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

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.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)¹ 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 replication, the loading of additional MCM2–7 complexes onto origins of replication is inactivated by redundant mechanisms after passage into S phase (9).The MCM complex plays a crucial role in determining the replication potential of cells, but recent work suggests that MCM proteins are not only targets of the S-phase checkpoints, but they also interact directly with components of the checkpoint and repair pathways (10, 11). In yeast, temperature sensitive MCM cells at restrictive temperature contain numerous foci recognized by the phosphorylated histone H2AX antibody (12), suggesting a role in the repair of DNA double-strand breaks. Although, in principle, only two DNA helicase activities are required to establish a bidirectional replication fork from each origin, a relatively large excess of MCM complexes are loaded at origins of replication and distributed along the chromatin (13). Their function is not well understood, and most of them are displaced from the DNA during S-phase, apparently without having played an active role in DNA replication. The “MCM paradox” refers to the fact that, at least in yeast, Xenopus, Drosophila, and mammalian cells, it is possible to reduce the concentration of MCM proteins by more than 90% without impairing DNA replication (14–18) and also refers to the observation that the majority of MCM complexes do not localize to the sites of DNA synthesis in mammalian cells, further suggesting a potential role for the MCM proteins beyond DNA replication.Using a combination of stable isotope labeling with amino acids in cell culture (SILAC)–based quantitative proteomics (19) with immunoprecipitation of green fluorescent protein (GFP)-tagged fusion proteins (20), we identified differences in protein binding partners with the MCM complex following DNA damage. Stable cell lines expressing GFP-tagged MCM2 and MCM5 were used in immunoprecipitation experiments from cells that were either mock treated, or treated with Etoposide for 15, 60, and 240 min. Etoposide is an antitumor drug that stabilizes a covalent complex between the DNA topoisomerase II and DNA by interfering with the cleavage-ligation reaction of the topoisomerase (21). This revealed specific interaction between the MCM complex and several proteins such as Nucleophosmin, BAG2, UPP1, and HDAC10. Interestingly, the MCM complex showed dynamic changes in interaction with Importin7 and the histone chaperone ASF1, and a decrease in interaction with the Chromodomain helicase DNA binding protein 3 (CHD3) resulting from the treatment with etoposide. This increase in interaction with ASF1 was followed by an enrichment of histone proteins, suggesting a novel role for the MCM proteins in histone deposition on chromatin following DNA damage.