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.

Methods
In the uploaded files, we present mass photomether data from the main and supplementary figures. The uploaded files include all the information required to plot the graphs as indicated in the attached main and supplementary figures.

Mass photometric characterization of protein complexes

Mass photometry measurements were conducted using a TwoMP mass photometer (Refeyn Ltd). Borosilicate microscope glass coverslips (No. 1.5 H thickness, 24 x 50 mm, VWR) were cleaned by soaking them sequentially in Milli-Q-water, isopropanol, and Milli-Q-water and then drying them with a stream of gaseous nitrogen. Next, silicone gaskets (CultureWell Reusable Gasket, Grace Bio-Labs) were placed on the clean glass coverslips to create defined wells. To convert optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMark Unstained Protein Standard, Invitrogen) was measured. For mass measurements, wells were filled with 18 ml measurement buffer (25 mM Tris-HCl pH 7.5, 75 mM NaCl) to facilitate focusing the microscope onto the glass plate surface. Next, respective proteins/protein complexes were added into the well, and sample binding to the glass coverslip surface was monitored for 1 min using the AcquireMP software (Refeyn Ltd). Data analysis was carried out using DiscoverMP software (Refeyn Ltd).

All Data were named according to how they appear in the results section. Main figures 3d, and Supplementary Figures 4b, 5f, 6b and 7c are mass photometry data.