Understanding the Biological Function of MCM 10

了解 MCM 10 的生物学功能

• 批准号: 8106727
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 27.57万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8106727
• 关键词: Applications Grants; Binding; Biochemical; Biological Process; Boxing; Camptothecin; Cell Cycle; Cells; Complex; DNA; DNA Damage; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Eukaryotic Cell; Event; Fission Yeast; Functional disorder; Funding; Genetic; Genome; Genome Stability; Genomics; Goals; Homo; Human; Laboratories; Learning; MCM10 gene; Maintenance; Malignant Neoplasms; Mammals; Maps; Molecular; Monitor; Mutation; N-terminal; Organism; Pathway interactions; Pattern; Play; Proliferating Cell Nuclear Antigen; Proteins; Regulation; Role; S Phase; Saccharomyces cerevisiae; Saccharomycetales; Site; Structure; System; Temperature; Testing; Ubiquitin; Ubiquitination; cancer therapy; design; genome wide association study; genome-wide; helicase; inhibitor/antagonist; insight; member; mutant; novel; prevent; repaired; response; tumorigenesis; ubiquitin ligase

DESCRIPTION (provided by applicant): During the last decade, it has become evident that S phase progression and faithful genome inheritance strongly rely on the stability of replication forks. Eukaryotic cells actively monitor replication fork progression, and temporary fork stalling elicits an S phase checkpoint response designed to prevent replication fork collapse and concomitant DNA damage. The evolutionarily conserved minichromosome maintenance protein 10 (Mcm10) plays a key role in controlling replication fork stability. A recent genome-wide screen for factors that maintain chromosomal integrity identified Mcm10 as one of only a few replication proteins that are highly effective in preventing DNA damage. However, how Mcm10 contributes to unperturbed S phase progression has remained elusive. Our studies in S. cerevisiae suggest that Mcm10 is an integral part of the eukaryotic replication fork and coordinates DNA unwinding with DNA synthesis through a multitude of interactions with other fork members. These include the replicative helicase, DNA polymerase (pol)- 1/primase and the homo-trimeric replication clamp, proliferating cell nuclear antigen (PCNA). Thus, Mcm10 may prevent replication fork collapse indirectly by properly regulating diverse fork activities. However, recent findings from our laboratory suggest that Mcm10 may also contribute more directly to eliciting an S phase checkpoint response through the binding of the hetero-trimeric checkpoint clamp 9-1-1 (Rad9/Rad1/Hus1 in S. pombe and mammals and Ddc1/Rad17/Mec3 in S. cerevisiae). Although PCNA and the 9-1-1 checkpoint clamp are structurally related, their interaction with Mcm10 is rather distinct. Importantly, ubiquitination of Mcm10 plays a crucial role in promoting the binding to one clamp (PCNA), but not the other (9-1-1). In the first part of this grant application, we seek to continue our ongoing structure-function analysis of Mcm10 in budding yeast. During the previous funding period, we identified an evolutionarily conserved PCNA interacting protein (PIP) box in Mcm10 and demonstrated that this PIP box is required for the binding between ubiquitinated Mcm10 and PCNA in S. cerevisiae. Whereas the interaction between Mcm10 and the 9-1-1 complex partially depends on a functional PIP box, it is independent of ubiquitination. Our hypothesis is that ubiquitination provides a molecular switch to target Mcm10 to PCNA. In the second part of this application, we will start to explore the pathways that connect MCM10 to the cellular genome integrity network. To this end, we have conducted a genome-wide synthetic lethality screen with the temperature sensitive mcm10-1 mutant. The top hits of this screen will be validated and further examined. The overall goal of this application is to gain a mechanistic understanding of how Mcm10 promotes the maintenance of genome stability and how cells counteract DNA damage at Mcm10-defective forks. These studies should ultimately help to dissect the molecular events that can cause genomic alterations, which contribute to tumorigenesis in humans. PUBLIC HEALTH RELEVANCE: DNA replication is a crucial step in the eukaryotic cell cycle. Proteins at the replication fork have to be tightly regulated to avoid spontaneous mutations and replication fork breakage. We are exploring the networks that connect replication forks with the DNA damage avoidance system of eukaryotic cells. Lessons learnt from simple organisms will be applied to the human system to understand the molecular pathways of cancer.

描述（由申请人提供）：在过去的十年中，S期进展和忠实的基因组遗传强烈依赖于复制叉的稳定性已经变得很明显。真核细胞主动监测复制叉的进展，并且暂时性的叉停滞激发S期检查点反应，旨在防止复制叉崩溃和伴随的DNA损伤。进化上保守的微小染色体维持蛋白10（Mcm 10）在控制复制叉稳定性中起着关键作用。最近的一项基因组范围的筛选维持染色体完整性的因素，确定Mcm 10是为数不多的复制蛋白质之一，在防止DNA损伤方面非常有效。然而，Mcm 10如何有助于不受干扰的S期进展仍然难以捉摸。我们在S.酿酒酵母的研究表明，Mcm 10是真核生物复制叉的组成部分，并通过与其他叉成员的大量相互作用协调DNA解旋与DNA合成。这些包括复制解旋酶、DNA聚合酶（pol）-1/引物酶和同源三聚体复制钳、增殖细胞核抗原（PCNA）。因此，Mcm 10可以通过适当地调节不同的分叉活动来间接地防止复制分叉崩溃。然而，我们实验室最近的研究结果表明，Mcm 10也可能通过与异源三聚体检查点钳9-1-1（S. pombe和哺乳动物中的Ddc 1/Rad 17/Mec 3。cerevisiae）。虽然PCNA和9-1-1检查点钳在结构上相关，但它们与Mcm 10的相互作用是相当不同的。重要的是，Mcm 10的泛素化在促进与一个钳（PCNA）结合而不是另一个钳（9-1-1）结合方面起着至关重要的作用。在本资助申请的第一部分，我们寻求继续我们正在进行的结构-功能分析芽殖酵母中的Mcm 10。在上一个资助期间，我们在Mcm 10中鉴定了一个进化上保守的PCNA相互作用蛋白（PIP）盒，并证明该PIP盒是S.啤酒。而Mcm 10和9-1-1复合物之间的相互作用部分取决于功能PIP框，它是独立的泛素化。我们的假设是，泛素化提供了一个分子开关，靶向Mcm 10的PCNA。在本申请的第二部分中，我们将开始探索将MCM 10连接到细胞基因组完整性网络的途径。为此，我们进行了全基因组的合成致死性筛选与温度敏感mcm 10 -1突变体。将验证并进一步检查此屏幕的最高命中率。该应用程序的总体目标是获得对Mcm 10如何促进基因组稳定性的维持以及细胞如何抵消Mcm 10缺陷叉的DNA损伤的机械理解。这些研究最终应该有助于剖析可能导致基因组改变的分子事件，这些分子事件有助于人类肿瘤的发生。 公共卫生相关性：DNA复制是真核细胞周期中的关键步骤。复制叉上的蛋白质必须受到严格的调控，以避免自发突变和复制叉断裂。我们正在探索连接复制叉与真核细胞DNA损伤避免系统的网络。从简单生物体中吸取的教训将应用于人类系统，以了解癌症的分子途径。