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..., 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..., # 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...,

MLH1-MLH3（MutLγ）的核酸内切酶活性可被MSH4-MSH5（MutSγ）、EXO1以及RFC-PCNA激活，以将减数分裂重组中间体如双霍利迪连接体（double Holliday junctions，HJs）解析为交换产物。本研究证实，EXO1可通过自身的MIP基序以及新发现的、以EXO1-I403为中心的结构域与MLH1直接相互作用。出乎意料的是，破坏该相互作用仅能部分抑制MutLγ的活性。本研究同时发现，EXO1可与MutSγ直接结合。至关重要的是，EXO1上的单点突变W371E会破坏其与MSH4的相互作用，并完全消除其激活MutLγ切割DNA的能力，但不会影响其自身的核酸酶功能。最后，破坏EXO1核酸酶结构域中负责结合镁离子的氨基酸残基，并不会影响MutSγ-MutLγ复合物的活性；而EXO1中介导与双链DNA（double-stranded DNA，dsDNA）相互作用的氨基酸残基的完整性则是必需的。本研究结果表明，EXO1是减数分裂解析酶复合物中不可或缺的结构组分，相关研究结果支撑了……。本研究上传的文件包含了主图及补充图中所用到的质量光度测定数据，上传的文件涵盖了绘制正文中主图与补充图所需的全部图表绘制信息。

蛋白质复合物的质量光度表征
质量光度测定采用TwoMP质量光度仪（Refeyn有限公司出品）完成。实验所用硼硅酸盐显微镜盖玻片（规格为1.5 H厚度，24×50 mm，购自VWR）需依次经Milli-Q水、异丙醇、Milli-Q水浸泡清洗，随后用氮气吹干。随后将硅胶垫圈（CultureWell可重复使用垫圈，购自Grace Bio-Labs）置于洁净盖玻片上，以形成预设凹槽。为将光学反射干涉对比度转换为分子质量，需使用已知的蛋白质分子量标准品（NativeMark未染色蛋白质标准品，购自Invitrogen）进行校准。进行质量测定时，凹槽内加入18 μL测定缓冲液（25 mM Tris-HCl pH 7.5……）

# EXO1通过与MLH1、MSH4及DNA的保守相互作用促进减数分裂相关的MLH1-MLH3核酸内切酶活性
数据集DOI：10.5061/dryad.6m905qgbn

## 数据与文件结构说明
本数据集包含了论文《EXO1通过与MLH1、MSH4及DNA的保守相互作用促进减数分裂相关的MLH1-MLH3核酸内切酶活性》中主图与补充图所对应的质量光度测定数据。上传的文件包含了绘制正文中主图与补充图各子图所需的全部信息，以及对应的文件名。

## **质量光度测定数据**
本数据集包含：
* 开放HDF5格式的原始质量光度测定文件（`.mp`）
* 由质量光度仪采集的实时成像视频文件（`.mp4`）
每个`.mp`文件及其对应的`.mp4`文件均与论文主图及补充图中的单组实验结果对应，包含正文中标注的特定蛋白质构建体相关信息……