DOI: 10.5061/dryad.6m905qgbn
title: EXO1 promotes the meiotic MLH1-MLH3 endonuclease through conserved interactions with MLH1, MSH4 and DNA

description: 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.
description: 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. <strong>Mass
 photometric characterization of protein complexes</strong>
 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.
description: # EXO1 promotes the meiotic MLH1-MLH3 endonuclease through conserved
 interactions with MLH1, MSH4 and DNA Dataset DOI:
 [10.5061/dryad.6m905qgbn](10.5061/dryad.6m905qgbn) ## Description of the
 data and file structure Here, we present mass photometer data from the
 main and supplementary figures of the manuscript, “EXO1 promotes the
 meiotic MLH1-MLH3 endonuclease through conserved interactions with MLH1,
 MSH4 and DNA”. The files uploaded include all the information needed to
 plot the panels as indicated in the attached main and supplementary
 figures and the file names. ## **Mass photometer data** The dataset
 comprises: * **Raw mass photometry files** (`.mp`) in the open HDF5 file
 format. * **Video files** (`.mp4`) showing the real-time movies captured
 by the mass photometer. Each `.mp` file and its corresponding `.mp4` file
 are associated with an individual result shown in the main and
 supplementary figures of the manuscript, including the specific protein
 construct(s) indicated in each description. However, these files may be
 viewed, analyzed, or repurposed independently of the manuscript as well.
 ## File Information **Main Figures** 1. **Figure 3d top: EXO1
 (D173A)-FLAG** * Main_Fig_3d_top.mp (raw mass photometry data) *
 Main_Fig_3d_top.mp4 (movie) 2. **Figure 3d middle: MutSγ
 (MSH4-Strep-MSH5-His)** * Main_Fig_3d_middle.mp (raw mass photometry data)
 * Main_Fig_3d_middle.mp4 (movie) 3. **Figure 3d bottom: EXO1 (D173A)-FLAG
 and MutSγ complex** * Main_Fig_3d_bottom.mp (raw mass photometry data) *
 Main_Fig_3d_bottom.mp4 (movie) **Supplementary Figures** 1.     
 **Supplementary Figure 4b left: EXO1-1-390-FLAG** * Sup_Fig_4b_right.mp
 (raw mass photometry data) * Sup_Fig_4b_right.mp4 (movie) 2.     
 **Supplementary Figure 4b right: EXO1-1-352-FLAG** * Sup_Fig_4b_right.mp
 (raw mass photometry data) - Sup_Fig_4b_right.mp4 (movie) 3.     
 **Supplementary Figure 5f left: EXO1 (W371E Y375E)** * Sup_Fig_5f_left.mp
 (raw mass photometry data) - Sup_Fig_5f_left.mp4 (movie) 4.     
 **Supplementary Figure 5f right: EXO1 1-390 (W371E Y375E)** *
 Sup_Fig_5f_right.mp (raw mass photometry data) - Sup_Fig_5f_right.mp4
 (movie) 5.      **Supplementary Figure 6b top left: MSH4 (F288E)-MSH5** *
 Sup_Fig_6b_top_left.mp (raw mass photometry data) -
 Sup_Fig_6b_top_left.mp4 (movie) 6.      **Supplementary Figure 6b top
 right: MSH4 (F225E F288E)-MSH5** * Sup_Fig_6b_top_right.mp (raw mass
 photometry data) - Sup_Fig_6b_top_right.mp4 (movie) 7.     
 **Supplementary Figure 6b bottom left: MSH4 (T238E Y244D F288E)-MSH5** *
 Sup_Fig_6b_bottom_left.mp (raw mass photometry data) -
 Sup_Fig_6b_bottom_left.mp4 (movie) 8.      **Supplementary Figure 6b
 bottom right: MSH4 (F225E)-MSH5** * Sup_Fig_6b_bottom_right.mp (raw mass
 photometry data) - Sup_Fig_6b_bottom_right.mp4 (movie) 9.     
 **Supplementary Figure 7c left: EXO1-KD-KD-FLAG** * Sup_Fig_7c_left.mp
 (raw mass photometry data) - Sup_Fig_7c_left.mp4 (movie) 10. 
  **Supplementary Figure 7c right: EXO1 1-390-KD-KD-FLAG** *
 Sup_Fig_7c_right.mp (raw mass photometry data) - Sup_Fig_7c_right.mp4
 (movie)  In total, there are **13 `.mp` files** and **13 `.mp4` files**.
 ## Opening and Viewing the Data ### Raw Mass Photometry Files (`.mp`) *
 The `.mp` files follow the open HDF5 file format. * **Recommended
 software**: * **DiscoverMP** (Refeyn Ltd) is the proprietary software used
 to analyze the data and export the videos. * **Open-source options**: Any
 HDF5-compatible viewer such as [HDFView (from the HDF
 Group)](https://portal.hdfgroup.org/display/support/Download+HDFView)
 Using DiscoverMP is recommended for reproducing exact mass assignments and
 visualization of the molecular count histograms. ### Movie Files (`.mp4`)
 * Standard video files that can be opened with most media players (e.g.,
 VLC, QuickTime, Windows Media Player). * Each `.mp4` is a 1-minute video
 of molecules binding to the coverslip surface as recorded by the mass
 photometer. ## Interpreting the Data When opened in DiscoverMP or
 HDF5-compatible software, `.mp` files contain: * **Reflection-interference
 contrast** measurements for individual molecules over time. * **Mass
 (kDa)**: Determined by calibrating the reflection signal with a protein
 standard (NativeMark™ Unstained Protein Standard, Invitrogen). *
 **Counts**: The number of molecules detected within a specific mass range
 or throughout the movie. The typical unit for molecular mass in these
 files is **kilodaltons (kDa)**. The “counts” are unitless (simply the
 accumulated counts of detected particles). ## Additional Notes * **Data
 Collection**: AcquireMP (Refeyn Ltd, version 2023 Rl.l) was used to
 collect all raw data. * **Data Analysis**: DiscoverMP (Refeyn Ltd, version
 v2023 Rl.2) was used to analyze the `.mp` files and to create the
 histogram plots. * For reproducibility, open the `.mp` files in DiscoverMP
 to obtain molecular mass distributions and corresponding counts. * The
 `.mp4` videos serve as a visual reference of molecules interacting with
 the coverslip surface during the 1-minute recording