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

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.

Keywords: APEX2; DNA replication; MCM2; proximity labeling.

Figure 1

Construction of fusion protein plasmids and validation of stably transfected cell lines. (a) Schematic representation of the functional elements in the MCM2-V5-APEX2 fusion protein plasmid. (b) Validation of MCM2 protein and V5 tag expression. MCM2-APEX2: cells stably overexpressing the MCM2-V5-APEX2 fusion protein; WT: ESC and NIH/3T3 wild-type cells. (c) Immunofluorescence images show co-immunostaining of the DAPI (blue), V5 tag (green) and streptavidin (red) in Mcm2-APEX2 cells derived from ESC and NIH/3T3 cell lines, with or without hydrogen peroxide treatment. Localization is observed in the nucleus. The inset displays a magnified portion of the cell. DAPI: 4′,6-diamidino-2-phenylindole; scale bar: 5 μm; H₂O₂+: treated with hydrogen peroxide; H₂O₂−: untreated with hydrogen peroxide. (d) Diagram illustrating the formation of the DNA replication initiation complex and the APEX2 labeling process. The DNA replication initiation complex containing MCM2 is assembled during the G1-G1/S phase. At the G1/S transition, BP and H₂O₂ are added and incubated for 1 min to induce biotinylation of proteins within 20 nm of APEX2. LC-MS/MS: liquid chromatography–tandem mass spectrometry; pre-RC: pre-replication complex; pre-IC: pre-initiation complex. All experiments were conducted with three biological replicates.

Figure 2

FACS analysis of cell cycle synchronization and APEX2 proximity labeling. (a,b) FACS analysis of PI-stained synchronized and unsynchronized ESC and NIH/3T3 MCM2-APEX2 cell lines. The values indicated on the bar chart represent the mean percentages of each cell cycle phase, calculated from three independent biological replicates. Detailed data can be found in Additional file S2. (c,d) Streptavidin–HRP Western blot detection of H₂O₂-induced protein biotinylation in cell lysates from ESC or NIH/3T3 MCM2-APEX2 cells. Asterisks denote endogenous biotinylated proteins. Syn: synchronized; AS: asynchronous.

Figure 3

Analysis of MCM2-APEX2 proximal proteome data. (a) The Venn diagram illustrates the proximal proteins of the DNA replication initiation complex identified from synchronized ESC-MCM2-APEX2 and NIH/3T3-MCM2-APEX2 cells. (b) The heatmap shows how the expression levels of shared proteins varied among various samples. Log-transformed proximal protein data were processed using MaxQuant after the addition of H₂O₂ (H₂O₂+). The experiment without H₂O₂ (H₂O₂−) served as a control. Two biological replicates were used for each sample (R1 and R2). Expression data were normalized and presented using log₁₀ transformation. (c) GO enrichment analysis of overlapping MCM2 proximal proteins in ESC and NIH/3T3 based on biological processes, with bubble color indicating −log₁₀ p-values of enrichment terms. (d) Network visualization of selected GO biological processes associated with the MCM2 proximal proteome, with individual proteins represented as nodes and interactions as edges. Interactions were retrieved from the STRING database with a confidence score > 0.4. Node colors represent the biological processes involved. (e) Venn diagram comparing cell cycle-dependent proteins from the Human Protein Atlas (HPA) database with 147 overlapping proteins. (f) Interaction network of Mcm2 and potential interacting proteins analyzed using STRING, with interaction scores > 0.4.

Figure 4

Visualization of MCM2 protein interactions with potential interacting proteins in living cells using BiFC analysis. The inset displays a magnified portion of the cell. DAPI (blue): 4′,6-diamidino-2-phenylindole; green: immunofluorescence staining with MCM2 antibody; red: BiFC-mScarlet3 fluorescent protein.