The Saccharomyces cerevisiae Mlh1-Mlh3 heterodimer is an endonuclease that preferentially binds to Holliday junctions.
MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX2EX4E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.
From the Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Lepakshi
L
Ranjha
Roopesh
R
Anand
Petr
P
Cejka
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D055503:Protein Multimerization / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D045566:DNA, Cruciform; D004271:DNA, Fungal; C088859:MLH1 protein, S cerevisiae; C492408:MLH3 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003850:Deoxyribonuclease I; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins
DNA Endonuclease; DNA Mismatch Repair; DNA Repair; DNA-binding Protein; Holliday Junction; Homologous Recombination; Meiosis; Mlh1; Mlh3; MutLγ