Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage*□S

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 -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. Molecular & Cellular Proteomics 14: 10.1074/ mcp.M115. 048991, 2002–2013, 2015. DNA replication during the S phase necessitates that the entire genome be duplicated with the minimum of errors. Thousands of replication forks are involved in this process and they must be coordinated to ensure that every section of DNA is only replicated once. Errors in DNA replication are likely to be a major cause of the genetic instability that can lead to cancer (1). Cells are able to prevent duplicate replication of DNA by having a distinct stage that occurs during the G1 phase when replication origins are “licensed” for replication, a process that involves the preloading of several proteins involved in DNA replication (2). As DNA is replicated at each origin, these proteins are removed, thereby ensuring that each origin fires only once during each S phase. DNA damage response kinases activated by the stalled forks prevent the replication machinery from being activated in new chromosome domains, indicating a tight relationship between the DNA damage response and the DNA replication pathways (3, 4). The first step of the replication licensing mechanism is the loading of the minichromosome maintenance (MCM) proteins on to replication origins along with origin recognition complex proteins, Cdt6 and Cdt1 (5). The eukaryotic MCM complex consists of six paralogs that form a heterohexameric ring. All eukaryotic organisms possess six homologous proteins (MCM2-MCM7) that form a heterohexameric ring that belong to the family of AAA (ATPase associated with various cellular activities) proteins and share similarities to other hexameric helicases (6). Even though additional MCM proteins have been identified in higher eukaryotes, the MCM2-MCM7 complex remains the prime candidate for the role of replicative helicase (7). MCM2–7 is required for both initiation and elongation of DNA replication, with its regulation at each stage being an essential player of eukaryotic DNA replication (8). As a critical mechanism to ensure only a single round of DNA From the ‡Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada Received February 10, 2015, and in revised form, April 28, 2015 Published, MCP Papers in Press, May 11, 2015, DOI 10.1074/ mcp.M115. 048991 Author’s Choice—Final version free via Creative Commons CC-BY license. Author contributions: R.D. , M. Douziech, and F.B. designed research; R.D. , M. Dubois, M. Douziech, and F.B. performed research; R.D. and F.B. analyzed data; F.B. wrote the paper. 1 The abbreviations used are: MCM, minichromosome maintenance; SILAC, stable isotope labeling with amino acids in cell culture; GFP, green fluorescent protein; MCMBP, MCM binding protein. Research