EXO1 promotes the meiotic MLH1-MLH3 endonuclease through conserved interactions with MLH1, MSH4 and DNA

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Meiotic recombination generates genetic diversity.
• Resolving Holliday junctions (HJs) into crossovers is essential for accurate chromosome segregation.
• The MutSγ (MSH4-MSH5) and MutLγ (MLH1-MLH3) complexes, along with EXO1, are implicated in HJ resolution.

Purpose of the Study:

• To elucidate the role of EXO1 in the meiotic resolvase complex.
• To investigate the interactions between EXO1, MutSγ, and MutLγ.
• To determine how EXO1's nuclease activity and interaction domains contribute to Holliday junction resolution.

Main Methods:

• Co-immunoprecipitation assays to study protein interactions.
• Site-directed mutagenesis to disrupt specific EXO1 interaction domains and nuclease activity.
• In vitro assays to assess DNA nicking and Holliday junction resolution activities.

Main Results:

• EXO1 directly interacts with both MLH1 (MutLγ) and MSH4 (MutSγ).
• A specific mutation (W371E) in EXO1 abolished its ability to activate MutLγ nicking by disrupting MutSγ interaction, without affecting EXO1's intrinsic nuclease function.
• Disrupting EXO1's dsDNA binding residues impaired MutSγ-MutLγ activity, while altering nuclease domain residues had no effect.
• EXO1's interaction with dsDNA and MutSγ is critical for activating MutLγ's endonuclease activity.

Conclusions:

• EXO1 acts as a structural scaffold, tethering the MutSγ-MutLγ complex to double-stranded DNA downstream of Holliday junction recognition.
• EXO1's role in activating MutLγ-mediated DNA cleavage is primarily through its interaction domains, not its intrinsic nuclease function.
• These findings highlight EXO1 as an integral component of the meiotic resolvase machinery, essential for promoting crossover formation.