The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism

Mol Cell. 2003 Sep;12(3):747-59. doi: 10.1016/s1097-2765(03)00342-3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cells, Cultured
  • DNA / genetics
  • DNA / metabolism*
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism
  • DNA Replication / genetics*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Gene Expression Regulation, Enzymologic / genetics
  • Gene Expression Regulation, Fungal / genetics
  • Macromolecular Substances
  • Mutation / genetics
  • Nucleic Acid Conformation
  • Proteins / metabolism
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces / enzymology*
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism*
  • Substrate Specificity

Substances

  • DNA-Binding Proteins
  • Macromolecular Substances
  • Proteins
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces pombe Proteins
  • xeroderma pigmentosum group F protein
  • DNA
  • DNA Polymerase I
  • Eme1 protein, S pombe
  • Endonucleases
  • MUS81 protein, S cerevisiae