), was used to determine whether the presence of this plasmid in TC382 would confer temperature sensitivity to this dasC, dnaA46 strain. Cultures of TC382 either lacking a plasmid, transformed with pJG31, or with the vector pBR322 were tested for the ability to grow at 30°C and 39°C. The dasC, dnaA46 strain containing pJG31 was more temperature sensitive than either TC382 lacking a plasmid, or TC382 containing pBR322 (Table 3). This result indicated 64 Table 3 Interference of dasC function by multicopy number plasmids containing wild type chromosomal DNA. E E I E . E] 'l EEfi' EEl' -- 0.75 pBR322 0.63 pJG3l 3.5 x 10-2 pJGAKpn 0.81 pRH023 2 x10‘2 pRHOll7 1 x10’4 pTXA2 7 x10“3 E E I E . El .1 Effi' EE] . pTXA2 4.1 x 10-2 pJGAKpn 0.71 pTXA114 1.0 42TXA115 0.45 The dasC, dnaA46 strain TC382 was transformed by the indicated plasmids. Ampicillin-resistant transformants grown at 30°C were then plated after dilution onto LB plates containing 30 ug/ml ampicillin and incubated at 30°C and 39°C (Expt. A) or at 30°C and 40°C (Expt. B) for 1 day. TC382 lacking a plasmid was plated on LB plates. The data are expressed as the efficiency of plating at 39°C or 40°C relative to 30°C. In comparable experiments, the efficiency of plating of TC187, the isogenic dnaA46 strain not suppressed by the dasC mutation, was <10‘5 at 40°C relative to 30°C. 65 that this 7 kb chromosomal fragment contained in pBR322 was responsible for the increase in temperature sensitivity. Derivatives of p163] were then constructed to determine the region within this 7 kb chromosomal fragment essential for reversing the suppressive effect of dasC on the dnaA46 mutation. The recombinant plasmid, pJGAKpn, was constructed which lacks sequences encoding thioredoxin and rho protein (Figure 1B). TC382 (dasC, dnaA46) containing this plasmid was able to grow at 30°C and 39°C as well as either the untransformed strain or this strain harboring pBR322 (Table 3). This result indicates that rho and/or trxA are required for reversing the suppressive effect of the dasC mutation. It is also possible that an unknown gene product encoded by this region was responsible for the observed phenotype. The rho and mm genes from this region were subcloned from pJG31 to form the plasmid pRHOZ3 (Figure 1B). As was observed with transformants of TC382 containing pJG31, this dasC, dnaA46 strain harboring pRH023 was temperature sensitive for growth. This result indicates that the region lacking in pJGAKpn and contained in pRI-1023 was required for reversing the suppressive effect of dasC. Recombinants mutationally altered in the rho gene or lacking most of the rho coding sequence were prepared. These include pRH0117 containing a frame-shift mutation, and pTXA2 containing the N-terminal coding region of the rho gene (Figure 13). The indicated plasmids altered in the rho gene were able to reverse the suppressing effect of the dasC mutation (Table 3). This indicates that the wild type rho gene in intact form apparently is not required for the increase in temperature sensitivity of the plasmid-containing dasC, dnaA46 strain. Based on DNA sequence inforrrration (34, 35) and restriction analysis, the recombinant pTXA2 contains the entire thioredoxin gene in active forrrr (see below), the rho promoter, and coding sequences for 13 amino acids of the N—terminal region of the rho polypeptide. Upon transformation into TC382 (dasC, dnaA46), pTXA2 was able to counteract 66 the suppressive effect of the dasC mutation (Table 3). The coding region for thioredoxin in pTXA2 was altered by cleavage with Clal restriction endonuclease, and conversion of the cohesive termini to blunt ends by the large fragment of DNA polymerase I. The blunt ends were religated together which is expected to produce a frame-shift mutation to inactivate thioredoxin (see below). This construction also interrupts an open reading frame predicted to direct the synthesis from the coding strand of 01A of a polypeptide of 132 amino acids. As this coding sequence which would be complementary to the mat message lacks even an infrequently used initiating codon (36), it is unlikely that this open reading frame, if transcribed, is translated. The resultant plasmids pTXA114 and pTXA115, as well as the vector pBR322 were unable to reverse the suppressive effect of the dasC mutation when introduced into TC382 (dasC, dnaA46) (Table 3). Thioredoxin is the only intact gene known to be present in the inserted fragment of pTXA2. These results indicate that the dasC gene is allelic with 11704, the gene for thioredoxin. When contained in MC1061, a strain wild type for the dasC and dnaA genes, plasmids pJGBl, pJGAKpn, and pRH023 did not affect the growth of this strain at either 30°C or 39°C (data not shown). This indicates that conferral of temperature sensitivity by plasmids containing trxA is specific for the strain TC382. The dasC mutation supports 77 and M13 growth . Thioredoxin has been shown to be essential for M13 and T7 phage propagation (37, 38). The ability of these phages to form plaques on the dasC, dnaA46 strain was determined in an attempt to detect an alteration in thioredoxin activity. '17 phage plated with approximately the same efficiency on TC382 (dasC, dnaA46), the isogenic parent strain TC187 (dnaA46), and on C600 (Table 4). Table 4 67 Growth of phages T7 and M13Gori1 on dasC, and WM mutant strains. Relevant S . :1 .1: Cr EH El El TC187 ch+ TC382 dasC C600 ch+ JM103 ch+ A307 trxA307 A307(pJG3l, tut/1+) Ach307 A307(pTXA2, trxA+) AtrxA307 A307(pTXA114, trxA+) AtrxA307 A307(pTXA115, trxA+) AtrxA307 53013233322) Amm307 T7 MIBGoril + +* + +* + ND ND + +** + + + T7 or the filamentous phage M13Gori1 were plated on the strains listed and on A307 containing the indicated plasmids. LB plates were incubated overnight at 33°C for the indicated strains except for A307 which was incubated at 37°C. ND, not determined; +, sensitive to plaque formation; -, insensitive to plaque formation (<10‘5 for T7, <10'8 for MIBGori 1). *, F1::Tn10 derivatives of TC187 and TC382 were used. **, T7 plaque formation on this strain was 50-fold lower relative to C600. 68 The ability of M13 to form plaques was tested after introduction of an F factor from K603 into TC382 and the parent strain TC187 (Experimental Procedures). The filamentous phage M13Goril was able to form plaques with approximately equal efficiencies on F factor—containing derivatives of TC382 (dasC, dnaA46), TC187 (dnaA46), and on JM103. Both '17 and M13Goril were not able to form plaques on the thioredoxin deletion mutant A307 unless this strain harbored the recombinant plasmid pJGBl, or pTXA2 (Table 4). The mat deletion mutant A307 containing pTXA114 or pTXAl 15 altered by a 2 bp insertion in the mat coding region remained insensitive to plaque formation with either M13 or T7 phage. These results indicate that only recombinant plasmids containing an intact trxA gene could complement the m deletion mutation to support T7 and M13 phage growth. The result that both T7 and M13Goril can form plaques with approximately equal efficiencies on a wild type strain and on the dasC, dnaA46 strain TC382 indicates that the dasC mutation does not dramatically decrease the ability of thioredoxin to function in T7 or M13 growth. DISCUSSION We have determined that a mutation, dasC, capable of suppressing the temperature- sensitive phenotype of the dnaA46 mutation maps in the gene encoding thioredoxin, a protein initially isolated as a cofactor for the reduction of ribonucleoside diphosphates by ribonucleotide reductase (39). This conclusion is based on the inability to separate dasC from a mots/can mutation by P1 transduction and on the ability of multicopy number plasmids containing the trxA gene to reverse the suppressive effect of dasC on the dnaA46 mutation. In contrast, alteration of the (HA coding region in the plasmid by a frameshift mutation abolished the ability of the recombinant plasmid to interfere with dasC function in suppression. Thioredoxin is required for T7 phage growth as an essential subunit of T7 DNA polymerase (37, 40). The phage-encoded gene 5 protein and thioredoxin in a 1 to 1 stoichiometry comprise the active form of this enzyme. Whereas T7 cannot be propagated in mutants of E. coli lacking thioredoxin, trxA mutants which produce thioredoxins lacking redox activity will support T7 phage growth (33). Biochemical studies with mutant thioredoxin-gene 5 protein complexes indicate that while the reduced form of thioredoxin is necessary to reconstitute T7 DNA polymerase, DNA synthesis by this enzyme does not require further reductant (41). Thioredoxin is also required as a host factor for filamentous phage assembly (38). Studies indicate that mutant forms of thioredoxin which are inactive as redox proteins will support filamentous phage growth (42). Thioredoxin is not apparently required as a reductant for phage assembly. The studies reported here indicate that the dasC mutation in trxA does not alter the ability of this protein to function in growth of either T7 or M13. Russel and Model (42) described an in vivo assay of thioredoxin redox function. Mutants of thioredoxin inactive as redox proteins cannot grow on media containing methionine sulfoxide when the normal 6 9 7O methionine generating pathway is blocked. TC3 82 containing mutations in metB and dasC plated with equal efficiency on minimal media supplemented with either methionine sulfoxide or methionine and other required amino acids indicating that the dasC mutation does not appear to affect the redox activity of thioredoxin (unpublished data). Preliminary studies with partially purified preparations of thioredoxin from this dasC strain also indicated that it is active in reduction of 5,5'-dithiobis-(2-nitrobenzoic acid). Eklund et al. (43) have described domains of thioredoxin which may be involved in interactions between thioredoxin and other proteins in oxido-reduction. Mutations in mm which affect filamentous phage assembly have been mapped at or near these domains (42). Some of these mutations encode proteins which are active in redox reactions indicating a functional interaction between these mutant forms of thioredoxin and thioredoxin reductase. It is not known whether the dasC mutation maps in one of these domains. Of its many activities, thioredoxin has been described as a protein disulfide reductase to regulate activity of enzymes involved in 002 fixation in plants (20). No evidence exists to indicate that the activity of dnaA protein is influenced by the redox state of its thiol groups. The existence of viable E. coli mutants lacking thioredoxin argues against a role of thioredoxin in modulating the activity of dnaA protein unless this effect is not absolutely required or unless another activity in E. coli can replace the function of thioredoxin. With regard to the latter possibility, it is thought glutaredoxin and glutaredoxin reductase function in the synthesis of deoxyribonucleotides in the absence of thioredoxin (44). Thioredoxin has also been observed to function as an efficient protein disulfide isomerase to catalyse the refolding of denatured RNase (45). While no evidence exists to indicate that the native conformation of monomeric dnaA protein is stabilized by disulfide bonds or that its cooperative binding to dnaA protein binding sites requires disulfide bond formation between monomers of dnaA protein, it is possible that the form of thioredoxin encoded by the dasC mutation effectively maintains dnaA46 protein in an active form at the normally nonperrnissive temperature. 71 We have not been able to transduce the dasC mutation with phenotypic expression from TC382 into isogenic and nonisogenic E. coli strains harboring the dnaA46 allele. This inability suggests that an unlinked mutation unstable in the absence of selection is also required for dasC to function as a suppressor. Attempts to identify the position of the dasC mutation by DNA sequence analysis have not been successful. Based on a cloning strategy described by Russel and Model (31), chromosomal DNA from TC382 (dasC, dnaA46) was inserted into an M13 vector followed by transformation of the thioredoxin deletion mutant A307 and selection for plaque formation. This cloning strategy requires that the trxA gene in the M13 recombinant complements the host trxA deficiency to allow phage growth. Sequence analysis of several recombinants obtained by this method indicated that the promotor and trxA coding region were identical to the wild type sequence. Under conditions which did not select for thioredoxin function in M13 phage growth, M13 recombinants identified by plaque hybridization (46) and analyzed by DNA sequencing or by restriction enzyme mapping lacked the C-terminal coding region of the trxA gene as well as a portion of the vector at the site of insertion. These results indicate that the deletion event occurred during propagation of the recombinant phage. Under identical conditions, the wild-type ch gene inserted into M13 was not deleted. These results suggest that the dasC mutation reverts to wild type under conditions of selection. In the absence of selection, dasC may be deleterious to cell growth when present in multicopy. Cells capable of maintaining M13 recombinants may arise by deletion of a portion of the m coding region. >399???" 10. 11. 12. 13. 14. 15. 16. 17. 18. 19 20. 21. REFERENCES Hirota, Y., Mordoh, J., and Jacob, F. (1970) J Mol. Biol. 53: 369-387. Kogoma, T. (1976) J. Mol. Biol. 103: 191-197. Zyskind, J.W. and Smith, D.W. (1977) J. Bact. 129: 1476-1486. Filutowicz, M. and Jonczyk, P. (1983) Mol. Gen. Genet. 191: 282-287. Lark, KG. (1972) J. Mol. Biol. 64: 47-60. Zyskind, J.W., Deen, T., and Smith, D.W. (1977) J. Bact. 129: 1466-1475. Fuller, RS. and Kornberg, A. (1983) Proc. Natl. Acad. Sci. USA. 80: 5817- 5821. Chakraborty, T., Yoshinaga, K., Lother, H., and Messer, W. (1982) The EMBO Journal 1: 1545-1549. 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The biochemical characteristics of dnaA5 protein include: 1) a high affinity for DNA containing oriC and the dnaA promoter, 2) an ability to form proper protein-DNA contacts at the dnaA boxes within the mall and dnaA promoters, 3) the formation of an altered complex with oriC, 4) inactivity in oriC unwinding assays, 5) inactivity in oriC plasmid replication systems containing purified enzymes, 6) inhibition of dnaA protein replication activity suggesting that mixed complexes which form are inactive and 7) therrnolabile replication activity in oriC plasmid replication systems containing a crude enzyme fraction. 76 INTRODUCTION The initiation of DNA replication in E. coli is a highly regulated event occurring at regularly timed intervals within the cell cycle (1). The activity of the initiator protein, dnaA, is essential for replication initiating from the chromosomal origin, oriC, both in vivo (2) and in vitro (3). Other enzymes involved in the initiation stage have been identified genetically and biochemically, and include dnaB protein, dnaC protein, DNA gyrase, primase, RNA polymerase, t0poisomerase I, and RNase H (4). A dnaK mutant defective in the initiation of DNA replication in vivo has been identified (5), indicating that there is a requirement for the dnaK gene product during initiation. The biochemical characterization of the dnaA dependent initiation process has led to the reconstitution of purified enzymes that function to replicate oriC plasmids in vitro (3, 6, 7). Early events in the replication process result in the conversion of supercoiled oriC plasmid DNA into topologically altered forms by the actions of dnaA protein, and other initiation proteins (8), demonstrating the specific function provided by dnaA protein during a stage prior to DNA synthesis. Additional studies have demonstrated that dnaA protein can function as a site-specific helicase to locally unwind DNA at oriC (9), allowing for the formation of single-stranded regions capable of being bound by pre-primosomal enzymes. The individual biochemical activities of dnaA protein which are required for its replication activity include 1) sequence specific binding to DNA containing the consensus sequence TTAT (C/A)CA (A/C)A, (10), 2) ATP binding with a Kd of 0.03 uM (11) which converts it to a form active for replication, 3) ADP binding which inhibits its replication activity, and 4) cardiolipin activation of the ADP form of dnaA protein (12, 13). The initial event in initiation involves dnaA protein binding to oriC through the interaction with four dnaA boxes to form a nucleoprotein complex which precedes its function in the ATP dependent duplex strand unwinding stage (14, 15). The sequence specific DNA binding activity of dnaA protein is also involved in transcriptional repression of certain genes. dnaA 77 78 protein binds to the dnaA box within its own promoter autoregulating its transcription (16- 18) and to two dnaA boxes within the rpoH promoter to inhibit transcription initiating from rpoI-I3P and rpoH4P (19). The identification of conditionally lethal dnaA mutants that are defective in the initiation of replication has led to the genetic mapping of the mutations and the positions were shown to be clustered within three regions of the dnaA coding region (20). The dnaA mutants were placed into three classes and the specific phenotypes associated with each class were categorized in order to associate a domain of the dnaA gene product with a specific function. The dnaA mutants that were categorized are temperature sensitive for cell growth presumably due to the inactivation of the dnaA gene product at elevated temperatures. Some of these conditionally lethal mutants, which include dnaA46 and dnaA5, are cold sensitive in the merodiploid state and asynchronous in the frequency of initiation of DNA replication at permissive temperatures (21). In a rapidly growing cell there is more than one initiation event during the cell cycle which results in an increase in the number of oriC origins. In response to the signal demanding that replication begin, initiations at all of the oriC origins occur simultaneously indicating that initiation is synchronous. In some dnaA mutants, initiation does not occur at each oriC origin as it does in the wild type dnaA cell indicating that the mutant has lost the ability to ensure that all oriC sites are simultaneously utilized. This un-coordinated initiation is termed asynchronous. The altered timing of initiation, under conditions in which the mutant protein is active, may reflect an intrinsic defect in its initiation activity or a regulatory deficiency. The biochemical characterization of these mutant forms of dnaA protein may lead to a greater understanding of the role of the dnaA gene product in initiation of DNA replication. The purification and characterization of the biochemical deficiencies of dnaA5 protein are reported with the goal of understanding the mechanisms related to the physiological defects associated with dnaA5 cells. EXPERIMENTAL PROCEDURES Reagents and chemicals. Reagents were obtained from the following sources: ribonucleotides, tRNA, polyvinyl alcohol (PVA) (type II), phosphocreatine, heparin, ampicillin, L-arabinose and calf thymus DNA, Sigma; deoxynucleotides and sepharose 4- B, Pharmacia-PL Biochemicals; HEPES, Tris Base, and dithiothreitol (DTI‘), Calbiochem- Behring; (a— 32?) ATP (800 Cilmmol or 3000Ci/mmol and (7-32P) ATP (6000 Ci/mmol), DuPont-New England Nuclear Corp.; (3H) TTP, ICN Radiochemicals; hydroxylapatite, Biorad; acrylamide, Boehringer Manheirn; TB media contains per liter, 12 g yeast extract (Difco), 24 g tryptone (Difco), 100 mM sodium phosphate (pH 7.0), and 4% glycerol; DNA cellulose was prepared according to the published methods (22) ; and Blue Dextran agarose was a gift from Dr. J. Wilson of this University. Enzymes and proteins. Enzymes and proteins were obtained from the following sources: bovine serum albumin, Sigma; T4 polynucleotide kinase, restriction endonucleases AvaI, Hinfl, ClaI, and Tan, New England Biolabs; calf intestinal phosphatase (CIP), Klenow fragment, restriction endonucleases Ach and EcoRI, Boehringer Manheirn; restriction endonuclease BamHI, Bethesda Research Laboratories. Highly purified replication proteins were obtained as described (23), dnaA protein (fraction IV, 2 x 105 units/mg); dnaA5 protein (fraction V, 6 x 104 units/mg) (Chapter III); dnaB protein (fraction V, 6 x 105 units/mg) ; dnaC protein (fraction VI, 3 x 106 units/mg); primase (fraction V, 2 x 106 units/mg); single stranded DNA binding protein (SSB) (fraction IV 4 x 104 units/mg); DNA polymerase III holoenzyme (fraction v, 2 x 105 units/mg); DNA gyrase subunit A (fraction 111, 2 x 105 units/mg); DNA gyrase B subunit (fraction V, 1 x 105 units/mg) and RNA polymerase (fraction V, 250 munits/mg). RN ase H (fraction 1v, 8 x 105 units/mg); topoisomerase I ( fraction IV, 5 x 104 units/mg) and HU protein (fraction IV, 5 x 104 units/mg) were gifts from Dr. A. Kornberg, Stanford 79 80 University. repA protein (fraction v, 8 x 10 5 units/mg) (29) was a gift from Dr. A. Abeles. Bacterial strains and plasmid DNAs. Escherichia coli WM433 was obtained from Dr. W. Messer (24); K37 HfrC supD (lambda) is a laboratory stock; TD2, is a derivative of MC1061 containing maA::7N10 and the dnaA5 allele; M13oriC26 (25) contains oriC and adjacent E. coli chromosomal DNA in M13Gori1; M13oriC2LB5 (3) contains oriC on a 345 bp EcoRI fragment in M13Goril; pTSOl82 (27) contains oriC and adjacent chromosomal DNA on a HaellI fragment in pBR322 ; M13Plori49 contains the P1 phage replication origin cloned into M13mp18 (31); pDS 105 (23) contains the dnaA5 gene cloned under the control of the araB promoter in the vector pINGl (26); pBF1509 contains the dnaA gene cloned into the BamI-II site of pAD329 (10); and pFN42 contains the promoter of the mall gene and a portion of the N-terminal coding region of rpoH cloned into the PstI site of pBR322 (19). Plasmid DNAs were prepared by the cleared lysis method followed by sedimentation in CsClz-ethidium bromide gradients (32). DNA replication assays. DNA replication reactions that employ a crude enzyme fraction deficient in dnaA protein were performed as previously described (28). DNA replication assays using oriC plasmid DNA and a crude enzyme fraction capable of sustaining DNA synthesis upon the addition of dnaA protein were performed in a 25 ul reaction volume containing: 25 mM HEPES-KOH, pH 7.8 ; ATP, 2 mM; CI'P, UTP, and GTP, each at 0.5 mM; dATP, dCTP, dGTP, and (3H) m (25 to 40 cpm/pmol), each at 100 uM; magnesium acetate, 11 mM; PVA, 8 % (w/V); phosphocreatine, 40 mM; creatine kinase, 100 ug/ml; supercoiled M13oriC26, 200 ng; and Fraction II from WM433 (28), 160 ug to 200 ug of protein. The reaction mixtures were assembled at 0°C and subsequently incubated at 30°C for 25 minutes unless otherwise indicated The incorporation of tritiated nucleotide into trichloroacetic acid precipitable DNA was quantitated by liquid scintillation counting (23). One unit of replication activity represents one pmol of nucleotide incorporated into DNA per minute at the indicated temperature. 81 The conditions used to demonstrate DNA replication initiating from P1 phage origin DNA templates involved the use of a replication system that employs a crude enzyme fraction, dnaA protein and phage encoded repA protein as previously described (30). The conditions used were identical to those used for the oriC plasmid replication assay employing WM433 Fraction II as the crude enzyme source, except 100 ng of M13Plori49 plasmid DNA and purified repA protein were used. DNA replication reactions using purified enzymes were performed as previously described (23). The 25 ul reactions contained: HEPES-KOH, pH 7.8, 25 mM; Tris-HCl, pH 7.5, 20 mM; sucrose 4% (w/V); ATP, 2mM; CTP, GTP, and UTP, each at 0.5 mM; dATP, dCI'P, dGTP, and (3H) TI‘P (25 to 40 cpm/pmol) each at 100 uM; magnesium acetate, 11 mM; phosphocreatine, 2 mM; DTI‘, 5 mM; creatine kinase, 100 ug/ml; BSA, 0.08 mg/ml; SSB, 220 ng; HU, 25 ng; t0poisomerase I, 2.5 units, and RNase H, 3 ng, were used only in the RNA polymerase dependent reactions; gyrase A subunit, 470 ng; gyrase B subunit, 600 ng; primase, 10 ng; dnaB protein, 100 ng; dnaC protein, 25 ng; RNA polymerase, 500 ng, where noted; DNA polymerase III holoenzyme, 270 ng; M13oriC2LB5 supercoiled DNA, 200 ng; and the indicated amounts of dnaA prorein or dnaA5 protein. The reactions were assembled at 0°C and the incubations were performed at 30°C for 30 minutes unless otherwise indicated. The total nucleotide incorporation was measured as previously described (26) DNA binding assays. DNA fragment retention assays on nitrocellulose filters followed by gel analysis to visualize the DNA have been previously described (10). The binding of dnaA protein or dnaA5 protein to DNA fragments was performed by the addition of the indicated amounts of protein at 0°C in a 25 ul reaction volume containing; 100 ng of pTSOl 82 DNA restricted with Tan endonuclease and end-labeled with (a—32P) dCTP and DNA polymerase I (large fragment); 40 mM HEPES-KOH, pH 7.8; 5 mM MgC12; 2 mM DTT; and 50 mM KCl. Following the incubation at 30°C or 38°C for 10 minutes, the samples were filtered through Millipore HAWP nitrocellulose filters (0.22um pore size) 82 and washed with 250 111 of the reaction buffer. The DNA bound to the filters was eluted by incubating the filter in elution buffer (0.5% (w/v) SDS, 500 mM magnesium acetate, 50 mM Tris-HCl (pH 8.0), and 10 mM EDTA) at 65°C for 20 minutes. The DNA was precipitated from the fractions contained in the flow through and bound samples by the addition of tRNA to 100 ug/ml, NaCl to 100 mM, ethanol to 70% (v/v), and incubation at - 70°C for 10 minutes. The samples were centrifuged at 12, 000 x g for 15 minutes and the pellets were resuspended in 25 ul of gel loading buffer (10% glycerol, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)). Portions of each reaction were separated by electrophoresis in a 6% polyacrylamide gel in TBE. The gels were dried on a Hoefer Scientific gel dryer (model SE 540) and exposed to Kodak XAR-S film at -70°C using a Crona Quanta III intensifying screen. The binding affinity of dnaA5 protein for DNA containing the dnaA promoter and for oriC, in comparison to dnaA protein, involved quantitating the amount of DNA bound to nitrocellulose filters following the methods which are described above. A 463 bp AvaI DNA fragment from M13oriC26, which contains oriC , and a 390 bp BamHI/EcoRI DNA fragment from pBF1509 containing the dnaA promoter fragment were separately treated with CIP and end-labeled with (7-32P) ATP and T4 polynucleotide kinase. In the binding reaction, 15 fmol of either end-labeled DNA fragments was incubated at 30°C or 40°C for 10 minutes in a 25 111 volume containing; 150 fmol of unlabeled pBR322 DNA restricted with Hian; 40 mM HEPES-KOH (pH 8.0); 5 mM MgC12; 2 mM DTT; 25 mM (NH4)ZSO4 and the indicated amounts of dnaA or dnaA5 proteins. Following the incubations, the samples were filtered through Millipore HAWP nitrocellulose membranes and washed with 250 ul of pre-warmed reaction buffer. The radioactive DNA bound to the filter was quantitated by liquid scintillation counting. ATP binding assays. The conditions used to demonstrate ATP binding by dnaA protein have been previously described (1 l, 31). A 25 ul reaction volume contained: 2 pmol of dnaA protein or dnaA5 protein; 0.5 uCi of (ct-32m ATP; the indicated concentrations of 83 ATP; 0.5 mM magnesium acetate; 5 mM DTT; 15 % (v/V) glycerol; 0.01% (v/v) triton X- 100; and 50 mM Tris-HCl, pH 8.0. The incubations were performed at 0°C for 15 minutes, filtered through Millipore HAWP nitrocellulose membranes and the filters were washed with 500 111 of the reaction buffer. The radioactive ATP bound to the nitrocellulose filter was quantitated by liquid scintillation counting. DNase I protection assays. The conditions used to perform DNase I protection experiments have been previously described (10). A standard 10 ul reaction mixture contained; DNase I binding buffer (10 % glycerol (WV), 40 mM Tris-HCl (pH 8.0), 10 mM MgC12, 0.2 mM EDTA (pH 7.0), 2 mM DTI‘, and 100 ug/ml BSA), 50 mM (NH4)2SO4 unless otherwise indicated, 25 fmol of (a-32P) end-labeled DNA (oriC was a 463 bp AvaI restriction fragment from Ml3oriC26 end-labeled and restricted with Hian; the dnaA promoter was a 390 bp BamI-lI/EcoRI fragment from pBF1509 end-labeled and restricted with Hian; and the rpoH promoter was labeled as described (19)) and the indicated amounts of dnaA protein or dnaA5 protein. The incubations were performed at 30°C for 10 minutes, after which 0.25 ul of a DNase I solution (prepared by diluting a 2 ug/ml solution of DNase I 200 fold in DNase I binding buffer containing 200 mM N aCl) was added. The incubations were continued at 30°C for 30 to 60 seconds and the reactions were quenched by the addition of 12 ul of DNase I stop buffer (0.1 % (w/v) SDS, 300 mM sodium acetate, 20 mM EDTA, and 100 ug/ml tRNA). The reaction products were precipitated by the addition of two volumes of ethanol and incubation at -70°C for 15 minutes. Following centrifugation at 12, 000 x g, the pellets were resuspended in DNase I gel loading buffer (80 % forrnarrride (v/v), 10 mM NaOH, 0.01% (w/v) Bromophenol Blue, and 1 mM EDTA). The samples were boiled for two minutes before electrophoresis in a 7 M meal 6 % (w/v) polyacrylamide sequencing gel. The gels were dried under a vacuum and exposed to Kodak XAR-S X-ray film at room temperature. Immunological methods. The ELISA was performed as previously described (33) in order to localize dnaA5 protein during chromatography. Rabbit serum containing antibodies to 84 dnaA protein was obtained after the injection of the protein (Mono 8 fraction V) into the popliteal lobes of rabbits as previously described (33). Portions of the fractions that eluted dming chromatography were incubated for one hour at room temperature in microtiter wells (Nunc) containing 100 ul of 50 mM sodium borate (pH 9.0). The wells were washed four times with PBS containing 0.05 % (v/v) tween 20, 100 ul of a 1:2000 dilution of dnaA anti-serum (in PBS containing 0.05 % (v/v) tween 20 and 0.2 % (w/v) BSA) was added to the microtiter wells and the incubations were continued for one hour at room temperature. The wells were washed four times with PBS containing 0.05 % (v/v) tween 20, 100 111 of a 1:1000 dilution of goat-anti rabbit HRP (Biorad) (in PBS containing 0.05 % (v/v) tween 20 and 0.2 % (w/v) BSA) was added to the microtiter wells and the incubations were continued for one hour at room temperature. The wells were washed four times with PBS containing 0.05% (v/v) tween 20 and HRP activity was detected as previously described (33). Protein determination. Protein determinations were performed by the method of Bradford (23). RESULTS Overproduction of dnaA5 protein. dnaA5 protein was purified from TD2 cells (dnaA5, tnaA::TN10) harboring the plasmid pDS 105 which contains the dnaA5 gene cloned into the protein overproducing vector pINGl (26). This vector contains a cloning site downstream of the araB control region and the induction of cloned genes can occur after the addition of arabinose to the cell culture. The initial experiments involved maximizing the expression of dnaA5 protein in mid- log phase cell cultures containing pDS 105 by the addition of arabinose to 0.75 % (w/v). A time course of the induction of dnaA5 protein was performed to determine the time required for maximal expression. The extent of dnaA5 protein overproduction was quantitated by immunological detection of antigenic material from induced whole cell lysates (data not shown) and by determining the levels of dnaA5 protein dependent replication activity present in crude lysates obtained fiom the induced cells. dnaA5 protein activity in replication systems that utilize a crude enzyme fraction was detected in lysates obtained from cells which were grown in TB media, induced with arabinose and grown for an additional 3 to 4 hours (data not shown). Activity was not detected in lysates obtained from cells grown and induced in LB media under identical conditions, demonstrating the importance of the media and the state of the cell in obtaining elevated quantities of dnaA5 protein (data not shown). Purification of dnaA5 protein. The chromatographic steps utilized during the purification of dnaA5 protein were similar to those used during the purification of dnaA protein (23). Presumably, the rnissense mutation in dnaA5 protein would not have altered its chromatographic characteristics in comparison to dnaA protein. The purification of dnaA protein involves: 1) lysis of cells 8 5 86 which contain the dnaA gene cloned into plNG to obtain a soluble protein fraction; 2) chromatography on heparin agarose; 3) chromatography on gel filtration resins; and 4) anion exchange chromatography on Mono S. Of these resins, only heparin agarose could be used in the purification of dnaA5 protein because its elution characteristics were drastically different in comparison to dnaA protein. In general, dnaA5 protein eluted broadly from the resins examined and sufficient purification was not achieved. Alternative methods were used for the purification of dnaA5 protein are summarized in Table 1. TD2 cells harboring the plasmid pDSlOS were grown in 12 L of TB media, containing 30 ug/ml of ampicillin, at 30°C to an O.D.595 of 0.5 and arabinose was added to a final concentration of 0.75 % (w/v). The cells were grown for an additional 3.5 to 4 hours with vigorous aeration and harvested by centrifugation at 5,000 x g for 15 rrrinutes. The cells were resuspended in TS buffer (50 mM Tris-HCl (pH 8.0) and 25% sucrose (w/v)) to an OD. 595 of 200 to 250 and frozen in liquid nitrogen. The purification listed in Table 1 was initiated using two 12 L preparations of cells. The frozen cells were thawed at 4°C and the following components were added to facilitate lysis: KCl to 500 mM, sperrnidine-HCI to 20 mM, DTT to 5 mM, and lysozyme to 0.3 mg/ml. After the addition of these reagents, the cells were incubated at 0°C for 30 minutes followed by quick-freezing in liquid nitrogen. The lysate was thawed at 2°C, centrifuged at 15, 000 x g for 12 minutes, and the soluble protein fraction was recovered (Fraction I). Fraction I was diluted five fold with Buffer A (40 mM HEPES-KOH (pH 7.6), 20 % (v/V) glycerol, and 5 mM DTT) to a conductivity equivalent to Buffer A containing 130 mM KCl. The protein was batch adsorbed to the heparin agarose by adding 200 ml of pre-equilibrated resin to the protein solution and the protein was allowed to bind to the resin by stirring for one hour at 4°C. The resultant slurry was packed into a solid column support and washed with Buffer A containing 130 mM KCl. The bound protein was eluted with a ten column volume linear gradient extending from 130 mM to l M KCl in Buffer A. The physical presence of dnaA5 protein was detected using an ELISA (see 87 Table 1 A summary of dnaA5 protein purification fraction volume protein activity specific activity activity yield (ml) (mg) (Mira—(um) (%l I. Lysate 51.0 1926 240, 000 127 (100) II. heparin agarose“ 72.0 76.0 62,000 842 26 III. Blue Dextran agarose 26.4 8.2 66,000 8,250 27 IV. DNA Cellulose 23.2 0.32 ND. ND. ND. mute 1.21 0.24 14. 080 64.000 5.8 . ND. not determined * One half of fraction II was used for subsequent chromatographic steps. The values listed for fractions HI-V are corrected for by a factor of two. 88 Experimental Procedures). The fractions containing dnaA5 protein were pooled (Fraction II) and assayed for replication activity. The units recovered and the specific activity of the fractions are summarized in Table 1. The heparin agarose fractions of dnaA5 protein were dialyzed against Buffer A until the conductivity was equivalent to Buffer A containing 150 mM KCl. The protein was applied to a Blue Dextran agarose column (5 ml), equilibrated in Buffer A containing 150 mM KCl and the bound protein was eluted with a linear gradient extending from 150 mM KCl to l M KCl in Buffer A. A substantial purification of dnaA5 protein was achieved because most of the proteins present in Fraction II flowed through the resin. dnaA5 protein was detected in the gradient eluent fractions using an ELISA and it was found to elute broadly in fractions with a conductivity equivalent to Buffer A containing 450 mM KCl. The fractions containing elevated levels of dnaA5 protein were pooled (Fraction III) and assayed to determine the recovery of replication activity (Table l). The fractions of dnaA5 protein that eluted from the Blue Dextran agarose column were 25% to 40% pure. A DNA cellulose column was used to further purify dnaA5 protein to near homogeneity. Fraction II] was dialyzed against Buffer A until the conductivity was equivalent to Buffer A containing 100 mM KC] and it was applied to a single-stranded DNA cellulose column, equilibrated in the same buffer, at 5 mg of protein per ml of resin. The protein that bound to the resin was eluted with a 20 column volume linear gradient extending from 100 mM to 1 M KCl in Buffer A. An ELISA was used to localize dnaA5 protein and it was found to elute broadly during the later part of the gradient (data not shown). It is possible that this purification was efficient because dnaA5 protein had a tendency to aggregate, which effectively retarded its elution from the resin compared to the bulk of the contaminants. dnaA5 protein obtained from this column was too dilute to assay and it was concentrated using hydroxylapatite. The fractions containing dnaA5 protein (Fraction IV) were loaded directly onto a 0.2 ml hydroxylapatite column, equilibrated with Buffer A containing 50 mM KCl, and the bound protein was eluted using Buffer A 89 containing 500 mM (NH4)2SO4. Hydroxylapatite fractions (Fraction V) were sufficiently concentrated to assay directly in replication reactions and the specific activity of the purified protein was determined to be 64,000 units/mg (T able 1). The purity of the individual fractions obtained during the chromatography is shown in Figure 1. dnaA5 protein is temperature sensitive in the replication of oriC plasmids in a system containing a crude enzyme fraction. An in vitro oriC plasmid replication system which specifically initiates DNA synthesis from oriC has been previously reported (29). The activity of dnaA protein in this replication system can be measured using a crude enzyme fraction deficient in dnaA protein. A titration of dnaA protein in this assay is shown in Figure 2A. The addition of 50 ng of 200 ng of dnaA protein resulted in initiation activity as measured by DNA synthesis. dnaA protein dependent replication activity was observed over temperatures ranging from 25°C to 38°C (Figure 2A), with an increasing temperature giving rise to increasing extents of DNA synthesis. Purified dnaA5 protein (Fraction V) was active in this replication system at temperatures of 25°C and 30°C (Figure 2B). The specific activity of dnaA5 protein at 30°C was reduced 2 to 4 fold compared to the specific activity of dnaA protein and the dependence upon recombinant plasmids containing oriC was observed (data not shown). Genetic studies demonstrated that cells harboring the dnaA5 allele exhibit, among other characteristics, a temperature sensitive defect in the initiation of DNA replication (20). Cells are able to initiate DNA synthesis at 30°C, but initiation is blocked at 40°C, presumably due to the thermal inactivation of the dnaA5 polypeptide. The ability of purified dnaA5 protein to initiate DNA synthesis at higher temperatures was tested to determine if it exhibited a thermolabile initiation defect in vitro. Although 100 ng or 200 ng of dnaA5 protein could sustain DNA synthesis at 25°C or 30°C, DNA synthesis was not 90 Figure 1. SDS polyacrylamide gel electrophoresis of dnaA5 protein fractions. Various fractions of dnaA5 protein obtained after chromatography were separated by electrophoresis in a 10% polyacrylamide gel. The fractions which are represented include; lane 1, molecular weight markers, gyrase A subunit (105 kd), dnaB protein (54 kd), and SSB (l8 kd); lane 2, dnaA protein (fraction IV); lane 3, dnaA5 protein (fraction 11); lane 4, dnaA5 protein (fraction 11]); and lane 5, dnaA5 protein (fraction V). 91 gyrA dnaS dnaB 92 observed after adding dnaA5 protein into the replication assays that were incubated at 35°C or 38°C (Figure 2B). This indicates that the therrnolabile defect associated with dnaA5 cells is due directly to the thermolability of dnaA5 protein in initiation. dnaA5 protein is active in the replication of PI phage origin containing plasmids. An in vitro DNA replication system has been developed for P1 phage (30). The reaction requires the presence of the P1 origin on a recombinant plasmid, phage encoded repA protein, host encoded dnaA protein, and a crude enzyme fraction capable of sustaining DNA synthesis. Other enzymes required for the replication of P1 phage DNA, which are present in the crude enzyme fraction, include DNA gyrase, RNA polymerase, DNA polymerase III holoenzyme, dnaB, dnaC, dnaJ, dnaK, grpE, and dnaG proteins (34). A titration of repA protein in this system is shown in Figure 3A. The addition of 50 n g to 200 ng of purified repA protein did not result in DNA synthesis in the absence of exogenously added dnaA protein. In the presence of a presumed excess of dnaA protein (90 ng), the addition of repA protein resulted in increasing extents of DNA synthesis until approximately 20 % of the input DNA template was replicated. The dependence of Plori plasmid replication upon dnaA protein is shown in Figure 3B. The reaction contained saturating levels of repA protein (200 n g) as determined from the assay described in Figure 3A. The addition of increasing levels of dnaA protein resulted in increasing extents of DNA synthesis demonstrating the dnaA protein dependence in the reaction. Assays were performed in parallel in which dnaA protein was titrated into an oriC plasmid replication system to examine the relative amounts required for initiation, in comparison to the levels of dnaA protein required for observing DNA synthesis in the Plori replication system (Figure 3B). The similar amounts of dnaA protein required for 93 observing high levels of DNA synthesis in both reactions indicates that dnaA protein may provide the same function in both systems. It is known that the ATP form of dnaA protein is active in oriC plasmid replication (11) while the ADP form of dnaA protein is inactive. In the Plori plasmid replication system it is not known if differing biochemical activities of dnaA protein are required in comparison to the oriC plasmid replication system. It is possible that dnaA5 protein may represent a form of dnaA protein which is deficient in the biochemical properties required for the replication of oriC plasmids, but not Plori plasmids. The replication system dependent upon the P1 origin was examined to see if dnaA5 protein was active in the initiation of DNA synthesis (Figure 4A). The titration of dnaA protein in the presence of saturating levels of repA protein resulted in the production of up to 140 pmol of DNA synthesis. The titration of dnaA5 protein in this system resulted in up to 85 pmol of DNA synthesis indicating that it is capable of initiating DNA synthesis from the Plori templates. As with its activity in oriC plasmid replication, dnaA5 protein activity in Plori plasmid replication is 2 to 4 fold lower in comparison to dnaA protein. In an attempt observe a biochemical distinction between dnaA5 protein and dnaA protein function in the Plori plasmid replication system, the temperature dependence of DNA synthesis was examined. If dnaA5 protein activity is not thermolabile in Plori plasmid replication, like it is in oriC plasmid replication, then this would indicate that a biochemical function utilized during the oriC dependent initiation reaction is not used during the initiation of Plori plasmid replication. dnaA protein can function in the initiation of DNA replication of oriC plasmids over a wide temperature range, while dnaA5 protein is temperature sensitive (Figure 2B). dnaA protein remained active in the Plori dependent replication system at elevated temperatures (Figure 4B), while dnaA5 protein was inactive. The thermolability exhibited by dnaA5 protein indicates that a labile activity operates during the initiation stage of both oriC and Plori plasmid replication. 94 Figure 2. The temperature dependent replication activity of dnaA5 and dnaA proteins. DNA replication reactions employing a crude enzyme fraction were assembled according to the experi- mental procedures. dnaA protein (A) or dnaA5 protein (B) was titrated into the reactions and in- cubations were performed at 25°C (a), 30°C (0), 35°C (I), or 38°C (a) for 25 minutes. The extents of DNA synthesis were quantitated according to the experimental procedures. 95 200 150 100 50 100 ' a .m 3 2 400 case ....556 :eao._oc:EE_ warm 30 25 0 Z Fraction number 167 Figure 6. SDS polyacrylamide gel of grpE protein containing fractions. Protein samples were separated by electrophoresis in a 12% polyacrylamide gel and stained with Coomasie Blue. The fractions represented are: lane 1, molecular weight markers, gyrase subunit A (105 kd), dnaB protein (54 kd) and SSB, (18 kd); lane 2, dnaK protein (Fraction HI); lane 3, purified grpE pro- tein from Dr. M. Zylicz; lane 4, Mono Q fraction 25 containing the second activating protein; lane 5, Superose 12 fraction number 28 containing the second activating protein; lane 6, Fraction II from a grpE overproducing cell strain; and lane 7, grpE protein eluted from a dnaK protein af- finity column. gyrA dnaB SSB 169 Figure 7. grpE protein replaces a Superose 12 fraction in the activation of dnaA5 protein. Replication reactions containing purified enzymes, 90 ng of dnaA5 protein, and 2 ug of dnaK protein were assembled according to the experimental procedures. grpE protein (O) or Superose 12 fraction number 28 (O ) was added and the incubations were continued at 30°C for 60 min- utes. The extents of DNA synthesis were quantitated according to the experimental procedures. DNA Synthesis (pmol) 170 200 150 100 50 O I l l 0 250 500 750 1000 protein (nanograms) 171 Figure 8. (A). dnaK protein titration in activation reactions. dnaK protein was added into repli- cation reactions containing purified enzymes and 90 ng of dnaA5 protein without grpE protein (0), or with 100 ng of grpE protein (O ). (B) grpE protein titration in activation reactions. grpE protein was titrated into replication reactions containing purified enzymes and 90 ng of dnaA5 protein without dnaK protein (0 ), with 1.2 ug of dnaK protein (I ), or with 2.5 ug of dnaK protein (O ). The reactions were assembled according to the experimental procedures and incubated at 30°C for 60 minutes. The extents of DNA synthesis were quantitated according to the experimental procedures 172 A. , 200 E 3 150 0 ‘3 O 5 100 2 > to ‘2‘ 50 o o I l l l A 0.0 1.0 2.0 3.0 4.0 5.0 dnaK protein (ug) B. 200 g 150 - 3 m '3 . c 100 E >. (D < . z 50 o 2.1 o l I j 0 50 100 150 200 grpE protein (nanograms) 173 Table 1 The activation of dnaA5 protein by WM433 Fraction H WCCL Fraction H (ug) DNA synthesis (12ml) dnaA5 - 19 Fraction H + dnaA5 25 18 Fraction H + dnaA5 50 70 Fraction H + dnaA5 100 184 Fraction H 50 8 fiction H 100 7 DNA replication reactions containing purified enzymes and 90 ng of dnaA5 protein, where indicated, were assembled according to the experimental procedures. The indicated components were added and incubations were performed at 30°C for 45 minutes. The extents of DNA synthesis were quantitated according to the experimental procedures. Table 2 174 The activating factor is heat sensitive H (diluted) H (diluted) IHa IHa IHb IHb IHc ch Or. in _‘ in ' u‘-.i-‘ D ‘ nhi r101 - 6 0°C 36 0°C 102 55°C 79 55°C 151 71°C 9 71°C 10 91°C 6 91°C 5 WM433 Fraction H was diluted two fold with Buffer A and portions were incubated at the indicated temperatures for 7 minutes. Insoluble material was removed by centrifugation at 12, 000 x g for 5 minutes and the indicated amounts of protein were added to replication reactions containing purified enzymes and 90 n g of dnaA5 protein. The incubations were performed at 30°C for 45 minutes. The extents of DNA synthesis were quantitated according to the experimental procedures. 175 Table 3 dnaA5 protein is required to observe Fraction HI dependent replication activity t in n Fr i nHI DN nth i m l + - 19 + 6 77 + 12 139 - 6 4 - 12 5 Replication reactions containing purified enzymes were assembled according to the experimental procedures. The indicated components were added and incubations were performed at 30°C for 45 minutes. The extents of DNA synthesis were quantitated according to the experimental procedures. 176 Table 4 The activating factor is trypsin sensitive t ._‘ to-m F 'n I in than IN‘ hi . l + - - 20 + + buffer 201 + + try/ti 4 + + ti/try 119 Fraction 1H (15 ul) was treated with Buffer A only, 2.5 ug of trypsin (try) or 2.5 ug of trypsin inhibitor (ti) and incubated at 30°C for 10 minutes. The reactions were then treated, respectively, with Buffer A only, 2.5 ug of trypsin inhibitor, or 2.5 ug of trypsin and incubated at 30°C for 10 minutes. The indicated amounts of the treated fractions were added to replication reactions containing purified enzymes and 90 ng of dnaA5 protein. The reactions were incubated at 30°C for 60 minutes and the extents of DNA synthesis were quantitated according to the experimental procedures. 177 Table 5 The second activating protein is heat sensitive F u in) tro in _: in _o-_nn - tr :14- DN‘ . nhi toml - - - 48 IHa 12 55°C 197 IIIb 12 60°C 169 HIc 12 65°C 95 um 12 70°C 46 WM433 Fraction H was diluted two fold with Buffer A and portions were incubated at the indicated temperatures for 7 minutes. The insoluble material was removed by centrifugation at 12, 000 x g for 5 minutes. The indicated components were added to replication reactions containing purified enzymes, 90 ng of dnaA5 protein, and 2 ug of dnaK protein. The reactions were incubated at 30°C for 60 minutes and the extents of DNA synthesis were quantitated according to the experimental procedures. Table 6 178 The partial purification of the second activating protein Fraction volume protein units specific activity unit yield fold purification (ml) (mg) x 103 (units/mg) (%) I 62.0 1220 ND. ND. N .D. ND. 11 7.50 795 48 60 ND ND 111 29.0 272 369 1354 (100) 22 IV 16.5 64.4 286 4440 77 74 V* 66.0 38.1 267 6915 67 115 * 1/33 of fraction IV was used to produce Fraction V and the values obtained were corrected for by a factor of 33. WM433 Fraction H was diluted two-fold with Buffer A, DNase I was added to a final concen- tration of 0.01 ug/ml, and an incubation was performed at 37°C for 20 minutes. The inclusion of DNase I resulted in the degradation of contaminating DNA that interfered with the replication assay after Mono Q chromatography (data not shown). The fraction was incubated at 55°C for 7 minutes, followed by centrifugation to remove insoluble material. An additional heat step was incorporated to remove additional contaminating protein as well as to inactivate residual DNase I. The resultant soluble fraction, Fraction HI, was diluted to a conductivity equivalent to 50 mM KCl in Buffer A and applied to a Mono Q column (8 cm x 1 cm) equilibrated in the same buffer. Bound protein was eluted with a twenty ml linear gradient extending from 50 mM KCl to 600 mM KCl in Buffer A. Every fifth fraction was assayed in the reconstituted enzyme system con- taining dnaA5 protein and 2 ug of dnaK protein. The protein and activity profiles are summa- 179 rized in Figure 4. The activity eluted between 250 mM and 350 mM KCl. A 90% yield of activ- ity was obtained (Fraction IV) with a 3-4 fold purification. The second activating protein did not bind to DNA cellulose, heparin agarose, Mono 8, or Blue Dextran agarose (data not shown). Gel filtration was performed in order to determine the molecular weight of the protein as well as to aid in its purification. Fraction number 25 from Mono Q was injected onto a Superose 12 gel fil- tration column equilibrated in Buffer A containing 250 mM KCl. The activity profile is shown in Figure 5A. The fractions containing activity are not highly enriched for one particular poly- peptide (Figure 6) and the purification was only two-fold compared to the starting material. The recovery of units was 85% (Fraction V) and the activity detected was also dependent upon exog- enousely added dnaK protein (data not shown). DISCUSSION dnaA5 protein is inactive in replication systems containing purified enzymes (19). By contrast, it is active in a replication system employing a crude enzyme fraction suggesting that an activating factor in the crude enzyme fiaction is absent in the reconstituted enzyme system. The extended lag prewding DNA replication dependent on dnaA5 protein in the crude enzyme system may reflect the time required for this activation to occur (19). An assay was developed which detects the ability of a factor from a crude enzyme fraction to activate dnaA5 protein in the replication system containing purified enzymes. dnaA protein involvement in the in vitro replication of oriC plasmids was that demonstrated using crude enzyme fractions obtained from dnaA mutants (32). A feature of this system was the requirement for an ATP regenerating system and PVA. Such crude enzyme fractions, deficient in dnaA protein activity, could support bidirectional initiation of DNA replication fiom oriC upon the addition of dnaA protein (33); this was used as an assay to purify dnaA protein (34). Reconstitution of the replication system containing a crude enzyme fraction into purified components led to the formation of a system composed of 12 highly purified enzyme fractions, including RNA polymerase, topoisomerase I, and RNase H (23). This reconstituted enzyme system supports oriC plasmid replication dependent upon dnaA protein. Simplification of this replication system dependent upon RNA polymerase has led to the discovery of an RNA polymerase independent replication system, where primase is the sole priming enzyme (30, 31). Further refinement resulted in the discovery that dnaA protein functions to unwind the double helix at A-T rich regions in the vicinity of oriC (4), facilitating the formation of a pre-priming intermediate including dnaB and dnaC proteins (3). The replication system containing RNA polymerase, t0poisomerase I, and RNase H is thought to be more physiologically relevant (28). The influence of these proteins on dnaA 1 8 0 181 protein activity is not completely understood. A transcriptional event by RNA polymerase is thought to alter the topology of DNA through transcriptional activation (35). This topological alteration may facilitate dnaA protein dependent unwinding of oriC. Topoisomerase I and RNase H maintain oriC dependence by preventing nonspecific transcription that leads to oriC independent initiation of DNA synthesis (23). The activation of dnaA5 protein was not observed in RNA polymerase dependent replication assays in the absence of PVA, in replication systems where primase is the sole priming enzyme, or in oriC unwinding assays ( FI* formation) (unpublished data). The assay used to detect activation of dnaA5 protein was the RNA polymerase dependent replication system containing PVA. The requirement for RNA polymerase in the replication reaction may indicate that the activated form of dnaA5 protein requires RNA polymerase to facilitate unwinding of oriC. grpE and dnaK heat shock proteins are both required for activation of dnaA5 protein replication activity. Initially, purification of the activator was unsuccessful. The demonstration that one of the activators was dnaK protein allowed for the partial purification of the second factor, subsequently shown to be grpE protein. The observation that the second protein required for the activation of dnaA5 protein was grpE protein was deduced from the following evidence: 1) a 24 Rd protein, isolated by gel filtration and resolved in a SDS polyacrylamide gel stained with Coomasie Blue, was coincident with activity, 2) the active fractions obtained by gel filtration were coincident with a protein that cross reacted to grpE antibody and 3) purified fractions of grpE protein replaced a Superose 12 fiaction which activated dnaA5 protein with a similar efficiency. The heat shock proteins dnaK, dnaJ, and grpE (36) function at an early step in lambda phage DNA replication (37-39). The dnaK protein involvement in initiation of lambda DNA replication occurs at a stage after the formation of a lambda O-lambda P-dnaB-dnaJ initiation complex at the origin (4044). Its action results in the displacement of lambda P protein from dnaB protein (46-49), permitting dnaB protein to function as a helicase. grpE 182 protein is dispensable in the in vitro lambda replication system composed of purified enzymes as its role is to reduce the levels of dnaK protein required by 10-fold (45). grpE and dnaK proteins form a complex (54) that is dissociated by ATP (29). The levels of ATP present in the lambda replication reaction are sufficiently high to maintain them in the dissociated state. grpE protein may function to reduce the level of dnaK protein required for initiation of lambda DNA replication by sequestering free lambda P protein to prevent its reassociation with dnaB helicase (50). In the absence of grpE protein, the elevated levels of dnaK protein may be required to, similarly, prevent reassociation of lambda P protein with the dnaB helicase. The activation of dnaA5 protein absolutely requires both grpE and dnaK protein; the addition of either protein alone, even up to 10 ug of dnaK protein (unpublished data), results in no dnaA5 protein activation. This seems to differ from the involvement of dnaK and grpE proteins in lambda DNA replication, as dnaK protein can function in the absence of grpE protein. The activation of dnaA5 protein may proceed through a readily reversible process during which dnaA5 protein can assume the inactive form. dnaK protein alone may be unable to prevent the reaction reversal, and by analogy to the lambda DNA replication system, grpE protein may prevent the activation reaction from reversing and thus maintain dnaA5 protein in its active state. grpE and dnaK mutants are temperature sensitive for E. coli growth (51, 52) demonstrating their essential role in cellular metabolism. A dnaK mutant defective in initiation of DNA replication (22) indicates its participation in this process in vivo. Until recently, no direct biochemical evidence existed which demonstrated a role for dnaK protein in oriC plasmid replication in vitro. A form of wild type dnaA protein that is inactive in oriC DNA replication systems composed of purified enzymes, elutes as an aggregate upon gel filtration (34) and contains elevated levels of phospholipid (53). This form of dnaA protein becomes active upon incubation with dnaK protein or phospholipase A2, indicating that dnaK protein may regulate the pool of dnaA protein in the cell by 183 converting it from the aggregate to the monomeric state by the dissociation of phospholipid (53). dnaA5 protein was not activated in replication by phospholipase A2 (data not shown), indicating the mechanism of activation may be different from the activation of the aggregated form of dnaA protein . The physiological defects associated with dnaA5 cells may relate to the activation of dnaA5 protein by heat shock proteins. The mechanism involving activation and its relation to physiological defects will be discussed in Chapter V (24). y—s 99:5?!" ." 10. ll. 12. 13. 14. 15. l6. l7. 18. 19. 20. 21. 22. 23. REFERENCES . Sekimizu, K., Bramhill, D., and Kornberg, A. (1988) Cell 50: 259-265. Fuller, R.S., Funnell, B.B., and Kornberg, A. (1984) Cell 38: 889-900. Baker, T.A., Sekimizu, K., Funnell, B.B., and Kornberg, A. (1986) Cell 45: 53-64. Bramhill, D. and Kornberg, A. (1988) Cell 52: 743-755. Yung, B.Y. and Kornberg, A. (1988) J. Biol. Chem. 264: 6146-6150. Sekimizu, K., Bramhill, D., and Kornberg, A. (1988) J. Biol. Chem. 263: 7124- 7130. Sekimizu, K. and Kornberg, A. (1988) J. Biol. Chem. 263: 7131-7135. Jacq, A., Kern, R., Tsugita, A., and Kohiyama, M. (1989) J. Bact. 171: 1409-1414. Yung, B.Y. and Kornberg, A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 7202-7205. Donachie, WD. (1968) Nature 219: 1077-1079. Hansen, F.G. and Rasmussen, K.V. (1977) Mal. Gen. Genet. 155: 219-225. Sakakibara, Y, and Yuasa, S. (1982) Mol. Gen. Genet. 186: 87-94. Chiaramello, A. and Zyskind, J.W. (1989) J. Bact. 171: 4272-4280. Skartstad, K,. Lobner-Olesen, A., Atlung, T., and von Meyenburg, K. (1989) Mol. Gen. Genet. 218: 50-56. Atlung, T., Lobner-Olesen, A., and Hansen, F.G. (1987) Mol. Gen. Genet. 206: 51-59. Hansen, B.B., Atling, T., Hansen, F.G., Skovgaard, 0., and von Meyenburg, K. (1984) Mol. Gen. Genet. 196: 387-396. Skarstad, K., Steen, H.B., and Boye, E. (1985) J. Bact. 163: 661-668. Hwang, D.S. and Kaguni, J.M. (1988) J. Biol. Chem. 263: 10625-10632. Hupp, T., Ph. D. Thesis, Chapter HI. Hwang, D.S. and Kaguni, J.M. (1988) J. Biol. Chem. 263: 10633-10640. Hwang, D.S., Carr, K.M., and Kaguni, J .M. (1990) manuscript in preparation. Sakakibara, Y. (1988) J. Bact. 170: 972-979. Kaguni, J.M. and Kornberg, A. (1984) Cell 38: 183-190. 1 8 4 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. . Ander, LA. and Klein, A. Nucl. Acids Res. 10: 1733-1740. 41. 42. 43. 185 Hupp, T., Ph. D. thesis, Chapter V. Zylicz, M. and Georgopoulos, C. (1984) J. Biol. Chem. 259: 8820-8 825. Lipinska, B., King, J., Ang, D., Georgopoulos, C. (1988) Nucl. Acids Res. 16: 7545-7562. Harlow, E. and Lane, D. (1988) Antibodies Cold Spring Harbor Laboratory. Cold Spring Harbor, New York. Ogawa, T. and Okazaki, T. (1984) Mol. Gen. Genet. 193: 231-237. Zylicz, M., Ang, D., and Georgopoulos, C. (1987) J. Bial.Chem. 262: 17437- 17442. van der Ende, A., Baker, T.A., Ogawa, T., and Kornberg, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 4078-4084. Ogawa, T., Baker, T.A., van der Ende, A., and Kornberg, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 3536-3566. Fuller, R.S., Kaguni, J.M., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U.S.A.78: 7370-7374. Kaguni, J.M., Fuller, R.S., and Kornberg, A. (1982) Nature 296: 623-627. Fuller, RS. and Kornberg, A. (1983) Proc. Natl. Acad. Sci. USA. 80: 5817-5820. Baker, T.A. and Kornberg, A. (1988) Cell 55: 113-123. Neidhart, EC. and VanBogelen, RA. (1982) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology American Society for Microbiology, Washington. Georgopoulos, C. (1977) Mol. Gen. Genet. 151: 35-39. Saito, H. and Uchida, H. (1977) J. Mol. Biol. 113: 1-25. Saito, H. and Uchida, H. (1978) Mol. Gen. Genet. 164: 1-8. Tsurimoto, T. and Matsubora, K. (1982) Proc. Natl. Acad. Sci. USA. 79: 7639- 7643. Wold,M., Mallory, J., Roberts, J., LeBowitz, J., and McMacken, R. (1982) Proc. Natl. Acad. Sci. USA. 79: 6176—6180. LeBowitz, J.H., Zylicz, M., Georgopoulos, C., and McMacken, R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82: 4678-4682. Dodson, M., Echols, H., Wickner, S., Alfano, C., Mensa-Wilmot, K., Games, B., LeBowitz, J ., Roberts, J.D., and McMacken, R. (1986) Proc. Natl. Acad. Sci. 45. 47. 48. 49. 51. 52. 53. 54. 1 8 6 U.S.A. 83: 7638-7642. Zylicz, M., Ang, D., Libereck, K., and Georgopoulos, C. (1989) The EMBO Journal 8: 1601-1608. Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264: 10709-10718. Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264: 10719- 10725. Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264: 10699-100708. Libereck, K., Georgopoulos, C., and Zylicz, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85 : 6632-6636. Zylicz, M., Ang, D., Libereck, K., Yamamoto, T., and Georgopoulos, C. (1988) Biachim Biophys. Acta 951: 344-350. Bukav, B. and Walker, G.C. (1989) J. Bact. 171: 6030-6038. Ang, D. and Georgopoulos, C. (1989) J. Bact. 171: 2748-2755. Hwang, D.S. and Kornberg, A., personal communication. Johnson, C., Chandrasakhar, G.N., and Georgopoulos, C. (1989) J. Bact. 171: 1590-1596. Chapter V THE Escherichia coli dnaA5 PROTEIN IS ACTIVATED BY grpE AND dnaK HEAT SHOCK PROTEINS PRIOR TO ITS INTERACTION W'ITH oriC DNA 187 ABSTRACT The replication activity of dnaA5 protein in a reconstituted enzyme system is restored through the combined actions of grpE and dnaK heat shock proteins (Chapter IV). The activation was divided into two stages which separated the activation event from DNA synthesis. The minimal components necessary during Stage I for the efficient activation of dnaA5 protein included: ATP at concentrations greater than 0.25 mM, PVA at levels of 8% or glycerol at concentrations of at least 10%, 50 ng to 100 ng of grpE protein, and dnaK protein at concentrations of at least 0.1 mg/ml. The subsequent addition of the activated form of dnaA5 protein to the reconstituted enzyme system (Stage H) resulted in high levels of replication with a lag preceding DNA synthesis characteristic to that of wild type dnaA protein. Although grpE and dnaJ proteins stimulate the ATPase activity of dnaK protein ten-fold, the presence of grpE and dnaA5 proteins had no effect on its ATPase activity. Using a monoclonal antibody that differentiates dnaA5 from dnaA protein a conformational change in dnaA5 protein was detected in the ATP dependent activation reaction. The interaction between dnaA5 protein and the heat shock proteins was thermolabile suggesting that the thermolability of dnaA5 cells may be related to the interaction of the dnaA5 gene product with the heat shock proteins. 188 INTRODUCTION The initiation of DNA replication, in viva (1) and in vitro (2), requires the function of the dnaA initiator protein. dnaA protein functions at an early stage in the initiation of replication of oriC plasmids, during which duplex DNA in the vicinity of oriC is converted to a single-stranded form (3). This intermediate is capable of supporting RNA priming and DNA synthesis (4). The individual biochemical properties of dnaA protein required for its replication activity include ATP binding (5) and sequence specific DNA binding to oriC (6). The dnaA dependent initiation of replication occurs at a specific time within the cell cycle (7) indicating that chromosomal DNA replication is tightly coupled to cell growth. It is not known if or how the replication activity of dnaA protein is modulated in viva, but accumulating evidence suggests that it may be under some form of regulation. dnaA protein bound to ADP is inactive in replication (8). The regulation of dnaA protein activity in viva has been proposed to occur through the phospholipid reactivation of the ADP form of dnaA protein (9). The concentration of dnaA protein affects the initiation of replication in vivo in that overproduction of dnaA protein from expression vectors containing the dnaA gene increases the rate of initiation of DNA synthesis (10), suggesting that dnaA protein is the rate-limiting factor in initiation. However, other rate-limitin g factors may exist as these initiation events are abortive (11). The concentration of dnaA protein per cell increases linearly with the growth rate (12), possibly on the demand for increasing the rate of initiation events in more rapidly growing cells. Clearly, there is a correlation between the levels of dnaA protein in the cell and the frequency of initiations, but the mechanism responsible for the coupling of dnaA protein activity in chromosomal DNA replication to cell growth is not known. Cells harboring mutations in dnaA, including dnaA5 and dnaA46, are temperature sensitive (13) and are asynchronous in the initiation of replication (14). An understanding 189 190 of the biochemical deficiencies associated with mutant forms of dnaA protein may allow for a greater understanding of initiation of DNA replication and its regulation. For this reason, mutant forms of dnaA protein, including dnaA5 (15) and dnaA46 (17, 18), have been purified and characterized in comparison to wild type dnaA protein. These mutant forms of dnaA protein are unable to bind ATP and interact inefficiently with oriC DNA resulting in their inability to initiate DNA synthesis in replication systems composed of purified enzymes (15, 17, 18). However, dnaA5 and dnaA46 proteins were observed to be active in replication systems employing a crude enzyme fraction and it was thought that components in the crude fractions were able to restore replication activity to the defective proteins. This hypothesis was confirmed by designing an assay which measured the activation of dnaA5 protein in replication systems composed of purified enzymes (16). Further purification of these activators has demonstrated that two heat shock proteins, grpE and dnaK, are required for the activation of dnaA5 protein. DNA replication initiating fiom the larrrbda origin, ariL, requires phage encoded lambda O and P proteins (19, 20) and host encoded heat shock proteins including grpE, dnaJ, and dnaK (21). Biochemical studies on the replication of plasmids containing ariL has resulted in the reconstitution of the enzymes and proteins required for the process. The lambda O initiator protein binds to its consensus sequence within ml and unwinds the duplex DNA in the vicinity of the origin (22). Lambda P protein interacts with lambda 0 protein and forms a complex with dnaB protein at the lambda origin (23). The requirement for dnaJ and dnaK heat shock proteins occurs after events leading to the formation of a lambda O- lambda P-dnaB nucleoprotein complex at ariL. In the presence of dnaJ protein, dnaK protein dissociates the lambda P protein fiom the initiation complex (24) in an ATP dependent manner to activate the helicase activity of dnaB protein (25). grpE protein is dispensable in this replication system containing purified enzymes (21) as its function is to reduce the level of dnaK protein required for initiation of DNA replication (26). 191 The identification of a dnaK mutant which is conditionally defective in initiation of DNA replication in viva (27) indicates that dnaK protein is involved in the initiation of replication from oriC. The demonstration that dnaK and grpE proteins are required for activation of dnaA5 protein in oriC plasmid replication (16) provides a new system for the study of the function of heat shock proteins and a possible direction in understanding the regulation of DNA replication. This study extends the characterization of the heat shock protein mediated activation of dnaA5 protein by providing evidence that dnaK and grpE proteins function prior to dnaA5 protein association with oriC DNA. This interaction is temperature sensitive indicating that the thermolability in cell growth is related to the interaction between the dnaA5 gene product and the dnaK and grpE gene products and not with an inherent instability of dnaA5 protein at elevated temperatures. The timing defect associated with dnaA5 and dnaA46 cells may be related to the inherent inactivity of the mutant initiator protein in initiating DNA replication. A rate limiting reaction involving the grpE and dnaK heat shock proteins may be required for the accumulation of initiation potential in viva giving rise to an asynchronous initiation phenotype. EXPERIMENTAL PROCEDURES Reagents and enzymes. Commercial enzymes, solvents, chemicals, and radioactive isotopes were obtained as described (15). Polyethyleneimine TLC plates were obtained from Macherey-Nagel. dnaJ protein was a gift of Dr. M. Zylicz, University of Utah. DNA replication assays. RNA polymerase dependent oriC plasmid replication assays which include PVA at 8% (w/v)were performed as previously described (16). The protein fractions used include; dnaA protein (fraction v, 2 x 10 5 units/mg) (17); dnaA5 protein (fiaction H1, (15)); dnaK protein (fraction IV, (16)); and grpE protein (fraction IH, (16)). Staging activation. The activation of dnaA5 protein in the RNA polymerase dependent replication system containing PVA was divided into two stages. Stage I contained the following components in a 5 111 volume: PVA at 8%, 40 mM HEPES-KOH (pH 7.6), 40 mM creatine phosphate, 0.4 mg/ml creatine kinase, 2 mM ATP, 10 mM magnesium acetate and, unless otherwise indicated, 100 ng of dnaA5 protein (fraction IH or IV, (15)), 2 ug of dnaK protein (fraction IV, (16)), and 100 ng of grpE protein (fiaction IH, (16)). The reactions were incubated at 30°C for one hour, unless otherwise indicated. During Stage I incubations, Stage H components were assembled at 0°C in a 20 ul volume and contained the following reagents: PVA (8%) (w/v), 40 mM HEPES-KOH (pH 7.6), 40 mM creatine phosphate, 2 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 0.5 mM UTP, 1 mM dNTP (3H- dNTP at 20 to 40 cpm/pmol nucleotide), 0.4 mg/ml creatine kinase, 10 mM magnesium acetate, 200 ng of M130riC2LB5 plasmid, and purified replication enzymes and proteins as described in Chapter H1 (15). After Stage I, the Stage I reactions were placed on ice and 20 ul of a mixture containing Stage H components were added. The incubations were continued at 30°C for 25 minutes unless otherwise indicated. The extents of DNA synthesis were determined according to published procedures (15). ATPase assays. The methods used for assembling the reactions were previously described (47 ) and are indicated in Figure 7. The products of the reaction were chromatographed on 1 9 2 193 polyetlryleneimine TLC plates by spotting a one 111 aliquot onto the origin, drying under a heat lamp, and developing the chromatogram in 1 M Formate I 500 mM LiCl. After the TLC plates were dried, the positions of the radioactive species were visualized by exposing the plates to X-ray film. The radioactive products were excised and the radioactivity was quantitated by liquid scintillation counting. Immunological methods. Monoclonal antibodies to dnaA protein were produced (data not shown) and purified on protein A Superose resin according to standard procedures (46). Quantitating the levels of monoclonal antibody bound to its antigen in an ELISA was performed by the incubation of a 1:1000 dilution of goat anti-mouse HRP (Biorad) in PBS buffer containing 0.05% tween 20 and 0.2% BSA for one hour at room temperature. Following four washes with PBS containing 0.05% tween 20, the levels of goat anti- mouse HRP present were quantitated as previously described (46). RESULTS The time course of DNA synthesis in activation reactions. The activation of dnaA5 protein in replication depends upon grpE and dnaK proteins (Chapter IV (16)). This coupled assay measures the activation of dnaA5 protein as detected by its ability to initiate DNA synthesis dependent upon other replication enzymes. An extended time course of activation (Figure 1A) revealed that a lag of about 30 minutes preceded DNA synthesis, then proceeded linearly for up to 100 minutes. Presumably, the 30 minute lag preceding DNA synthesis reflects the time required for activation of dnaA5 protein by the combined action of grpE and dnaK proteins. This extended lag is not characteristic of DNA synthesis initiated by dnaA protein (2). The initiation of DNA synthesis dependent upon dnaA protein (Figure 1B) occuned much earlier in this replication system, which is expected, since it is thought that dnaA protein is in an active form when isolated from the cell and does not need grpE and dnaK proteins for activity. Once replication was initiated by both the dnaA protein dependent and the dnaA5 protein dependent systems, DNA synthesis proceeded linearly for up to 60 minutes. Staging the activation of dnaA5 protein. The extended lag required for dnaA5 protein to initiate DNA synthesis in a replication system composed of purified enzymes (Figure 1A) may reflect the time required for activation to occur. Presumably, the assay measures two events that are independent; the activation of dnaA5 protein by the heat shock proteins and subsequent initiation of replication catalyzed by the activated form of dnaA5 protein. The reaction was initially divided into two stages which involved the incubation of dnaA5 protein with a presumed minimal complement of proteins and reaction substrates. In the first stage (Stage I), dnaA5 protein was incubated with grpE and dnaK proteins at 30°C for 1 hour in a 5 ul reaction mixture as described in the Experimental Procedures. The second stage (Stage H) involved 1 9 4 195 the addition of a 20 ul reaction mixture containing oriC DNA, 3H-dNTP, and replication enzymes to the Stage I mixture followed by incubations at 30°C for 30 minutes. The results are summarized in Table 1A. Control reactions containing only dnaA5 protein, or a sub-set of the three proteins (dnaA5, grpE and dnaK), showed that DNA synthesis did not occur. The addition of dnaA5, grpE, and dnaK proteins to Stage I resulted in high levels of DNA synthesis. The control reactions which involved the omission of dnaA5, grpE and dnaK proteins in Stage I but their addition to Stage H did not result in DNA synthesis. This is expected since the activation of dnaA5 protein in the coupled system requires that at least 30 rrrinutes elapse before any detectable DNA synthesis occurs (Figure 1 A). These results indicate that grpE and dnaK proteins activate dnaA5 protein at a stage preceding binding to oriC DNA and can occur in the absence of general replication enzymes. It is possible that one of the two heat shock proteins function during Stage I and the other functions during an early part of Stage H. To demonstrate that grpE and dnaK proteins are both required during Stage 1, experiments were performed which involved the incubation of dnaA5 protein with one of the two heat shock proteins in Stage I and subsequent addition of the omitted component to Stage H (Table 1B). The incubation of dnaA5 protein with grpE or dnaK proteins in a Stage I reaction mixture and subsequent addition of either dnaK or grpE proteins to Stage H, respectively, did not result in DNA synthesis. The control reaction in which all three components were incubated together dming Stage 1 resulted in high levels of DNA synthesis. These results indicate that one of the two heat shock proteins cannot function individually during Stage I, but both grpE and dnaK proteins are required to observe activation of dnaA5 protein. The time course of the staged activation of dnaA5 protein. To determine the time course of DNA synthesis (Stage II), dnaA5, grpE and dnaK proteins were incubated in a Stage I reaction mixture at 30°C for 60 minutes, the time required for efficient activation of dnaA5 protein. Stage 11 components were added to the 196 Stage I reactions and the incubations were performed at 30°C from 0 to 60 minutes (Figure 2A). A lag of 5 minutes preceded DNA synthesis, which is linear for up to 20 minutes. This short lag phase preceding DNA synthesis is similar to that observed with dnaA protein in the same replication system (Figme 1B) (2). The 30 to 40 nrinute reduction in the lag phase preceding dnaA5 protein dependent initiation of DNA synthesis in the staged system compared to the coupled activation reaction (Figrne 1B) demonstrates that efficient and complete activation of dnaA5 protein can occur before its interaction with the general set of replication enzymes and oriC DNA. The extents of DNA synthesis observed also indicate that the specific activity of the activated form of dnaA5 protein is similar to dnaA protein. The time required for the activation of dnaA5 protein to occur during Stage I is described in Figure 2B. dnaA5 protein was incubated with dnaK and grpE proteins at 30°C in a Stage I reaction mixture for the indicated times, after which Stage H components were added and incubations were continued at 30°C for 25 minutes. The activation of dnaA5 protein is linear from 10 to 30 minutes with a low level of increased activity observed with extended times of incubation. dnaK protein exhibits a concentration dependence in staged activation reactions. The optimal amounts of dnaK and grpE proteins required for dnaA5 protein activation in the coupled activation reaction (Chapter IV, (16)) were 5 ug and 120 ng, respectively. The reduction in volume from 25 111 in the coupled activation reaction to 5 ul in Stage I reactions may result in a change in the levels of the heat shock proteins required for activation to proceed. Preliminary experiments demonstrated that five fold less dnaK protein was required for the activation of dnaA5 protein in the staged activation reaction (data not shown). Stage I incubations are performed in a 5 111 volume, which is a five fold lower volume than that used during the coupled activation reaction. Therefore, the observation that five fold less dnaK protein was required during the staged activation of dnaA5 protein may be due to the five fold volume reduction. 197 The replication observed after the titration of dnaK protein into differing volumes of a Stage I reaction and subsequent addition to Stage H is shown in Figure 3A. Maximal activation of dnaA5 protein during Stage I required only 0.5 ug of dnaK protein in 2.5 ul reaction volumes. Doubling the volumes of the Stage I reaction up to 5 111 and 10 ul resulted in increasing amounts of dnaK protein required for the activation of dnaA5 protein. This differs from the amount of dnaK protein required during the coupled activation reaction up to as much as 10 fold. Five ug of dnaK protein are required for maximal activation of dnaA5 protein in the coupled activation reaction (Chapter IV, (16)), while as little as 0.5 ug are required for maximal activation in the staged activation reactions. Under these conditions, there is a decrease in the ratio of dnaK protein to dnaA5 protein from 40:1 to 4:1. These data indicate that the relative amount of dnaK protein required for the activation of dnaA5 protein is not critical, but that the concentration may be important. grpE protein is required in staichiametric levels during the activation of dnaA5 protein. The amount of grpE protein required for the staged activation reaction is shown in Figure 3B. grpE protein dependence was observed from 30 ng to 120 ng of protein added. This level of grpE protein required for the activation of dnaA5 protein is the same as that required in the coupled activation reaction (Chapter IV, (16)). This indicates that the relative molar quantity of grpE protein is essential for activation and is independent of the reaction volume. The amounts of grpE protein required for maximal activation of dnaA5 protein suggest that two molecules of grpE protein are required for the activation of one molecule of dnaA5 protein. The temperature dependence of the activation reaction. dnaA5 protein is thermolabile in initiation of DNA synthesis in a replication system containing a crude enzyme fraction (15) and in viva (13). The replication activity of dnaA5 protein depends upon dnaK and grpE proteins in systems containing purified enzymes. It 198 was of interest to determine whether the thermolabile defect of dnaA5 protein was during Stage I in the interaction with grpE and dnaK proteins or whether it became manifest during Stage H when dnaA5 protein functioned in replication. The complete Stage I activation reaction (containing dnaA5, grpE, and dnaK proteins), was incubated for one hour at temperatures ranging from 25°C to 38°C followed by Stage H incubations at 30°C for 25 minutes. Increasing the temperature of the Stage I incubations resulted in decreasing replication activity observed during Stage H (Figure 4A). This suggests that the interaction between dnaA5 protein and the heat shock proteins is temperature sensitive. If the above conclusion is valid, then the activated form of dnaA5 protein should become thermally insensitive. The complete Stage I activation reaction (containing dnaA5, grpE, and dnaK proteins), was incubated at 30°C for one hour, during which complete activation of dnaA5 protein should occur. Aliquots from the Stage I reactions were titrated into Stage H reactions and incubations were performed at 30°C and 38°C for 25 minutes (Figure 4B). The activated form of dnaA5 protein resulted in normal extents of DNA synthesis at 30°C and reasonably high levels of replication activity were observed at 38°C. Although the specific activity of dnaA5 protein was reduced three fold at 38°C compared to 30°C, there was substantial replication activity at elevated temperatmes, confirrnin g that the thermolabile deficiency is in the interaction between dnaA5 protein and the heat shock proteins. The influence afsalvents an the activation reaction. The activation reaction, whether staged or coupled, has included 8% PVA. The initial inclusion of PVA resulted from the knowledge that activation of dnaA5 protein could not be detected in its absence (data not shown). The replacement of PVA by solvents known to stabilize proteins, like glycerol, was initiated in order to understand the affects of solvents on the activation of dnaA5 protein. Although less efficient than PVA, glycerol at 20% 199 could replace PVA in Stage I (Table 2). The activation of dnaA5 protein in the presence of 20% glycerol was dependent upon grpE and dnaK proteins. The addition of glycerol in Stage I reactions resulted in background levels of DNA synthesis at concentrations less than or equal to 5% (Figure 5). DNA synthesis increased linearly at levels of glycerol ranging from 10 % to 30%, demonstrating the requirement of stabilizing solvents during Stage I. Attempts at replacing PVA in Stage H have not been successful (Table 3). dnaA5 protein, activated during Stage I reactions which contain PVA or glycerol was active in Stage H reactions containing PVA (Table 3, experiments 1 and 2). However, dnaA5 protein that was activated in Stage I reactions containing PVA or glycerol was not able to initiate DNA synthesis in Stage H reactions in the absence of PVA (Table 3, experiments 3 and 4). There is some labile event, yet to be understood, which requires the inclusion of PVA during the initiation of DNA replication by the activated form of dnaA5 protein. ATP is required for the activation of dnaA5 protein. To determine whether ATP was required during activation, Stage I reactions were assembled according to the Experimental Procedm'es in the presence of various concentrations of ATP (Figure 6). In the absence of ATP, no activation was observed indicating that the reaction requires ATP. The maximal concentrations of ATP required for efficient activation were from 0.5 mM to 1 mM. The ATPase activity of dnaK protein is not stimulated by dnaA5 protein. The heat shock proteins, dnaJ and grpE, are both required to stimulate the ATPase activity of dnaK protein up to 10 fold (46), while grpE or dual proteins alone do not influence the ATPase activity of dnaK protein. The ATPase activity of uncoating ATPase, a 70 kd heat shock protein analogous to dnaK protein, is stimulated when clathrin is bound to coated vesicles. This indicates that a specific conformation is recognized by uncoating ATPase 200 before catalyzing protein disassembly (34, 35). By analogy, it is possible that dnaA5 protein serves as a substrate for dnaK protein to stimulate its ATPase activity. The effects of grpE and dnaJ proteins on the stimulation of the ATPase activity of dnaK protein, under conditions used during Stage I incubations, is shown in Figure 7A. The incubation of either grpE or dnaJ proteins with dnaK protein resulted in the level of ATPase activity similar to that catalyzed by dnaK protein alone. ATPase activity was not detectable with grpE and dnaJ proteins in the absence of dnaK protein. In contrast, the presence of both grpE and dnaJ proteins stimulated the rate of ATP hydrolysis by dnaK protein. The effects of dnaA5 protein on the ATPase activity of dnaK protein is shown in Figure 7B. dnaA5 protein alone did not exhibit ATPase activity and its addition to dnaK protein did not stimulate the ATPase activity of dnaK protein. grpE protein is also essential for activation, yet dnaA5 and grpE proteins did not stimulate the ATPase activity of dnaK protein. This suggests that the mechanism of dnaK, dnaJ, and grpE protein dependent disassembly of the ariL initiation complex may be different from the dnaK and grpE protein dependent activation of dnaA5 protein. dnaA5 protein and dnaA protein react differentially with monoclonal antibodies. Monoclonal antibodies that recognize dnaA protein were produced by standard procedures (unpublished results). An ELISA was performed to determine if dnaA5 protein exhibited a differential reactivity with a particular monoclonal antibody, when compared to dnaA protein. This would indicate a conformational alteration is associated with the mutant protein. The affinity of monoclonal antibody, M1, for dnaA and dnaA5 proteins was determined using an ELISA (Figure 8A). The monoclonal antibody, M1, had a similar affinity to dnaA and dnaA5 proteins, indicating that the epitope recognized by M1 is apparently identical in both proteins. In contrast, the monoclonal antibody, A4, exhibited a differential affinity to dnaA5 protein in comparison to dnaA protein. The monoclonal antibody, A4, reacted very 201 weakly with dnaA protein under these conditions, while it was five to ten-fold more reactive to dnaA5 protein. Presumably, there is a conformational change in dnaA5 protein that results in a greater affinity of A4 to its epitope. The activation of dnaA5 protein produces a conformational change in the protein. A prediction derived from these studies on the activation of dnaA5 protein is that the heat shock proteins can induce a conformational change in dnaA5 protein to allow it to assume a conformation more similar to its wild type counterpart. The monoclonal antibody, A4, exhibits a higher affinity for dnaA5 protein than dnaA protein in an ELISA (Figure 8A). If dnaA5 protein undergoes a conformational change after activation, then it may assume a conformation more closely related to dnaA protein and may bind with a lower affinity to monoclonal antibody, A4. As the activation of dnaA5 protein requires ATP (Figure 6), Stage I reactions (containing dnaA5, grpE and dnaK proteins) were assembled in the presence and absence of ATP and incubated at 30°C for 30 minutes. Presumably, the omission of ATP in Stage I would prevent the heat shock protein dependent activation of dnaA5 protein. An ELISA was performed on the activated and inactivated forms of dnaA5 protein using monoclonal antibody A4 (Figure 8B). In the presence of ATP, during which the presumed conformational change associated with activation of dnaA5 protein occurs, a decrease in the affinity of A4 to its epitope was observed relative to dnaA5 protein under similar conditions but in the absence of ATP. This suggests that a conformational change is induced within dnaA5 protein after activation and that this conformation is more closely related to dnaA protein resulting in a lower affinity to monoclonal antibody, A4. 202 Figure 1. (A). Time course of activation of dnaA5 protein. DNA replication reactions containing purified enzymes, 100 ng of dnaA5 protein, 2 ug of dnaK protein and 100 ng of grpE protein were assembled according to the experimental procedures. (B). Time course of DNA synthesis dependent upon dnaA protein. DNA replication reactions containing purified enzymes and 90 ng of dnaA protein were assembled according to the experimental procedures. Incubations were per- formed at 30°C for the indicated times and the extents of DNA synthesis were quantitated ac- cording to the experimental procedures. -, “‘5‘; their» 'L’ DNA Synthesis (pmol) DNA Synthesis (pmol) 400 300 200 203 500 100 o l l l l . l 0 20 40 60 80 100 120 Time (minutes) 200 150 100 50 o I l l 0 20 40 60 Time (minutes) 204 Figure 2 . Time course of staged activation of dnaA5 protein. (A). Stage I reactions containing 100 ng of dnaA5 protein, 2 ug of dnaK protein, and 100 ng of grpE protein were assembled ac- cording to the experimental procedures. The reactions were incubated at 30°C for the indicated times and, after the addition of Stage H components, the reactions were incubated at 30°C for 30 minutes. (B). Stage I reactions containing 100 ng of dnaA5 protein, 100 ng of grpE protein, and 2 ug of dnaK protein were assembled according to the experimental procedures and incubat- ed for one hour at 30°C. Stage 11 components were added and reactions were incubated at 30°C for the indicated times. The extents of DNA synthesis were quantitated according to the experi- mental procedures. 205 400 ..o. a 2 1 300 “ 2653 «.8556 m <20 glycerol (96) 212 Figure 6. The concentrations of ATP in Stage I required for the activation of dnaA5 protein. Staged activation reactions containing 100 ng of dnaA5 protein, 100 ng of grpE prOtein, and 2 ug of dnaK protein were assembled and processed according to the experimental procedures, ex- cept that differing levels of ATP were added during Stage I incubations. 213 15.0 10.0 mM ATP in Stage I 300 0U 00 0 0 2 1 case enosim