Analysis of DNA replication during the SOS response in Escherichia coli Thesis submitted for the degree of Doctor of philosophy at the University of Leicester by Mohammed A. Khidhir B.Sc. (University of Baghdad) University of Leicester, Department of Genetics March 1987 UMI Number: U000911 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Disscrrlation Publishing UMI U000911 Published by ProQuest LLC 2015. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Dedication : To my parents, my wife Nadia and my son Yasir Acknowledgement First and most, I would like to thank Professor I.E. Holland for his interesting style of supervision and encouragement. I am also grateful to Dr S. Casaregola for his invaluable source of information and important discussion, I would like to thank the people in the laboratory and everyone in the department for the friendly atmosphere during the work on this project. Accurate and efficient typing of the thesis was done by Sheila Mackley for which I appreciate. Finally I would like to thank my wife Nadia for her interminable support and patience. Abbreviations DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid DTT Dithio threitol EDTA Ethylanediaminetetra-acetic acid h hours Kb Kilobases KD Kilodaltons min minutes PEG Polyethylene glycol rpm revolutions per minutes SDS Sodium dodecyl sulphate TCA Trichloroacetic acid Tris Tris (hydroxy methyl) amino methane TEMED N,N,N’,N’, tetra methyl ethylene diamine Tet Tetracycline CAM Chloramphenicol UV Ultraviolet ci curies MP Minimally purified Hp Highly purified rif rifamycin/rifampicin Ts Temperature sensitive rNTP ribonucleoside triphosphate dNTP deoxy nucleoside triphosphate ATP Adenosine triphosphate Cpm Counts per minutes Abbreviations continued .... 2 X-gal 5-bromo-4 chloro-3-indolyl-g-D-galactoside MMS Methyl methane sulphonate dNMP deoxy nucleoside monophosphate. N = adenine, quanosine, thymidine, cytidine rNMP ribonucleoside monophosphate Moaps 3“(N-morpholino propane sulphonic acid) Contents Page Chapter 1. 1.1 A. Mechanisms of DNA replication in E. coli i. Genetics of DNA replication in E. coli 1 ii. Small replicons as tools for the study of DNA 3 replication iii. Origin and direction of replication 4 I.IB. The replication fork 5 i. Polymerase involved in DNA replication 5 ii. Discontinuous replication 7 iii. Nature of primer synthesis 9 iv. Unwinding and elongation of DNA chain iji vitro 10 V . Initiation of DNA replication at the 14 chromosomal origin vi. Stable DNA replication 18 1.2. Fidelity and proof reading activities of E. coli 21 DNA polymerase 1.3. Overview of different DNA repair processes 25 1.4. The SOS response 29 i. Induction of the synthesis of RecA protein 30 ii. Stable DnA replication 31 iii. Cell division 31 iv. Inducible error prone repair (SOS-repair) 33 V . The genetic control of the SOS response 34 vi. The inducing signal 36 vii. Identification of SOS inducible genes under lexA 38 control Page viii. The lexA binding sites 39 ix. The role of the umuD,C in UV and chemical 39 mutagenesis Chapter 2. Materials and Methods 2.1. Bacterial and bacteriophage strains 42 2.2. Media 42 2.3. Growth of bacterial cultures 43 2.4. UV-irradiation 43 2.5. Measurement of bacterial cell number 43 2.6(a). DNA synthesis 44 2.6(b). Rate of DNA synthesis 44 2.7. Measurement of stable DNA replication 44 2.8. Generalized transduction using PI vir 45 a. Preparation of lysatee b. PI-transduction 2.9. Transformation 46 a. The Rbcl method b. Hanahan method c. The calcium chloride method 47 2.10. Phenol extraction 48 2.11. Ethanol precipitation 48 2.12. Preparation of plasmid DNA 49 a. Rapid plasmid DNA preparation b. Large scale plasmid preparation 2.13. SDS-polyacrylamide gel electrophoresis 50 a. Preparation and running of gels b. Autoradiography 51 Page 2.14. Restriction enzyme digest 51 2.15. Preparation of cell-free extract for DNA replication 51 2 .16. Concentration of extract 52 2.17. Hydroxylamine treatment 52 2.18.' The J_n vitro replication essay 52 2.19. The measurement of the level of g-galactosidase 53 activity Chapter 3. The kinetics of DNA synthesis following UV 3.1. Effect of recA~ and lexA(ind~1 ) mutations an 55 inhibition of DNA synthesis in UV-irradiated bacteria 3.2. DNA synthesis in the recA44l mutant at the 57 restrictive temperature 3.3. DNA synthesis in lexA(ts) mutants at the 59 restrictive temperature 3.4. Effect of inhibition of protein synthesis on 6l inhibition of DNA synthesis after UV-irradiation 3.5. Discussion 62 Chapter 4. Mechanism of the recovery of DNA synthesis in 64 Uv-irradiated bacteria 4.1. Effect of a split UV dose on recovery of DNA 64 synthesis after UV-irradiation 4.2. Recovery of DNA synthesis following UV-irradiation 65 in E. coli K12 4 .3. Effect of a split dose on the rate of DNA synthesis 66 in E. coli K12 (AB1157) Page 4.4. The recovery of DNA synthesis following UV 67 irradiation in mutants defective in DNA repair or other SOS functions 4.5. Recovery of DNA synthesis in a umuC mutant 68 4.6. Discussion 69 Chapter 5. Further studies on the role of RecA protein in the recovery of DNA synthesis following UV-irradiation 5.1. The recovery of DNA synthesis following UV in 70 recA430 5.2. The recovery of DNA synthesis following UV in 71 recA453 (zab-53) 5.3. The recovery of DnA synthesis in a recA constit- 71 utive mutant 5.4. Requirement for RecA protein in the recovery of 72 DNA synthesis 5.5. Effect of a split dose upon DNA synthesis in the 73 recA temperature sensitive mutant (recA-200)^® 5.6. Effect of inhibition of protein synthesis on the 74 recovery of DnA synthesis in a recA constitutive mutant 5.7. Discussion 75 Chapter 6. Analysis of the role of stable DNA replication in recovery of DNA synthesis after UV 6.1. Introduction 77 6.2. UV-sensitivity of constitutive "stable" replication 78 (cSdr) Page 6.3* The recovery of DNA synthesis following UV- 78 irradiation in an isdr strain 6.4. Recovery of DnA synthesis following UV-irradiation 79 of an rnh, recA^c double mutant 6.5. Discussion 80 Chapter 7. In vitro mutagenesis 83 7.1. The jji vitro replication system 84 7.2. UV-sensitivity of DNA replication vitro 84 7 :3. Replication of plasmid DNA in extracts from SOS 85 induced cells 7.4. Development of systems for vitro mutagenesis 86 i . Selection based on the E. coli lac system 86 ii. Selection based on the reversion of mutations in 87 tet or cam antibiotic resistance genes 7.5. Optimization of the procedure for the detection of 88 mutated plasmids i . Transformation 88 ii. Isolation of replicated DNA for transformation 89 7.6 . Discussion 90 Chapter 8. 8. 1. In vivo mutagenesis of strain SC41 in the presence 92 or absence of pKMIOI 8.2. Attempts to mutagenise plasmid pMC7 carrying 92 laclQ vitro 8 .3. Determination of the level of g-galactosidase in 94 the lac+ transformants Page 8.4. Is the lac+ phenotype of strains isolated in 8.2 95 above due to a mutation in the pMC7 plasmid or in the bacterial chromosome 8.5. Curing of plasmid pMC7 from tet^ lac+ trans­ 96 formants of CSH26 8.6. Discussion 97 Chapter 9. In vitro mutagenesis : Analysis of reversion to tetracycline resistance 9.1 . Introduction 99 9.2. Production of antibiotic sensitive derivatives 99 of plasmid pBR325 Jji vitro 9.3. Attempts to mutagenise plasmid pLG7001 (tet^, 100 camR in vitro) 9.4. Discussion 101 Chapter 10. General discussion 102 10.1. The role of stable DNA replication and other 102 phenomena in the recovery of DNA synthesis following UV-irradiation 10.2. Model(s) for trans-dimer synthesis by-pass 104 mechanism 10.3. Dri vitro) mutagenesis 107 10.4. Involvement of Reca protein in SOS mutagenesis 108 10.5. The SOS system in relation to other stress 109 responses Future studies 111 Page References 113 Appendix I. Mutations affecting or involved in SOS functions used in this study I i. Mutations in the recA gene I ii. Mutations in the lexA gene II Appendix II. IV Chapter 1 Introduction 1.1 A Mechanism of DNA replication in E.coli i) Genetics of DNA replication in E.coli Contrary to earlier expectations that DNA synthesis required simply a polymerase, triphosphates and a single-strand template (reviewed in Kornberg, 1980), DNA replication i^ vivo has proved to be a surprisingly complex process. This stems primarily from the requirements for unwinding a double-stranded DNA molecule, the opposite chemical polarity of complementary strands and the inherent inability of Dna polymerases to initiate synthesis in the absence of a primer. This complexity was nevertheless indicated already in 1968 with the finding of Epstein et al. that bacteriophage T4 replication appeared to involve the activity of at least five complementation groups. Subsequent studies in E.coli confirmed this genetic complexity with several conditional lethal mutants being divided conveniently into delayed stop and fast stop classes with respect to DNA replication at high temperature C Gr o s s >197^ } Sevastopoulos et al., 1977; Wechsler, 1978).