Regulation of DNA Replication by MCM Proteins

MCM 蛋白对 DNA 复制的调节

• 批准号: 7582247
• 负责人: JEAN GAUTIER
• 金额: $ 32.2万
• 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 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7582247
• 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; G1 Phase; 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; Methanobacteria; Methanobacterium; Mutation; Oncogene Activation; Oncogenes; Output; Phase; 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; 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复制起始和叉进程的调节对于维持基因组稳定性至关重要。微型染色体维持（MCM）蛋白是重复的复合物（PRE-RC）组装，起源射击和重新分散进程所必需的。由于所有MCM蛋白对于生命都是必不可少的，因此很难评估它们以外的RC组装以外的作用和作用机制。我们已经纯化了活跃的重组MCM复合物，该复合物支持与MCM耗尽无细胞提取物中与DNA复制相关的所有已知MCM功能。该系统使我们能够绕过与MCM基本角色相关的实验限制。 MCM通过多种方式与基因组稳定性保持联系。 MCM蛋白是ATM/ATR激酶的靶标，MCM蛋白水平的下调会触发基因组不稳定性，而MCM结合蛋白MYC的起源活性增加，激活了DNA损伤反应，会产生损伤和基因组不稳定性。最后，MCM在多种肿瘤中异常表达，并用作肿瘤进展的预后标记。首先，我们将研究MCM活性的两个关键方面：DNA的机理以及从染色质中卸载MCM的调节。 CDC7蛋白激酶对MCM复合物的磷酸化是起源激活的重要步骤。接下来，我们要表征这些磷酸化事件并评估其生理后果。 DNA复制启动的一个惊人特征是，在染色质上加载了大量的MCM复合物，只有DNA放松的这些潜在位点的子集被指定为复制的功能起源。我们已经确定MYC原始癌基因与MCM蛋白结合并在原点规范中起作用。我们建议表征MYC的新功能。我们还将研究尚未指定为功能起源的MCM复合物在复制重新启动中的潜在作用。最后，我们将通过分析MCM蛋白的下调或外部复制起源的下调如何产生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AT​​M和ATR蛋白激酶对DNA复制过程中基因组稳定性维持的MCM磷酸化的贡献。我们预计这些研究将为MCM蛋白功能的机理提供重要的见解。特别是，探测S相过程中MCM，MYC和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力相关的癌症治疗干预靶标至关重要。 癌症可以看作是基因组不稳定性疾病。细胞保持基因组稳定性的最具挑战性时间是在DNA复制期间，当复杂的DNA交易使基因组的完整性处于危险之中时。该提案的重点是MCM蛋白，这在整个DNA复制过程中都是必需的，对于在正常条件下或遵循致癌基因依赖性的复制应力下维持基因组稳定性至关重要。