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

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Crossover formation is essential for genetic recombination in eukaryotes.
• The XPF-family endonuclease Mus81-Mms4 (Eme1) plays a significant role in this process.
• A central question concerns the molecular mechanism by which Mus81-Mms4 processes DNA structures like Holliday junctions.

Purpose of the Study:

• To investigate the oligomeric state and substrate preference of Saccharomyces cerevisiae Mus81-Mms4.
• To determine if Mus81-Mms4 multimerizes in vitro and in vivo.
• To elucidate the role of Cdc5 kinase in Mus81-Mms4 activation and substrate processing.

Main Methods:

• Biochemical assays to determine the solution structure of Mus81-Mms4.
• DNA binding experiments to assess substrate interaction.
• Immunoprecipitation to study Mus81-Mms4 multimerization in vivo.
• In vitro kinase assays with Cdc5.

Main Results:

• Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer in solution and when bound to DNA.
• Immunoprecipitation confirmed that Mus81-Mms4 does not multimerize in vivo or on chromatin.
• Cdc5 kinase activates Mus81-Mms4 on 3' flaps and Holliday junctions but does not induce multimerization or a preference for intact Holliday junctions.
• Mus81-Mms4 preferentially cleaves nicked recombination intermediates (e.g., D-loops, nicked Holliday junctions, 3' flaps) over intact Holliday junctions.

Conclusions:

• Mus81-Mms4 functions as a monomeric heterodimer.
• The enzyme processes nicked intermediates, not intact Holliday junctions, during crossover formation.
• Mus81-dependent crossing over likely occurs via a noncanonical mechanism not involving the cleavage of classic Holliday junctions.