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

Mol Cell Biol. 2012 Aug;32(15):3065-80. doi: 10.1128/MCB.00547-12. Epub 2012 May 29.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / metabolism
  • Chromatin / metabolism
  • DNA / genetics
  • DNA / metabolism
  • DNA, Cruciform / genetics
  • DNA, Cruciform / metabolism
  • DNA-Binding Proteins / metabolism*
  • Endonucleases / metabolism*
  • Flap Endonucleases / metabolism*
  • Protein Multimerization
  • Protein Serine-Threonine Kinases / metabolism
  • Recombination, Genetic
  • Recombinational DNA Repair*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Cell Cycle Proteins
  • Chromatin
  • DNA, Cruciform
  • DNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • DNA
  • Protein Serine-Threonine Kinases
  • CDC5 protein, S cerevisiae
  • Endonucleases
  • Flap Endonucleases
  • MUS81 protein, S cerevisiae
  • MMS4 protein, S cerevisiae