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Updated: Apr 12, 2026
Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response
Published on: August 2, 2024
Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular
Romain Drissi1, Marie-Line Dubois1, Mélanie Douziech1
1From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
Abstract:
The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.
Insights
The minichromosome maintenance (MCM) complex, crucial for DNA replication, dynamically interacts with proteins involved in DNA repair and chromatin remodeling following DNA damage. This suggests a novel role for MCM proteins beyond replication, extending into DNA damage response pathways.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- The minichromosome maintenance (MCM) complex (MCM2-7) is essential for DNA replication, functioning as a DNA helicase loaded onto chromatin.
- MCM proteins are involved in initiating DNA replication and are regulated by S-phase checkpoints.
- Emerging evidence suggests MCM proteins may also play roles in DNA repair pathways.
Purpose of the Study:
- To investigate the dynamic protein interactions of the MCM complex in response to DNA damage.
- To identify novel MCM-interacting proteins and characterize their functional significance in DNA damage response.
- To explore the potential involvement of MCM proteins in chromatin remodeling during DNA repair.
Main Methods:
- Utilized stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics.
- Employed immunoprecipitation of green fluorescent protein (GFP)-tagged MCM proteins to isolate interacting partners.
- Quantified changes in protein interactions upon DNA damage induction.
- Assessed MCM protein post-translational modifications (phosphorylation, ubiquitination) and co-localization with DNA damage markers (γ-H2AX).
Main Results:
- Identified dynamic changes in MCM complex interactions with proteins including Importin7, ASF1, and CHD3 following DNA damage.
- Observed increased phosphorylation and ubiquitination on specific MCM protein sites.
- Demonstrated increased co-localization of the MCM complex with γ-H2AX at sites of DNA damage.
- Confirmed the recruitment of MCM proteins to DNA damage sites.
Conclusions:
- The MCM complex exhibits dynamic protein interactions in response to DNA damage, indicating a role beyond DNA replication.
- MCM proteins are recruited to DNA damage sites and may participate in chromatin remodeling during DNA repair.
- These findings reveal a novel function for MCM proteins in the cellular DNA damage response.
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