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. 2000 Jun;155(2):487–497. doi: 10.1093/genetics/155.2.487

Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12.

S J Sandler 1
PMCID: PMC1461104  PMID: 10835375

Abstract

In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3'-5' helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.

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Selected References

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  1. Allen G. C., Jr, Kornberg A. Assembly of the primosome of DNA replication in Escherichia coli. J Biol Chem. 1993 Sep 15;268(26):19204–19209. [PubMed] [Google Scholar]
  2. Berlyn M. K. Linkage map of Escherichia coli K-12, edition 10: the traditional map. Microbiol Mol Biol Rev. 1998 Sep;62(3):814–984. doi: 10.1128/mmbr.62.3.814-984.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clark A. J., Sandler S. J. Homologous genetic recombination: the pieces begin to fall into place. Crit Rev Microbiol. 1994;20(2):125–142. doi: 10.3109/10408419409113552. [DOI] [PubMed] [Google Scholar]
  4. Colasanti J., Denhardt D. T. The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels. Mol Gen Genet. 1987 Sep;209(2):382–390. doi: 10.1007/BF00329669. [DOI] [PubMed] [Google Scholar]
  5. Cox M. M., Goodman M. F., Kreuzer K. N., Sherratt D. J., Sandler S. J., Marians K. J. The importance of repairing stalled replication forks. Nature. 2000 Mar 2;404(6773):37–41. doi: 10.1038/35003501. [DOI] [PubMed] [Google Scholar]
  6. Jones J. M., Nakai H. Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate. J Mol Biol. 1999 Jun 11;289(3):503–516. doi: 10.1006/jmbi.1999.2783. [DOI] [PubMed] [Google Scholar]
  7. Jones J. M., Nakai H. The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition. EMBO J. 1997 Nov 17;16(22):6886–6895. doi: 10.1093/emboj/16.22.6886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kim S., Dallmann H. G., McHenry C. S., Marians K. J. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell. 1996 Feb 23;84(4):643–650. doi: 10.1016/s0092-8674(00)81039-9. [DOI] [PubMed] [Google Scholar]
  9. Kogoma T., Cadwell G. W., Barnard K. G., Asai T. The DNA replication priming protein, PriA, is required for homologous recombination and double-strand break repair. J Bacteriol. 1996 Mar;178(5):1258–1264. doi: 10.1128/jb.178.5.1258-1264.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kowalczykowski S. C., Dixon D. A., Eggleston A. K., Lauder S. D., Rehrauer W. M. Biochemistry of homologous recombination in Escherichia coli. Microbiol Rev. 1994 Sep;58(3):401–465. doi: 10.1128/mr.58.3.401-465.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kuzminov A. Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol Mol Biol Rev. 1999 Dec;63(4):751-813, table of contents. doi: 10.1128/mmbr.63.4.751-813.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lane H. E., Denhardt D. T. The rep mutation. III. Altered structure of the replicating Escherichia coli chromosome. J Bacteriol. 1974 Nov;120(2):805–814. doi: 10.1128/jb.120.2.805-814.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lane H. E., Denhardt D. T. The rep mutation. IV. Slower movement of replication forks in Escherichia coli rep strains. J Mol Biol. 1975 Sep 5;97(1):99–112. doi: 10.1016/s0022-2836(75)80025-8. [DOI] [PubMed] [Google Scholar]
  14. Lee E. H., Kornberg A. Replication deficiencies in priA mutants of Escherichia coli lacking the primosomal replication n' protein. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3029–3032. doi: 10.1073/pnas.88.8.3029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lee E. H., Masai H., Allen G. C., Jr, Kornberg A. The priA gene encoding the primosomal replicative n' protein of Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4620–4624. doi: 10.1073/pnas.87.12.4620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu J., Marians K. J. PriA-directed assembly of a primosome on D loop DNA. J Biol Chem. 1999 Aug 27;274(35):25033–25041. doi: 10.1074/jbc.274.35.25033. [DOI] [PubMed] [Google Scholar]
  17. Liu J., Nurse P., Marians K. J. The ordered assembly of the phiX174-type primosome. III. PriB facilitates complex formation between PriA and DnaT. J Biol Chem. 1996 Jun 28;271(26):15656–15661. doi: 10.1074/jbc.271.26.15656. [DOI] [PubMed] [Google Scholar]
  18. Masai H., Asai T., Kubota Y., Arai K., Kogoma T. Escherichia coli PriA protein is essential for inducible and constitutive stable DNA replication. EMBO J. 1994 Nov 15;13(22):5338–5345. doi: 10.1002/j.1460-2075.1994.tb06868.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Michel B., Ehrlich S. D., Uzest M. DNA double-strand breaks caused by replication arrest. EMBO J. 1997 Jan 15;16(2):430–438. doi: 10.1093/emboj/16.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nurse P., DiGate R. J., Zavitz K. H., Marians K. J. Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4615–4619. doi: 10.1073/pnas.87.12.4615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nurse P., Liu J., Marians K. J. Two modes of PriA binding to DNA. J Biol Chem. 1999 Aug 27;274(35):25026–25032. doi: 10.1074/jbc.274.35.25026. [DOI] [PubMed] [Google Scholar]
  22. Nurse P., Zavitz K. H., Marians K. J. Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response. J Bacteriol. 1991 Nov;173(21):6686–6693. doi: 10.1128/jb.173.21.6686-6693.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sandler S. J., Marians K. J. Role of PriA in replication fork reactivation in Escherichia coli. J Bacteriol. 2000 Jan;182(1):9–13. doi: 10.1128/jb.182.1.9-13.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sandler S. J., Marians K. J., Zavitz K. H., Coutu J., Parent M. A., Clark A. J. dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12. Mol Microbiol. 1999 Oct;34(1):91–101. doi: 10.1046/j.1365-2958.1999.01576.x. [DOI] [PubMed] [Google Scholar]
  25. Sandler S. J. Overlapping functions for recF and priA in cell viability and UV-inducible SOS expression are distinguished by dnaC809 in Escherichia coli K-12. Mol Microbiol. 1996 Feb;19(4):871–880. doi: 10.1046/j.1365-2958.1996.429959.x. [DOI] [PubMed] [Google Scholar]
  26. Sandler S. J., Samra H. S., Clark A. J. Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC. Genetics. 1996 May;143(1):5–13. doi: 10.1093/genetics/143.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Seigneur M., Bidnenko V., Ehrlich S. D., Michel B. RuvAB acts at arrested replication forks. Cell. 1998 Oct 30;95(3):419–430. doi: 10.1016/s0092-8674(00)81772-9. [DOI] [PubMed] [Google Scholar]
  28. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stahl F. W. The Holliday junction on its thirtieth anniversary. Genetics. 1994 Oct;138(2):241–246. doi: 10.1093/genetics/138.2.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tougu K., Marians K. J. The extreme C terminus of primase is required for interaction with DnaB at the replication fork. J Biol Chem. 1996 Aug 30;271(35):21391–21397. doi: 10.1074/jbc.271.35.21391. [DOI] [PubMed] [Google Scholar]
  31. Uzest M., Ehrlich S. D., Michel B. Lethality of rep recB and rep recC double mutants of Escherichia coli. Mol Microbiol. 1995 Sep;17(6):1177–1188. doi: 10.1111/j.1365-2958.1995.mmi_17061177.x. [DOI] [PubMed] [Google Scholar]
  32. Wu T. T. A model for three-point analysis of random general transduction. Genetics. 1966 Aug;54(2):405–410. doi: 10.1093/genetics/54.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zavitz K. H., Marians K. J. ATPase-deficient mutants of the Escherichia coli DNA replication protein PriA are capable of catalyzing the assembly of active primosomes. J Biol Chem. 1992 Apr 5;267(10):6933–6940. [PubMed] [Google Scholar]
  34. Zavitz K. H., Marians K. J. Helicase-deficient cysteine to glycine substitution mutants of Escherichia coli replication protein PriA retain single-stranded DNA-dependent ATPase activity. Zn2+ stimulation of mutant PriA helicase and primosome assembly activities. J Biol Chem. 1993 Feb 25;268(6):4337–4346. [PubMed] [Google Scholar]
  35. Zavitz K. H., Marians K. J. Helicase-deficient cysteine to glycine substitution mutants of Escherichia coli replication protein PriA retain single-stranded DNA-dependent ATPase activity. Zn2+ stimulation of mutant PriA helicase and primosome assembly activities. J Biol Chem. 1993 Feb 25;268(6):4337–4346. [PubMed] [Google Scholar]

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