Regulation of DNA Replication by MCM Proteins

MCM 蛋白对 DNA 复制的调节

• 批准号: 7384287
• 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/7384287
• 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; Condition; DNA; DNA Damage; DNA Polymerase Inhibitor; DNA biosynthesis; DNA chemical synthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Disease; Down-Regulation; Employee Strikes; Event; Fire - disasters; 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; Protein Overexpression; Proteins; Proto-Oncogenes; Recombinants; Recruitment Activity; Regulation; Replication Initiation; Replication Origin; Restart; 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; human MCM2 protein; insight; novel; origin recognition 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（由申请方提供）：DNA复制起始和分叉进展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）蛋白是复制前复合体（pre-RC）组装、起始点启动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，因此它们在RC前组装之后的作用和作用机制一直难以评估。我们已经纯化了活性重组MCM复合物，其支持与MCM耗尽的无细胞提取物中的DNA复制相关的所有已知MCM功能。这个系统使我们能够绕过与MCM的基本作用相关的实验限制。MCM以几种方式与基因组稳定性的维持有关。MCM蛋白是ATM/ATR激酶的靶标，MCM蛋白水平的下调触发基因组不稳定性，并通过MCM结合蛋白Myc增加起始活性，激活DNA损伤反应，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解旋的机制和MCM从染色质卸载的调节。MCM复合物被CDC 7蛋白激酶磷酸化是起始点激活的重要步骤。接下来，我们想描述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，并且只有这些潜在的DNA解旋位点的一个子集被指定为复制的功能起点。我们已经确定，Myc原癌基因结合到MCM蛋白质，并在起源规范中发挥作用。我们建议描述Myc的这种新功能。我们还将调查MCM复合物的潜在作用，还没有被指定为功能的起源，在复制重新启动。最后，我们将通过分析MCM蛋白的下调或复制起点的非计划性激活如何产生DNA损伤来进一步探索MCM在维持基因组稳定性中的作用。我们还将确定ATM和ATR蛋白激酶对MCM磷酸化对DNA复制期间维持基因组稳定性的贡献。我们预计，这些研究将提供重要的见解MCM蛋白的功能机制。特别是，探测MCM，Myc和S期期间基因组稳定性的维持之间的联系将是至关重要的，以确定与癌基因激活和复制应激相关的癌症的治疗干预的目标。 癌症可以被视为一种基因组不稳定性疾病。细胞维持基因组稳定性最具挑战性的时刻是DNA复制期间，此时复杂的DNA处理会使基因组的完整性面临风险。该建议的重点是MCM蛋白，其在整个DNA复制过程中是必需的，并且对于在正常条件下或在癌基因依赖性复制应激之后维持基因组稳定性至关重要。