Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair

Erin K. Schwartz, William D. Wright, Kirk T. Ehmsen, James E. Evans, Henning Stahlberg, Wolf Dietrich Heyer

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)3065-3080
Number of pages16
JournalMolecular and Cellular Biology
Volume32
Issue number15
DOIs
StatePublished - Aug 2012

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ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Schwartz, E. K., Wright, W. D., Ehmsen, K. T., Evans, J. E., Stahlberg, H., & Heyer, W. D. (2012). Mus81-Mms4 functions as a single heterodimer to cleave nicked intermediates in recombinational DNA repair. Molecular and Cellular Biology, 32(15), 3065-3080. https://doi.org/10.1128/MCB.00547-12