Regulation of DNA replication initiation and fork progression is critical for the maintenance of genome stability. The mini-chromosome maintenance (MCM) proteins are uniquely required for pre-replicative complex (pre-RC) assembly, origin firing and replisome progression. Because all MCM proteins are essential for life, their role and mechanism of action beyond pre-RC assembly has been difficult to assess. We have purified active recombinant MCM complexes that support all known MCM functions associated with DNA replication in MCM- depleted cell-free extracts. This system allows us to bypass the experimental limitation associated with MCM's essential roles. MCM are connected with the maintenance of genome stability in several ways. MCM proteins are targets of the ATM/ATR kinases, down-regulation of MCM protein levels triggers genome instability and increased origin activity by the MCM-binding protein Myc, activates a DNA damage response, generates damage and genome instability. Finally, MCM are aberrantly expressed in a variety of tumor and are used as prognostic markers for tumor progression. First, we will investigate two key aspects of MCM activity: the mechanism of DNA unwinding and the regulation of MCM unloading from chromatin. Phosphorylation of the MCM complex by CDC7 protein kinase is an essential step in the activation of origins. Next, we want to characterize these phosphorylation events and assess their physiological consequences. A striking feature of initiation of DNA replication is that a vast excess of MCM complexes are loaded on chromatin and only a subset of these potential sites of DNA unwinding is specified to become functional origins of replication. We have determined that the Myc proto-oncogene binds to MCM proteins and plays a role in origin specification. We propose to characterize this novel function of Myc. We will also investigate the potential role of MCM complexes that have not been specified to be functional origins, in replication restart. Finally, we will probe further the role of MCM in the maintenance of genome stability by analyzing how down-regulation of MCM proteins or unscheduled activation of replication origins generates DNA damage. We will also determine the contribution of MCM phosphorylation by the ATM and ATR protein kinase to the maintenance of genome stability during DNA replication. We anticipate that these studies will provide important insights into the mechanism of MCM proteins functions. In particular, probing the connection between MCM, Myc and the maintenance of genome stability during S-phase will be critical to identify targets for therapeutic intervention in cancer associated with oncogene activation and replication stress. Cancer can be viewed as a disease of genome instability. The most challenging time for a cell to maintain genome stability is during DNA replication, when complex DNA transactions put the integrity of the genome at risk. The focus of this proposal is the MCM proteins, which are required throughout DNA replication and critical for the maintenance of genome stability under normal conditions or following oncogene-dependent, replication stress.
| Peterson, Shaun E; Li, Yinyin; Wu-Baer, Foon et al. (2013) Activation of DSB processing requires phosphorylation of CtIP by ATR. Mol Cell 49:657-67 |
| Williams, Hannah L; Gottesman, Max E; Gautier, Jean (2012) Replication-independent repair of DNA interstrand crosslinks. Mol Cell 47:140-7 |
| Peterson, Shaun E; Li, Yinyin; Chait, Brian T et al. (2011) Cdk1 uncouples CtIP-dependent resection and Rad51 filament formation during M-phase double-strand break repair. J Cell Biol 194:705-20 |
| Srinivasan, Seetha V; Gautier, Jean (2011) Study of cell cycle checkpoints using Xenopus cell-free extracts. Methods Mol Biol 782:119-58 |
| Kurth, Isabel; Gautier, Jean (2010) Origin-dependent initiation of DNA replication within telomeric sequences. Nucleic Acids Res 38:467-76 |
| Wang, Lily C; Gautier, Jean (2010) The Fanconi anemia pathway and ICL repair: implications for cancer therapy. Crit Rev Biochem Mol Biol 45:424-39 |
| Di Virgilio, Michela; Ying, Carol Y; Gautier, Jean (2009) PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin. DNA Repair (Amst) 8:1311-20 |
| Ben-Yehoyada, Merav; Wang, Lily C; Kozekov, Ivan D et al. (2009) Checkpoint signaling from a single DNA interstrand crosslink. Mol Cell 35:704-15 |
| Wang, Lily Chien; Stone, Stacie; Hoatlin, Maureen Elizabeth et al. (2008) Fanconi anemia proteins stabilize replication forks. DNA Repair (Amst) 7:1973-81 |
