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，因此它们在 RC 组装前的作用和作用机制很难评估。我们纯化了活性重组 MCM 复合物，支持与 MCM 耗尽的无细胞提取物中 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 通过多种方式与维持基因组稳定性相关。 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并增加 MCM 结合蛋白 Myc 的起源活性，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 解旋机制和 MCM 从染色质卸载的调节。 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 复制起始的一个显着特征是，染色质上加载了大量的 MCM 复合物，并且这些潜在 DNA 解旋位点中只有一部分被指定为功能性复制起点。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 的这一新功能。我们还将研究尚未指定为功能起点的 MCM 复合物在复制重启中的潜在作用。最后，我们将通过分析 MCM 蛋白的下调或复制起点的意外激活如何产生 DNA 损伤，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 ATM 和 ATR 蛋白激酶对 MCM 磷酸化对维持 DNA 复制过程中基因组稳定性的贡献。我们预计这些研究将为 MCM 蛋白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。 癌症可以被视为一种基因组不稳定的疾病。细胞维持基因组稳定性最具挑战性的时期是 DNA 复制期间，此时复杂的 DNA 事务会使基因组的完整性面临风险。该提案的重点是 MCM 蛋白，它是整个 DNA 复制过程中所必需的，对于正常条件下或在癌基因依赖性复制应激后维持基因组稳定性至关重要。