Regulation of DNA Replication by MCM Proteins

描述（由申请人提供）：DNA复制起始和分叉进展的调控是维持基因组稳定性的关键。迷你染色体维持（MCM）蛋白是复制前复合体（pre-RC）组装、起始激发和复制体进展所必需的。由于所有MCM蛋白都是生命所必需的，因此很难评估它们在pre-RC组装之外的作用和作用机制。我们纯化了活性重组MCM复合物，这些复合物支持所有已知的MCM功能，这些功能与MCM耗尽的无细胞提取物中的DNA复制相关。这个系统允许我们绕过与MCM的基本角色相关的实验限制。MCM以几种方式与基因组稳定性的维持有关。MCM蛋白是ATM/ATR激酶的靶点，MCM蛋白水平的下调触发基因组不稳定，MCM结合蛋白Myc的起源活性增加，激活DNA损伤反应，产生损伤和基因组不稳定。最后，MCM在多种肿瘤中异常表达，并被用作肿瘤进展的预后标志物。首先，我们将研究MCM活性的两个关键方面：DNA解绕的机制和MCM从染色质上卸载的调控。CDC7蛋白激酶磷酸化MCM复合体是激活起源的重要步骤。接下来，我们想要表征这些磷酸化事件并评估其生理后果。DNA复制起始的一个显著特征是，大量过量的MCM复合物被装载在染色质上，只有这些DNA解绕的潜在位点中的一部分被指定为复制的功能性起点。我们已经确定Myc原癌基因与MCM蛋白结合，并在起源规范中发挥作用。我们建议描述Myc的这种新功能。我们还将研究尚未被指定为功能起源的MCM复合物在复制重启中的潜在作用。最后，我们将进一步探讨MCM在维持基因组稳定性中的作用，通过分析MCM蛋白的下调或复制起点的非计划激活如何产生DNA损伤。我们还将确定ATM和ATR蛋白激酶对MCM磷酸化在DNA复制过程中维持基因组稳定性的贡献。我们期望这些研究将为MCM蛋白的功能机制提供重要的见解。特别是，探索MCM、Myc与s期基因组稳定性维持之间的联系，对于确定与癌基因激活和复制应激相关的癌症治疗干预靶点至关重要。

DESCRIPTION (provided by applicant): 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.