Abstract
The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.
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- 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]
- 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]
- 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]
- Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. A conserved NTP-motif in putative helicases. Nature. 1988 May 5;333(6168):22–22. doi: 10.1038/333022a0. [DOI] [PubMed] [Google Scholar]
- Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hodgman T. C. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. doi: 10.1038/333022b0. [DOI] [PubMed] [Google Scholar]
- Howard-Flanders P., West S. C., Stasiak A. Role of RecA protein spiral filaments in genetic recombination. Nature. 1984 May 17;309(5965):215–219. doi: 10.1038/309215a0. [DOI] [PubMed] [Google Scholar]
- Hsieh P., Camerini-Otero C. S., Camerini-Otero R. D. Pairing of homologous DNA sequences by proteins: evidence for three-stranded DNA. Genes Dev. 1990 Nov;4(11):1951–1963. doi: 10.1101/gad.4.11.1951. [DOI] [PubMed] [Google Scholar]
- 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]
- Kodadek T., Alberts B. M. Stimulation of protein-directed strand exchange by a DNA helicase. Nature. 1987 Mar 19;326(6110):312–314. doi: 10.1038/326312a0. [DOI] [PubMed] [Google Scholar]
- Koo H. S., Claassen L., Grossman L., Liu L. F. ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1212–1216. doi: 10.1073/pnas.88.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Lloyd R. G., Buckman C. Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair. J Bacteriol. 1991 Feb;173(3):1004–1011. doi: 10.1128/jb.173.3.1004-1011.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lloyd R. G. Conjugational recombination in resolvase-deficient ruvC mutants of Escherichia coli K-12 depends on recG. J Bacteriol. 1991 Sep;173(17):5414–5418. doi: 10.1128/jb.173.17.5414-5418.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lloyd R. G., Sharples G. J. Dissociation of synthetic Holliday junctions by E. coli RecG protein. EMBO J. 1993 Jan;12(1):17–22. doi: 10.1002/j.1460-2075.1993.tb05627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matson S. W., Kaiser-Rogers K. A. DNA helicases. Annu Rev Biochem. 1990;59:289–329. doi: 10.1146/annurev.bi.59.070190.001445. [DOI] [PubMed] [Google Scholar]
- Müller B., Burdett I., West S. C. Unusual stability of recombination intermediates made by Escherichia coli RecA protein. EMBO J. 1992 Jul;11(7):2685–2693. doi: 10.1002/j.1460-2075.1992.tb05334.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oh E. Y., Grossman L. Helicase properties of the Escherichia coli UvrAB protein complex. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3638–3642. doi: 10.1073/pnas.84.11.3638. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orren D. K., Selby C. P., Hearst J. E., Sancar A. Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I. J Biol Chem. 1992 Jan 15;267(2):780–788. [PubMed] [Google Scholar]
- 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]
- Parsons C. A., Tsaneva I., Lloyd R. G., West S. C. Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5452–5456. doi: 10.1073/pnas.89.12.5452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao B. J., Dutreix M., Radding C. M. Stable three-stranded DNA made by RecA protein. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):2984–2988. doi: 10.1073/pnas.88.8.2984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao B. J., Jwang B., Radding C. M. RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process. J Mol Biol. 1990 Jun 20;213(4):789–809. doi: 10.1016/S0022-2836(05)80264-5. [DOI] [PubMed] [Google Scholar]
- 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]
- Shiba T., Iwasaki H., Nakata A., Shinagawa H. SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8445–8449. doi: 10.1073/pnas.88.19.8445. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Taylor A., Smith G. R. Unwinding and rewinding of DNA by the RecBC enzyme. Cell. 1980 Nov;22(2 Pt 2):447–457. doi: 10.1016/0092-8674(80)90355-4. [DOI] [PubMed] [Google Scholar]
- Tsaneva I. R., Illing G., Lloyd R. G., West S. C. Purification and properties of the RuvA and RuvB proteins of Escherichia coli. Mol Gen Genet. 1992 Oct;235(1):1–10. doi: 10.1007/BF00286175. [DOI] [PubMed] [Google Scholar]
- Tsaneva I. R., Müller B., West S. C. ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli. Cell. 1992 Jun 26;69(7):1171–1180. doi: 10.1016/0092-8674(92)90638-s. [DOI] [PubMed] [Google Scholar]
- 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]
- Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- West S. C., Cassuto E., Howard-Flanders P. Homologous pairing can occur before DNA strand separation in general genetic recombination. Nature. 1981 Mar 5;290(5801):29–33. doi: 10.1038/290029a0. [DOI] [PubMed] [Google Scholar]
- West S. C., Howard-Flanders P. Duplex-duplex interactions catalyzed by RecA protein allow strand exchanges to pass double-strand breaks in DNA. Cell. 1984 Jun;37(2):683–691. doi: 10.1016/0092-8674(84)90401-x. [DOI] [PubMed] [Google Scholar]