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

The endonuclease activity of MLH1-MLH3 (MutLγ) is stimulated by MSH4-MSH5
(MutSγ), EXO1, and RFC-PCNA to resolve meiotic recombination intermediates
such as double Holliday junctions (HJs) into crossovers. We show that EXO1
directly interacts with MLH1 via the EXO1 MIP motif, and a newly
identified patch centered around EXO1-I403. Disrupting this interaction
unexpectedly only partially inhibited MutLγ. We found that EXO1 also
directly interacts with MutSγ. Crucially, a single point mutation in EXO1
(W371E) impairs its interaction with MSH4 and completely abolished its
ability to activate DNA nicking by MutLγ without affecting its intrinsic
nuclease function. Finally, disrupting magnesium coordinating residues in
the nuclease domain of EXO1 has no impact on MutSγ-MutLγ activity, while
the integrity of EXO1 residues mediating interactions with double-stranded
DNA (dsDNA) is important. Our findings suggest EXO1 is an integral
structural component of the meiotic resolvase complex, supported by
conserved interactions with MutSγ, MutLγ and dsDNA. We propose that EXO1
helps tether MutSγ-MutLγ to dsDNA downstream of HJ recognition to promote
DNA cleavage.