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Nucleic Acids Res. Apr 1, 1998; 26(7): 1560–1566.
PMCID: PMC147448

Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli.


Homologous recombination is a fundamental cellular process that shapes and reshapes the genomes of all organisms and promotes repair of damaged DNA. A key step in this process is the resolution of Holliday junctions formed by homologous DNA pairing and strand exchange. In Escherichia coli , a Holliday junction is processed into recombinant products by the concerted activities of the RuvA and RuvB proteins, which together drive branch migration, and RuvC endonuclease, which resolves the structure. In the absence of RuvABC, recombination can be promoted by increasing the expression of the RusA endonuclease, a Holliday junction resolvase encoded by a cryptic prophage gene. Here, we describe the DNA binding properties of RusA. We found that RusA was highly selective for branched molecules and formed complexes with these structures even in the presence of a large excess of linear duplex DNA. However, it does bind weakly to linear duplex DNA. Under conditions where there was no detectable binding to duplex DNA, RusA formed a highly structured complex with a synthetic Holliday junction that was remarkably stable and insensitive to divalent metal ions. The duplex arms were found to adopt a specific alignment within this complex that approximated to a tetrahedral conformation of the junction.

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Selected References

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  • Sharples GJ, Chan SN, Mahdi AA, Whitby MC, Lloyd RG. Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions. EMBO J. 1994 Dec 15;13(24):6133–6142. [PMC free article] [PubMed]
  • West SC. Processing of recombination intermediates by the RuvABC proteins. Annu Rev Genet. 1997;31:213–244. [PubMed]
  • Rafferty JB, Sedelnikova SE, Hargreaves D, Artymiuk PJ, Baker PJ, Sharples GJ, Mahdi AA, Lloyd RG, Rice DW. Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction. Science. 1996 Oct 18;274(5286):415–421. [PubMed]
  • Tsaneva IR, Müller B, West SC. 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. [PubMed]
  • 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. [PubMed]
  • Shah R, Bennett RJ, West SC. Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro. Cell. 1994 Dec 2;79(5):853–864. [PubMed]
  • Whitby MC, Bolt EL, Chan SN, Lloyd RG. Interactions between RuvA and RuvC at Holliday junctions: inhibition of junction cleavage and formation of a RuvA-RuvC-DNA complex. J Mol Biol. 1996 Dec 20;264(5):878–890. [PubMed]
  • Eggleston AK, Mitchell AH, West SC. In vitro reconstitution of the late steps of genetic recombination in E. coli. Cell. 1997 May 16;89(4):607–617. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Lloyd RG, Benson FE, Shurvinton CE. Effect of ruv mutations on recombination and DNA repair in Escherichia coli K12. Mol Gen Genet. 1984;194(1-2):303–309. [PubMed]
  • Sharples GJ, Benson FE, Illing GT, Lloyd RG. 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. [PubMed]
  • Mahdi AA, Sharples GJ, Mandal TN, Lloyd RG. Holliday junction resolvases encoded by homologous rusA genes in Escherichia coli K-12 and phage 82. J Mol Biol. 1996 Apr 5;257(3):561–573. [PubMed]
  • Mandal TN, Mahdi AA, Sharples GJ, Lloyd RG. 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. [PMC free article] [PubMed]
  • Lloyd RG, Sharples GJ. Dissociation of synthetic Holliday junctions by E. coli RecG protein. EMBO J. 1993 Jan;12(1):17–22. [PMC free article] [PubMed]
  • Whitby MC, Ryder L, Lloyd RG. 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. [PubMed]
  • Whitby MC, Lloyd RG. Branch migration of three-strand recombination intermediates by RecG, a possible pathway for securing exchanges initiated by 3'-tailed duplex DNA. EMBO J. 1995 Jul 17;14(14):3302–3310. [PMC free article] [PubMed]
  • McGlynn P, Al-Deib AA, Liu J, Marians KJ, Lloyd RG. The DNA replication protein PriA and the recombination protein RecG bind D-loops. J Mol Biol. 1997 Jul 11;270(2):212–221. [PubMed]
  • Chan SN, Harris L, Bolt EL, Whitby MC, Lloyd RG. Sequence specificity and biochemical characterization of the RusA Holliday junction resolvase of Escherichia coli. J Biol Chem. 1997 Jun 6;272(23):14873–14882. [PubMed]
  • Dunderdale HJ, Sharples GJ, Lloyd RG, West SC. Cloning, overexpression, purification, and characterization of the Escherichia coli RuvC Holliday junction resolvase. J Biol Chem. 1994 Feb 18;269(7):5187–5194. [PubMed]
  • Duckett DR, Murchie AI, Diekmann S, von Kitzing E, Kemper B, Lilley DM. The structure of the Holliday junction, and its resolution. Cell. 1988 Oct 7;55(1):79–89. [PubMed]
  • Picksley SM, Parsons CA, Kemper B, West SC. 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. [PubMed]
  • Lloyd RG, Sharples GJ. Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. Nucleic Acids Res. 1993 Apr 25;21(8):1719–1725. [PMC free article] [PubMed]
  • Parsons CA, Kemper B, West SC. Interaction of a four-way junction in DNA with T4 endonuclease VII. J Biol Chem. 1990 Jun 5;265(16):9285–9289. [PubMed]
  • Dunderdale HJ, Benson FE, Parsons CA, Sharples GJ, Lloyd RG, West SC. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature. 1991 Dec 19;354(6354):506–510. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Benson FE, West SC. Substrate specificity of the Escherichia coli RuvC protein. Resolution of three- and four-stranded recombination intermediates. J Biol Chem. 1994 Feb 18;269(7):5195–5201. [PubMed]
  • Takahagi M, Iwasaki H, Shinagawa H. Structural requirements of substrate DNA for binding to and cleavage by RuvC, a Holliday junction resolvase. J Biol Chem. 1994 May 27;269(21):15132–15139. [PubMed]
  • Bennett RJ, West SC. Structural analysis of the RuvC-Holliday junction complex reveals an unfolded junction. J Mol Biol. 1995 Sep 15;252(2):213–226. [PubMed]
  • Shah R, Cosstick R, West SC. The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts. EMBO J. 1997 Mar 17;16(6):1464–1472. [PMC free article] [PubMed]
  • Ariyoshi M, Vassylyev DG, Iwasaki H, Nakamura H, Shinagawa H, Morikawa K. Atomic structure of the RuvC resolvase: a holliday junction-specific endonuclease from E. coli. Cell. 1994 Sep 23;78(6):1063–1072. [PubMed]
  • Saito A, Iwasaki H, Ariyoshi M, Morikawa K, Shinagawa H. Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7470–7474. [PMC free article] [PubMed]
  • White MF, Lilley DM. The resolving enzyme CCE1 of yeast opens the structure of the four-way DNA junction. J Mol Biol. 1997 Feb 14;266(1):122–134. [PubMed]
  • Cooper JP, Hagerman PJ. Gel electrophoretic analysis of the geometry of a DNA four-way junction. J Mol Biol. 1987 Dec 20;198(4):711–719. [PubMed]
  • Pöhler JR, Giraud-Panis MJ, Lilley DM. T4 endonuclease VII selects and alters the structure of the four-way DNA junction; binding of a resolution-defective mutant enzyme. J Mol Biol. 1996 Aug 2;260(5):678–696. [PubMed]
  • White MF, Giraud-Panis MJ, Pöhler JR, Lilley DM. Recognition and manipulation of branched DNA structure by junction-resolving enzymes. J Mol Biol. 1997 Jun 27;269(5):647–664. [PubMed]
  • Lilley DM, Kemper B. Cruciform-resolvase interactions in supercoiled DNA. Cell. 1984 Feb;36(2):413–422. [PubMed]
  • Jensch F, Kemper B. Endonuclease VII resolves Y-junctions in branched DNA in vitro. EMBO J. 1986 Jan;5(1):181–189. [PMC free article] [PubMed]
  • Kleff S, Kemper B. Initiation of heteroduplex-loop repair by T4-encoded endonuclease VII in vitro. EMBO J. 1988 May;7(5):1527–1535. [PMC free article] [PubMed]
  • Solaro PC, Birkenkamp K, Pfeiffer P, Kemper B. Endonuclease VII of phage T4 triggers mismatch correction in vitro. J Mol Biol. 1993 Apr 5;230(3):868–877. [PubMed]
  • Al-Deib AA, Mahdi AA, Lloyd RG. Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12. J Bacteriol. 1996 Dec;178(23):6782–6789. [PMC free article] [PubMed]

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