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.