The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism.
Functional studies strongly suggest that the Mus81-Eme1 complex resolves Holliday junctions (HJs) in fission yeast, but in vitro it preferentially cleaves flexible three-way branched structures that model replication forks or 3' flaps. Here we report that a nicked HJ is the preferred substrate of endogenous and recombinant Mus81-Eme1. Cleavage occurs specifically on the strand that opposes the nick, resulting in resolution of the structure into linear duplex products. Resolving cuts made by the endogenous Mus81-Eme1 complex on an intact HJ are quasi-simultaneous, indicating that Mus81-Eme1 resolves HJs by a nick and counternick mechanism, with a large rate enhancement of the second cut arising from the flexible nature of the nicked HJ intermediate. Recombinant Mus81-Eme1 is ineffective at making the first cut. We also report that HJs accumulate in a DNA polymerase alpha mutant that lacks Mus81, providing further evidence that the Mus81-Eme1 complex targets HJs in vivo.
Gaillard
Pierre-Henri L
PHL
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Noguchi
Eishi
E
Shanahan
Paul
P
Russell
Paul
P
D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D046911:Macromolecular Substances; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C095888:xeroderma pigmentosum group F protein; D004247:DNA; D004256:DNA Polymerase I; C442837:Eme1 protein, S pombe; D004720:Endonucleases; C411624:MUS81 protein, S cerevisiae