title: Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.
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.
authors.affiliation: From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Romain
authors.initials: R
authors.lastname: Drissi
authors.affiliation: From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Marie-Line
authors.initials: ML
authors.lastname: Dubois
authors.affiliation: From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Douziech
authors.affiliation: From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada fm.boisvert@usherbrooke.ca.
authors.forename: François-Michel
authors.initials: FM
authors.lastname: Boisvert
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D049452:Green Fluorescent Proteins; D005047:Etoposide; D064110:Minichromosome Maintenance Proteins