Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair.
The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3' flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3' flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions.
Department of Microbiology, University of California, Davis, Davis, California, USA.
Erin K
EK
Schwartz
William D
WD
Wright
Kirk T
KT
Ehmsen
James E
JE
Evans
Henning
H
Stahlberg
Wolf-Dietrich
WD
Heyer
D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D045585:Flap Endonucleases / Q000378:metabolism*; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011995:Recombination, Genetic; D059767:Recombinational DNA Repair; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D017346:Protein Serine-Threonine Kinases; C081927:CDC5 protein, S cerevisiae; D004720:Endonucleases; D045585:Flap Endonucleases; C411624:MUS81 protein, S cerevisiae; C112337:MMS4 protein, S cerevisiae