# Identification of MCM2-Interacting Proteins Associated with Replication Initiation Using APEX2-Based Proximity Labeling Technology

## TL;DR

This study identifies proteins that interact with MCM2 during DNA replication initiation using a proximity labeling technique.

## Contribution

The novel use of APEX2-based proximity labeling reveals transient MCM2-interacting proteins in mouse cells.

## Key findings

- MCM2-interacting proteins CD2BP2, VRK1, and GTSE1 were identified in mouse ESCs and NIH/3T3 cells.
- Bimolecular fluorescence complementation confirmed interactions between MCM2 and the candidate proteins.
- The study provides insights into the transient regulatory network of the DNA replication initiation complex.

## Abstract

DNA replication is a crucial biological process that ensures the accurate transmission of genetic information, underpinning the growth, development, and reproduction of organisms. Abnormalities in DNA replication are a primary source of genomic instability and tumorigenesis. During DNA replication, the assembly of the pre-RC at the G1-G1/S transition is a crucial licensing step that ensures the successful initiation of replication. Although many pre-replication complex (pre-RC) proteins have been identified, technical limitations hinder the detection of transiently interacting proteins. The APEX system employs peroxidase-mediated rapid labeling with high catalytic efficiency, enabling protein labeling within one minute and detection of transient protein interactions. MCM2 is a key component of the eukaryotic replication initiation complex, which is essential for DNA replication. In this study, we fused MCM2 with enhanced APEX2 to perform in situ biotinylation. By combining this approach with mass spectrometry, we identified proteins proximal to the replication initiation complex in synchronized mouse ESCs and NIH/3T3. Through a comparison of the results from both cell types, we identified some candidate proteins. Interactions between MCM2 and the candidate proteins CD2BP2, VRK1, and GTSE1 were confirmed by bimolecular fluorescence complementation. This research establishes a basis for further study of the component proteins of the conserved DNA replication initiation complex and the transient regulatory network involving its proximal proteins.