Understanding the Biological Function of MCM 10

描述(申请人提供)：在过去的十年中，S的阶段进展和忠实的基因组遗传强烈依赖于复制分叉的稳定性。真核细胞主动监控复制分叉的进展，临时分叉停滞会引发S阶段的检查点反应，旨在防止复制分叉崩溃和伴随的dna损伤。进化上保守的微小染色体维持蛋白10(Mcm10)在控制复制叉稳定性方面起着关键作用。最近对维持染色体完整性的因素进行的全基因组筛查发现，Mcm10是少数几种在防止DNA损伤方面高度有效的复制蛋白之一。然而，Mcm10如何在平静的S期进展中发挥作用仍然是个未知数。我们在酿酒酵母中的研究表明，Mcm10是真核复制叉子的组成部分，通过与其他叉子成员的一系列相互作用来协调DNA解离和DNA合成。其中包括复制型解旋酶、DNA聚合酶(Pol1/Primase)和同源三聚体复制钳、增殖细胞核抗原(PC NA)。因此，Mcm10可能通过适当调节不同的分叉活动来间接防止复制分叉的崩溃。然而，我们实验室最近的发现表明，Mcm10也可能通过与异源三聚体检查点钳9-1-1结合(在庞贝和哺乳动物中为RAD9/RAD1/HUS1，在酿酒酵母中为DDc1/RAD17/MEC3)，而更直接地诱导S期的检查点反应。尽管增殖细胞核抗原和9-1-1检查点钳在结构上是相关的，但它们与Mcm10的相互作用是相当不同的。重要的是，Mcm10的泛素化在促进与一个钳子(增殖细胞核抗原)的结合中起着至关重要的作用，而不是与另一个(9-1-1)结合。在这项资助申请的第一部分，我们寻求继续我们正在进行的Mcm10在萌芽酵母中的结构和功能分析。在之前的资助期间，我们在Mcm10中发现了一个进化上保守的增殖细胞核抗原相互作用蛋白(PIP)盒，并证明该PIP盒是酿酒酵母泛素化的Mcm10与增殖细胞核抗原结合所必需的。虽然Mcm10与9-1-1复合体之间的相互作用部分依赖于一个功能性的PIP盒，但它不依赖于泛素化。我们的假设是泛素化提供了靶向Mcm10到增殖细胞核抗原的分子开关。在本申请的第二部分，我们将开始探索将MCM10连接到细胞基因组完整性网络的途径。为此，我们用温度敏感型mcm10-1突变体进行了全基因组合成致死性筛选。此屏幕的最高点击率将进行验证和进一步检查。这项应用的总体目标是从机械上了解Mcm10如何促进基因组稳定性的维持，以及细胞如何抵消Mcm10缺陷叉处的DNA损伤。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，这些改变有助于人类肿瘤的发生。 与公共卫生相关：DNA复制是真核细胞周期中的关键步骤。复制分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分叉断裂。我们正在探索将复制叉与真核细胞的DNA损伤避免系统连接起来的网络。从简单生物体中学到的知识将应用于人类系统，以了解癌症的分子途径。

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.