Regulation of DNA Replication by MCM Proteins

MCM 蛋白对 DNA 复制的调节

• 批准号: 8050089
• 负责人: JEAN GAUTIER
• 金额: $ 31.56万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050089
• 关键词: ATP phosphohydrolase; ATR protein kinase; Abbreviations; Address; Aphidicolin; Binding; Binding Proteins; Bypass; CDC7 gene; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chromosomes; Comb animal structure; Complex; DNA; DNA Damage; DNA Polymerase Inhibitor; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Disease; Down-Regulation; Employee Strikes; Event; Generations; Genetic; Genetic Materials; Genome; Genome Stability; Genomic Instability; Histone Deacetylase; In Vitro; Life; MCM2 gene; MOS pp39 Serine/Threonine Kinase; MYC gene; Maintenance; Malignant Neoplasms; Methanobacterium; Mutation; Oncogene Activation; Oncogenes; Output; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Pre-Replication Complex; Process; Prognostic Marker; Protein Kinase; Proteins; Proto-Oncogenes; Recombinants; Recruitment Activity; Regulation; Replication Initiation; Replication Origin; Risk; Role; S Phase; Single-Stranded DNA; Site; Source; Stress; System; Testing; Therapeutic Intervention; Time; To specify; ataxia telangiectasia mutated protein; helicase; histone acetyltransferase; human CDC7 protein; insight; novel; origin recognition complex; overexpression; protein complex; protein function; response; transmission process; tumor; tumor progression; tumorigenesis

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.

描述(由申请人提供)：DNA复制启动和分叉进程的调节对于维持基因组稳定性至关重要。微小染色体维持(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。由于所有的MCM蛋白都是生命所必需的，它们的作用和作用机制超出了RC前组装，因此很难评估。我们已经纯化了活性的重组MCM复合体，它们支持所有已知的与MCM耗竭的无细胞提取物中的DNA复制相关的MCM功能。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通过几种方式与维持基因组的稳定性有关。MCM蛋白是ATM/ATR激酶的靶标，MCM蛋白水平的下调触发了基因组的不稳定，并通过MCM结合蛋白Myc增加了起源活性，激活了DNA损伤反应，导致了损伤和基因组的不稳定。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解离的机制和MCM从染色质中卸载的调节。CDC7蛋白激酶对MCM复合体的磷酸化是起始蛋白活化过程中的重要步骤。接下来，我们想要描述这些磷酸化事件，并评估它们的生理后果。DNA复制启动的一个显着特征是大量过剩的MCM复合体负载在染色质上，只有这些潜在的DNA解离位点的子集被指定为复制的功能起点。我们已经确定Myc原癌基因与MCM蛋白结合，并在来源指定中发挥作用。我们建议对Myc的这一新功能进行描述。我们还将研究尚未被指定为功能起始的MCM复合体在复制重新启动中的潜在作用。最后，我们将通过分析MCM蛋白的下调或复制起始点的非计划激活如何产生DNA损伤来进一步探讨MCM在维持基因组稳定性中的作用。我们还将确定在DNA复制过程中，ATM和ATR蛋白激酶对维持基因组稳定性的作用。我们期待这些研究将为MCM蛋白的功能机制提供重要的见解。特别是，探索MCM、Myc与S期基因组稳定性的维持之间的联系，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。 癌症可以被视为一种基因组不稳定的疾病。对细胞来说，维持基因组稳定最具挑战性的时期是DNA复制期间，此时复杂的DNA交易将基因组的完整性置于危险之中。这项建议的重点是MCM蛋白，它在整个DNA复制过程中都是必需的，对于在正常条件下或在癌基因依赖的复制压力下维持基因组的稳定性至关重要。