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. 1994 Aug;176(16):5093–5100. doi: 10.1128/jb.176.16.5093-5100.1994

Chi-dependent formation of linear plasmid DNA in exonuclease-deficient recBCD+ strains of Escherichia coli.

M M Zaman 1, T C Boles 1
PMCID: PMC196349  PMID: 8051022

Abstract

Escherichia coli strains carrying mutations in sbcB (exonuclease I) or xthA (exonuclease III) accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids containing the chi sequence, 5'-GCTGGTGG-3'. Chi-dependent formation of high-molecular-weight plasmid DNA is dependent on recA and recF functions. In addition, chi stimulation occurs only in cis. Our data are consistent with models in which RecA and RecF proteins bind to and protect the DNA ends produced by RecBCD-chi interaction.

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Selected References

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  1. Bassett C. L., Kushner S. R. Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli. J Bacteriol. 1984 Feb;157(2):661–664. doi: 10.1128/jb.157.2.661-664.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernardi A., Bernardi F. Complete sequence of pSC101. Nucleic Acids Res. 1984 Dec 21;12(24):9415–9426. doi: 10.1093/nar/12.24.9415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Cheng K. C., Smith G. R. Distribution of Chi-stimulated recombinational exchanges and heteroduplex endpoints in phage lambda. Genetics. 1989 Sep;123(1):5–17. doi: 10.1093/genetics/123.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Churchward G., Belin D., Nagamine Y. A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984 Nov;31(1-3):165–171. doi: 10.1016/0378-1119(84)90207-5. [DOI] [PubMed] [Google Scholar]
  6. Cohen A., Clark A. J. Synthesis of linear plasmid multimers in Escherichia coli K-12. J Bacteriol. 1986 Jul;167(1):327–335. doi: 10.1128/jb.167.1.327-335.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dabert P., Ehrlich S. D., Gruss A. Chi sequence protects against RecBCD degradation of DNA in vivo. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12073–12077. doi: 10.1073/pnas.89.24.12073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Dixon D. A., Kowalczykowski S. C. Homologous pairing in vitro stimulated by the recombination hotspot, Chi. Cell. 1991 Jul 26;66(2):361–371. doi: 10.1016/0092-8674(91)90625-9. [DOI] [PubMed] [Google Scholar]
  10. Dixon D. A., Kowalczykowski S. C. The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme. Cell. 1993 Apr 9;73(1):87–96. doi: 10.1016/0092-8674(93)90162-j. [DOI] [PubMed] [Google Scholar]
  11. Ennis D. G., Amundsen S. K., Smith G. R. Genetic functions promoting homologous recombination in Escherichia coli: a study of inversions in phage lambda. Genetics. 1987 Jan;115(1):11–24. doi: 10.1093/genetics/115.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  13. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  14. Goldmark P. J., Linn S. Purification and properties of the recBC DNase of Escherichia coli K-12. J Biol Chem. 1972 Mar 25;247(6):1849–1860. [PubMed] [Google Scholar]
  15. Griffin T. J., 4th, Kolodner R. D. Purification and preliminary characterization of the Escherichia coli K-12 recF protein. J Bacteriol. 1990 Nov;172(11):6291–6299. doi: 10.1128/jb.172.11.6291-6299.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hollingshead S., Vapnek D. Nucleotide sequence analysis of a gene encoding a streptomycin/spectinomycin adenylyltransferase. Plasmid. 1985 Jan;13(1):17–30. doi: 10.1016/0147-619x(85)90052-6. [DOI] [PubMed] [Google Scholar]
  17. James A. A., Morrison P. T., Kolodner R. Genetic recombination of bacterial plasmid DNA. Analysis of the effect of recombination-deficient mutations on plasmid recombination. J Mol Biol. 1982 Sep 25;160(3):411–430. doi: 10.1016/0022-2836(82)90305-9. [DOI] [PubMed] [Google Scholar]
  18. Karu A. E., MacKay V., Goldmark P. J., Linn S. The recBC deoxyribonuclease of Escherichia coli K-12. Substrate specificity and reaction intermediates. J Biol Chem. 1973 Jul 25;248(14):4874–4884. [PubMed] [Google Scholar]
  19. Kobayashi I., Murialdo H., Crasemann J. M., Stahl M. M., Stahl F. W. Orientation of cohesive end site cos determines the active orientation of chi sequence in stimulating recA . recBC-mediated recombination in phage lambda lytic infections. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5981–5985. doi: 10.1073/pnas.79.19.5981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. LEHMAN I. R., NUSSBAUM A. L. THE DEOXYRIBONUCLEASES OF ESCHERICHIA COLI. V. ON THE SPECIFICITY OF EXONUCLEASE I (PHOSPHODIESTERASE). J Biol Chem. 1964 Aug;239:2628–2636. [PubMed] [Google Scholar]
  22. Laban A., Cohen A. Interplasmidic and intraplasmidic recombination in Escherichia coli K-12. Mol Gen Genet. 1981;184(2):200–207. doi: 10.1007/BF00272905. [DOI] [PubMed] [Google Scholar]
  23. Lam S. T., Stahl M. M., McMilin K. D., Stahl F. W. Rec-mediated recombinational hot spot activity in bacteriophage lambda. II. A mutation which causes hot spot activity. Genetics. 1974 Jul;77(3):425–433. doi: 10.1093/genetics/77.3.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lanzov V., Stepanova I., Vinogradskaja G. Genetic control of recombination exchange frequency in Escherichia coli K-12. Biochimie. 1991 Feb-Mar;73(2-3):305–312. doi: 10.1016/0300-9084(91)90217-o. [DOI] [PubMed] [Google Scholar]
  25. Lerner C. G., Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 1990 Aug 11;18(15):4631–4631. doi: 10.1093/nar/18.15.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Madiraju M. V., Clark A. J. Effect of RecF protein on reactions catalyzed by RecA protein. Nucleic Acids Res. 1991 Nov 25;19(22):6295–6300. doi: 10.1093/nar/19.22.6295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Madiraju M. V., Templin A., Clark A. J. Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6592–6596. doi: 10.1073/pnas.85.18.6592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Niki H., Ogura T., Hiraga S. Linear multimer formation of plasmid DNA in Escherichia coli hopE (recD) mutants. Mol Gen Genet. 1990 Oct;224(1):1–9. doi: 10.1007/BF00259444. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Prentki P., Krisch H. M. A modified pBR322 vector with improved properties for the cloning, recovery, and sequencing of blunt-ended DNA fragments. Gene. 1982 Feb;17(2):189–196. doi: 10.1016/0378-1119(82)90072-5. [DOI] [PubMed] [Google Scholar]
  32. RICHARDSON C. C., LEHMAN I. R., KORNBERG A. A DEOXYRIBONUCLEIC ACID PHOSPHATASE-EXONUCLEASE FROM ESCHERICHIA COLI. II. CHARACTERIZATION OF THE EXONUCLEASE ACTIVITY. J Biol Chem. 1964 Jan;239:251–258. [PubMed] [Google Scholar]
  33. Rosenberg S. M., Hastings P. J. The split-end model for homologous recombination at double-strand breaks and at Chi. Biochimie. 1991 Apr;73(4):385–397. doi: 10.1016/0300-9084(91)90105-a. [DOI] [PubMed] [Google Scholar]
  34. Silberstein Z., Cohen A. Synthesis of linear multimers of OriC and pBR322 derivatives in Escherichia coli K-12: role of recombination and replication functions. J Bacteriol. 1987 Jul;169(7):3131–3137. doi: 10.1128/jb.169.7.3131-3137.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Simmon V. F., Lederberg S. Degradation of bacteriophage lambda deoxyribonucleic acid after restriction by Escherichia coli K-12. J Bacteriol. 1972 Oct;112(1):161–169. doi: 10.1128/jb.112.1.161-169.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Smith G. R. Homologous recombination in procaryotes. Microbiol Rev. 1988 Mar;52(1):1–28. doi: 10.1128/mr.52.1.1-28.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith G. R., Kunes S. M., Schultz D. W., Taylor A., Triman K. L. Structure of chi hotspots of generalized recombination. Cell. 1981 May;24(2):429–436. doi: 10.1016/0092-8674(81)90333-0. [DOI] [PubMed] [Google Scholar]
  38. Stahl F. W. Roles of double-strand breaks in generalized genetic recombination. Prog Nucleic Acid Res Mol Biol. 1986;33:169–194. doi: 10.1016/s0079-6603(08)60023-9. [DOI] [PubMed] [Google Scholar]
  39. Taylor A. F., Smith G. R. RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5226–5230. doi: 10.1073/pnas.89.12.5226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Thaler D. S., Sampson E., Siddiqi I., Rosenberg S. M., Thomason L. C., Stahl F. W., Stahl M. M. Recombination of bacteriophage lambda in recD mutants of Escherichia coli. Genome. 1989;31(1):53–67. doi: 10.1139/g89-013. [DOI] [PubMed] [Google Scholar]
  41. Volkert M. R., Hartke M. A. Suppression of Escherichia coli recF mutations by recA-linked srfA mutations. J Bacteriol. 1984 Feb;157(2):498–506. doi: 10.1128/jb.157.2.498-506.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Willetts N. S., Clark A. J. Characteristics of some multiply recombination-deficient strains of Escherichia coli. J Bacteriol. 1969 Oct;100(1):231–239. doi: 10.1128/jb.100.1.231-239.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yamamoto K., Kusano K., Takahashi N. K., Yoshikura H., Kobayashi I. Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model. Mol Gen Genet. 1992 Jul;234(1):1–13. doi: 10.1007/BF00272339. [DOI] [PubMed] [Google Scholar]
  44. 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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