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. 1991 Jul 15;88(14):6063–6067. doi: 10.1073/pnas.88.14.6063

Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.

B Connolly 1, C A Parsons 1, F E Benson 1, H J Dunderdale 1, G J Sharples 1, R G Lloyd 1, S C West 1
PMCID: PMC52022  PMID: 1829835

Abstract

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benson F. E., Illing G. T., Sharples G. J., Lloyd R. G. Nucleotide sequencing of the ruv region of Escherichia coli K-12 reveals a LexA regulated operon encoding two genes. Nucleic Acids Res. 1988 Feb 25;16(4):1541–1549. doi: 10.1093/nar/16.4.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benson F., Collier S., Lloyd R. G. Evidence of abortive recombination in ruv mutants of Escherichia coli K12. Mol Gen Genet. 1991 Feb;225(2):266–272. doi: 10.1007/BF00269858. [DOI] [PubMed] [Google Scholar]
  3. Connolly B., West S. C. Genetic recombination in Escherichia coli: Holliday junctions made by RecA protein are resolved by fractionated cell-free extracts. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8476–8480. doi: 10.1073/pnas.87.21.8476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cox M. M., Lehman I. R. Enzymes of general recombination. Annu Rev Biochem. 1987;56:229–262. doi: 10.1146/annurev.bi.56.070187.001305. [DOI] [PubMed] [Google Scholar]
  5. Faulds D., Dower N., Stahl M. M., Stahl F. W. Orientation-dependent recombination hotspot activity in bacteriophage lambda. J Mol Biol. 1979 Jul 15;131(4):681–695. doi: 10.1016/0022-2836(79)90197-9. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Iwasaki H., Shiba T., Makino K., Nakata A., Shinagawa H. Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair. J Bacteriol. 1989 Oct;171(10):5276–5280. doi: 10.1128/jb.171.10.5276-5280.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kolodner R., Fishel R. A., Howard M. Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli. J Bacteriol. 1985 Sep;163(3):1060–1066. doi: 10.1128/jb.163.3.1060-1066.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lloyd R. G., Benson F. E., Shurvinton C. E. Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12. Mol Gen Genet. 1984;194(1-2):303–309. doi: 10.1007/BF00383532. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Lovett S. T., Kolodner R. D. Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2627–2631. doi: 10.1073/pnas.86.8.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Luisi-DeLuca C., Lovett S. T., Kolodner R. D. Genetic and physical analysis of plasmid recombination in recB recC sbcB and recB recC sbcA Escherichia coli K-12 mutants. Genetics. 1989 Jun;122(2):269–278. doi: 10.1093/genetics/122.2.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mahdi A. A., Lloyd R. G. Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair. Mol Gen Genet. 1989 Apr;216(2-3):503–510. doi: 10.1007/BF00334397. [DOI] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. Müller B., Jones C., Kemper B., West S. C. Enzymatic formation and resolution of Holliday junctions in vitro. Cell. 1990 Jan 26;60(2):329–336. doi: 10.1016/0092-8674(90)90747-3. [DOI] [PubMed] [Google Scholar]
  16. Otsuji N., Iyehara H., Hideshima Y. Isolation and characterization of an Escherichia coli ruv mutant which forms nonseptate filaments after low doses of ultraviolet light irradiation. J Bacteriol. 1974 Feb;117(2):337–344. doi: 10.1128/jb.117.2.337-344.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Parsons C. A., Kemper B., West S. C. Interaction of a four-way junction in DNA with T4 endonuclease VII. J Biol Chem. 1990 Jun 5;265(16):9285–9289. [PubMed] [Google Scholar]
  18. Picksley S. M., Parsons C. A., Kemper B., West S. C. Cleavage specificity of bacteriophage T4 endonuclease VII and bacteriophage T7 endonuclease I on synthetic branch migratable Holliday junctions. J Mol Biol. 1990 Apr 20;212(4):723–735. doi: 10.1016/0022-2836(90)90233-C. [DOI] [PubMed] [Google Scholar]
  19. Rosenberg S. M. Chi-stimulated patches are heteroduplex, with recombinant information on the phage lambda r chain. Cell. 1987 Mar 13;48(5):855–865. doi: 10.1016/0092-8674(87)90082-1. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Shurvinton C. E., Lloyd R. G., Benson F. E., Attfield P. V. Genetic analysis and molecular cloning of the Escherichia coli ruv gene. Mol Gen Genet. 1984;194(1-2):322–329. doi: 10.1007/BF00383535. [DOI] [PubMed] [Google Scholar]
  23. Shurvinton C. E., Lloyd R. G. Damage to DNA induces expression of the ruv gene of Escherichia coli. Mol Gen Genet. 1982;185(2):352–355. doi: 10.1007/BF00330811. [DOI] [PubMed] [Google Scholar]
  24. Taylor A. F., Smith G. R. Action of RecBCD enzyme on cruciform DNA. J Mol Biol. 1990 Jan 5;211(1):117–134. doi: 10.1016/0022-2836(90)90015-E. [DOI] [PubMed] [Google Scholar]
  25. Umezu K., Nakayama K., Nakayama H. Escherichia coli RecQ protein is a DNA helicase. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5363–5367. doi: 10.1073/pnas.87.14.5363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Van Houten B. Nucleotide excision repair in Escherichia coli. Microbiol Rev. 1990 Mar;54(1):18–51. doi: 10.1128/mr.54.1.18-51.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. West S. C., Cassuto E., Howard-Flanders P. Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein. Mol Gen Genet. 1982;187(2):209–217. doi: 10.1007/BF00331119. [DOI] [PubMed] [Google Scholar]
  28. Wiegand R. C., Beattie K. L., Holloman W. K., Radding C. M. Uptake of homologous single-stranded fragments by superhelical DNA. III. The product and its enzymic conversion to a recombinant molecule. J Mol Biol. 1977 Nov;116(4):805–824. doi: 10.1016/0022-2836(77)90272-8. [DOI] [PubMed] [Google Scholar]

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