Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Nov 1;13(21):5220–5228. doi: 10.1002/j.1460-2075.1994.tb06853.x

Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase.

M C Whitby 1, S D Vincent 1, R G Lloyd 1
PMCID: PMC395472  PMID: 7957087

Abstract

The product of the recG gene of Escherichia coli is needed for normal recombination and DNA repair in E. coli and has been shown to help process Holliday junction intermediates to mature products by catalysing branch migration. The 76 kDa RecG protein contains sequence motifs conserved in the DExH family of helicases, suggesting that it promotes branch migration by unwinding DNA. We show that RecG does not unwind blunt ended duplex DNA or forked duplexes with short unpaired single-strand ends. It also fails to unwind a partial duplex (52 bp) classical helicase substrate containing a short oligonucleotide annealed to circular single-stranded DNA. However, unwinding activity is detected when the duplex region is reduced to 26 bp or less, although this requires high levels of protein. The unwinding proceeds with a clear 3' to 5' polarity with respect to the single strand bound by RecG. Substantially higher levels of unwinding are observed with substrates containing a three-way duplex branch. This is attributed to RecG's particular affinity for junction DNA which we demonstrate would be heightened by single-stranded DNA binding protein in vivo. Reaction requirements for unwinding are the same as for branch migration of Holliday junctions, with a strict dependence on hydrolysis of ATP. These results define RecG as a new class of helicase that has evolved to catalyse the branch migration of Holliday junctions.

Full text

PDF
5220

Images in this article

5221Image
on p.5221
5222Image
on p.5222
5222Image
on p.5222
5223Image
on p.5223
5224Image
on p.5224
5225Image
on p.5225
5226Image
on p.5226
5227Image
on p.5227

Selected References

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

  1. Bennett R. J., Dunderdale H. J., West S. C. Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion. Cell. 1993 Sep 24;74(6):1021–1031. doi: 10.1016/0092-8674(93)90724-5. [DOI] [PubMed] [Google Scholar]
  2. Duckett D. R., Lilley D. M. The three-way DNA junction is a Y-shaped molecule in which there is no helix-helix stacking. EMBO J. 1990 May;9(5):1659–1664. doi: 10.1002/j.1460-2075.1990.tb08286.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dunderdale H. J., Benson F. E., Parsons C. A., Sharples G. J., Lloyd R. G., West S. C. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature. 1991 Dec 19;354(6354):506–510. doi: 10.1038/354506a0. [DOI] [PubMed] [Google Scholar]
  4. Iwasaki H., Takahagi M., Nakata A., Shinagawa H. Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration. Genes Dev. 1992 Nov;6(11):2214–2220. doi: 10.1101/gad.6.11.2214. [DOI] [PubMed] [Google Scholar]
  5. Iwasaki H., Takahagi M., Shiba T., Nakata A., Shinagawa H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 1991 Dec;10(13):4381–4389. doi: 10.1002/j.1460-2075.1991.tb05016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Johnson R. D., Symington L. S. Crossed-stranded DNA structures for investigating the molecular dynamics of the Holliday junction. J Mol Biol. 1993 Feb 20;229(4):812–820. doi: 10.1006/jmbi.1993.1087. [DOI] [PubMed] [Google Scholar]
  7. Kornberg A., Scott J. F., Bertsch L. L. ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork. J Biol Chem. 1978 May 10;253(9):3298–3304. [PubMed] [Google Scholar]
  8. 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]
  9. Lloyd R. G., Sharples G. J. Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. Nucleic Acids Res. 1993 Apr 25;21(8):1719–1725. doi: 10.1093/nar/21.8.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lohman T. M. Escherichia coli DNA helicases: mechanisms of DNA unwinding. Mol Microbiol. 1992 Jan;6(1):5–14. doi: 10.1111/j.1365-2958.1992.tb00831.x. [DOI] [PubMed] [Google Scholar]
  11. Lohman T. M. Helicase-catalyzed DNA unwinding. J Biol Chem. 1993 Feb 5;268(4):2269–2272. [PubMed] [Google Scholar]
  12. Lu M., Guo Q., Kallenbach N. R. Effect of sequence on the structure of three-arm DNA junctions. Biochemistry. 1991 Jun 18;30(24):5815–5820. doi: 10.1021/bi00238a001. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Panyutin I. G., Hsieh P. Formation of a single base mismatch impedes spontaneous DNA branch migration. J Mol Biol. 1993 Mar 20;230(2):413–424. doi: 10.1006/jmbi.1993.1159. [DOI] [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. 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]
  19. Parsons C. A., West S. C. Formation of a RuvAB-Holliday junction complex in vitro. J Mol Biol. 1993 Jul 20;232(2):397–405. doi: 10.1006/jmbi.1993.1399. [DOI] [PubMed] [Google Scholar]
  20. Ryder L., Whitby M. C., Lloyd R. G. Mutation of recF, recJ, recO, recQ, or recR improves Hfr recombination in resolvase-deficient ruv recG strains of Escherichia coli. J Bacteriol. 1994 Mar;176(6):1570–1577. doi: 10.1128/jb.176.6.1570-1577.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sharples G. J., Whitby M. C., Ryder L., Lloyd R. G. A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions. Nucleic Acids Res. 1994 Feb 11;22(3):308–313. doi: 10.1093/nar/22.3.308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Stasiak A., Tsaneva I. R., West S. C., Benson C. J., Yu X., Egelman E. H. The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7618–7622. doi: 10.1073/pnas.91.16.7618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Tsaneva I. R., Müller B., West S. C. RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1315–1319. doi: 10.1073/pnas.90.4.1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. West S. C., Cassuto E., Howard-Flanders P. Mechanism of E. coli RecA protein directed strand exchanges in post-replication repair of DNA. Nature. 1981 Dec 17;294(5842):659–662. doi: 10.1038/294659a0. [DOI] [PubMed] [Google Scholar]
  27. West S. C. Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem. 1992;61:603–640. doi: 10.1146/annurev.bi.61.070192.003131. [DOI] [PubMed] [Google Scholar]
  28. West S. C. The processing of recombination intermediates: mechanistic insights from studies of bacterial proteins. Cell. 1994 Jan 14;76(1):9–15. doi: 10.1016/0092-8674(94)90168-6. [DOI] [PubMed] [Google Scholar]
  29. Whitby M. C., Ryder L., Lloyd R. G. Reverse branch migration of Holliday junctions by RecG protein: a new mechanism for resolution of intermediates in recombination and DNA repair. Cell. 1993 Oct 22;75(2):341–350. doi: 10.1016/0092-8674(93)80075-p. [DOI] [PubMed] [Google Scholar]
  30. Wong I., Lohman T. M. Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding. Science. 1992 Apr 17;256(5055):350–355. doi: 10.1126/science.256.5055.350. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES