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. 1996 Jul;143(3):1101–1114. doi: 10.1093/genetics/143.3.1101

Recombination-Dependent Growth in Exonuclease-Depleted Recbc Sbcbc Strains of Escherichia Coli K-12

L Ryder 1, G J Sharples 1, R G Lloyd 1
PMCID: PMC1207382  PMID: 8807285

Abstract

Analysis of the aroLM-sbcCD interval of the Escherichia coli K-12 chromosome revealed a new gene (rdgC) encoding a function required for growth in recombination-deficient recBC sbcBC strains. Deletion of rdgC does not reduce viability, conjugational recombination, or DNA repair in rec(+), recA, recB, recF, or recJ mutants. However, it makes the growth of recBC sbcBC strains reliant on the RecA, RecF, and RuvC proteins and, to a large extent, on RuvAB. The recBC sbcBC ΔrdgC ruvAB construct forms colonies, but cell viability is reduced to <5%. A recBC sbcBC ΔrdgC derivative carrying the temperature-sensitive recA200 allele grows at 32° but not 42°. Multicopy rdgC(+) plasmids reduce the growth rate of recBC sbcBC strains, while multicopy sbcC(+) plasmids that reactivate SbcCD nuclease cannot be maintained without RdgC protein. The data presented are interpreted to suggest that exonuclease-depleted recBC sbcBC strains have difficulty removing the displaced arm of a collapsed replication fork and that this problem is compounded in the absence of RdgC. Recombination then becomes necessary to repair the fork and allow chromosome duplication to be completed. The possibility that RdgC is an exonuclease is discussed.

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Selected References

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  1. Angell S., Schwarz E., Bibb M. J. The glucose kinase gene of Streptomyces coelicolor A3(2): its nucleotide sequence, transcriptional analysis and role in glucose repression. Mol Microbiol. 1992 Oct;6(19):2833–2844. doi: 10.1111/j.1365-2958.1992.tb01463.x. [DOI] [PubMed] [Google Scholar]
  2. Burland V., Plunkett G., 3rd, Daniels D. L., Blattner F. R. DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication. Genomics. 1993 Jun;16(3):551–561. doi: 10.1006/geno.1993.1230. [DOI] [PubMed] [Google Scholar]
  3. Capaldo-Kimball F., Barbour S. D. Involvement of recombination genes in growth and viability of Escherichia coli K-12. J Bacteriol. 1971 Apr;106(1):204–212. doi: 10.1128/jb.106.1.204-212.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carra J. H., Schleif R. F. Variation of half-site organization and DNA looping by AraC protein. EMBO J. 1993 Jan;12(1):35–44. doi: 10.1002/j.1460-2075.1993.tb05629.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chalker A. F., Leach D. R., Lloyd R. G. Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome. Gene. 1988 Nov 15;71(1):201–205. doi: 10.1016/0378-1119(88)90092-3. [DOI] [PubMed] [Google Scholar]
  6. Clyman J., Cunningham R. P. Escherichia coli K-12 mutants in which viability is dependent on recA function. J Bacteriol. 1987 Sep;169(9):4203–4210. doi: 10.1128/jb.169.9.4203-4210.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Connelly J. C., Leach D. R. The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination. Genes Cells. 1996 Mar;1(3):285–291. doi: 10.1046/j.1365-2443.1996.23024.x. [DOI] [PubMed] [Google Scholar]
  8. DeFeyter R. C., Davidson B. E., Pittard J. Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):233–239. doi: 10.1128/jb.165.1.233-239.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dixon D. A., Churchill J. J., Kowalczykowski S. C. Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2980–2984. doi: 10.1073/pnas.91.8.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  11. Horiuchi T., Fujimura Y., Nishitani H., Kobayashi T., Hidaka M. The DNA replication fork blocked at the Ter site may be an entrance for the RecBCD enzyme into duplex DNA. J Bacteriol. 1994 Aug;176(15):4656–4663. doi: 10.1128/jb.176.15.4656-4663.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Horiuchi T., Fujimura Y. Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate. J Bacteriol. 1995 Feb;177(3):783–791. doi: 10.1128/jb.177.3.783-791.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Joseph J. W., Kolodner R. Exonuclease VIII of Escherichia coli. II. Mechanism of action. J Biol Chem. 1983 Sep 10;258(17):10418–10424. [PubMed] [Google Scholar]
  14. 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]
  15. Kusano K., Nakayama K., Nakayama H. Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant. Involvement of RecF recombination pathway genes. J Mol Biol. 1989 Oct 20;209(4):623–634. doi: 10.1016/0022-2836(89)90000-4. [DOI] [PubMed] [Google Scholar]
  16. Kushner S. R., Nagaishi H., Templin A., Clark A. J. Genetic recombination in Escherichia coli: the role of exonuclease I. Proc Natl Acad Sci U S A. 1971 Apr;68(4):824–827. doi: 10.1073/pnas.68.4.824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kuzminov A. Collapse and repair of replication forks in Escherichia coli. Mol Microbiol. 1995 May;16(3):373–384. doi: 10.1111/j.1365-2958.1995.tb02403.x. [DOI] [PubMed] [Google Scholar]
  18. Lloyd R. G., Buckman C., Benson F. E. Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme. J Gen Microbiol. 1987 Sep;133(9):2531–2538. doi: 10.1099/00221287-133-9-2531. [DOI] [PubMed] [Google Scholar]
  19. Lloyd R. G., Buckman C. Identification and genetic analysis of sbcC mutations in commonly used recBC sbcB strains of Escherichia coli K-12. J Bacteriol. 1985 Nov;164(2):836–844. doi: 10.1128/jb.164.2.836-844.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lloyd R. G., Porton M. C., Buckman C. Effect of recF, recJ, recN, recO and ruv mutations on ultraviolet survival and genetic recombination in a recD strain of Escherichia coli K12. Mol Gen Genet. 1988 May;212(2):317–324. doi: 10.1007/BF00334702. [DOI] [PubMed] [Google Scholar]
  21. Lovett S. T., Clark A. J. Genetic analysis of regulation of the RecF pathway of recombination in Escherichia coli K-12. J Bacteriol. 1983 Mar;153(3):1471–1478. doi: 10.1128/jb.153.3.1471-1478.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  23. Mandal T. N., Mahdi A. A., Sharples G. J., Lloyd R. G. Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations. J Bacteriol. 1993 Jul;175(14):4325–4334. doi: 10.1128/jb.175.14.4325-4334.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Minton N. P., Whitehead P. J., Atkinson T., Gilbert H. J. Nucleotide sequence of an Erwinia chrysanthemi gene encoding shikimate kinase. Nucleic Acids Res. 1989 Feb 25;17(4):1769–1769. doi: 10.1093/nar/17.4.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Médigue C., Viari A., Hénaut A., Danchin A. Escherichia coli molecular genetic map (1500 kbp): update II. Mol Microbiol. 1991 Nov;5(11):2629–2640. doi: 10.1111/j.1365-2958.1991.tb01972.x. [DOI] [PubMed] [Google Scholar]
  26. Nakayama K., Irino N., Nakayama H. The recQ gene of Escherichia coli K12: molecular cloning and isolation of insertion mutants. Mol Gen Genet. 1985;200(2):266–271. doi: 10.1007/BF00425434. [DOI] [PubMed] [Google Scholar]
  27. Naom I. S., Morton S. J., Leach D. R., Lloyd R. G. Molecular organization of sbcC, a gene that affects genetic recombination and the viability of DNA palindromes in Escherichia coli K-12. Nucleic Acids Res. 1989 Oct 25;17(20):8033–8045. doi: 10.1093/nar/17.20.8033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Phillips G. J., Prasher D. C., Kushner S. R. Physical and biochemical characterization of cloned sbcB and xonA mutations from Escherichia coli K-12. J Bacteriol. 1988 May;170(5):2089–2094. doi: 10.1128/jb.170.5.2089-2094.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Picksley S. M., Lloyd R. G., Buckman C. Genetic analysis and regulation of inducible recombination in Escherichia coli K-12. Cold Spring Harb Symp Quant Biol. 1984;49:469–474. doi: 10.1101/sqb.1984.049.01.053. [DOI] [PubMed] [Google Scholar]
  30. Rinken R., Thomas B., Wackernagel W. Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding. J Bacteriol. 1992 Aug;174(16):5424–5429. doi: 10.1128/jb.174.16.5424-5429.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Russell C. B., Thaler D. S., Dahlquist F. W. Chromosomal transformation of Escherichia coli recD strains with linearized plasmids. J Bacteriol. 1989 May;171(5):2609–2613. doi: 10.1128/jb.171.5.2609-2613.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schmidt M., Arnold W., Niemann A., Kleickmann A., Pühler A. The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase. Gene. 1992 Dec 1;122(1):35–43. doi: 10.1016/0378-1119(92)90029-o. [DOI] [PubMed] [Google Scholar]
  33. Sharples G. J., Benson F. E., Illing G. T., Lloyd R. G. Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination. Mol Gen Genet. 1990 Apr;221(2):219–226. doi: 10.1007/BF00261724. [DOI] [PubMed] [Google Scholar]
  34. Shinagawa H., Makino K., Amemura M., Kimura S., Iwasaki H., Nakata A. Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination. J Bacteriol. 1988 Sep;170(9):4322–4329. doi: 10.1128/jb.170.9.4322-4329.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Takahagi M., Iwasaki H., Nakata A., Shinagawa H. Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease. J Bacteriol. 1991 Sep;173(18):5747–5753. doi: 10.1128/jb.173.18.5747-5753.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Titgemeyer F., Reizer J., Reizer A., Saier M. H., Jr Evolutionary relationships between sugar kinases and transcriptional repressors in bacteria. Microbiology. 1994 Sep;140(Pt 9):2349–2354. doi: 10.1099/13500872-140-9-2349. [DOI] [PubMed] [Google Scholar]
  37. Umezu K., Nakayama H. RecQ DNA helicase of Escherichia coli. Characterization of the helix-unwinding activity with emphasis on the effect of single-stranded DNA-binding protein. J Mol Biol. 1993 Apr 20;230(4):1145–1150. doi: 10.1006/jmbi.1993.1231. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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