Understanding the Biological Function of MCM 10

描述（由申请人提供）：在过去的十年中，很明显，S期进展和忠实的基因组遗传强烈依赖于复制分叉的稳定性。真核细胞主动监测复制分叉的进展，而暂时的分叉停滞引发了S期检查点反应，旨在防止复制分叉崩溃和伴随的DNA损伤。进化上保守的小染色体维持蛋白10 （Mcm10）在控制复制叉的稳定性中起关键作用。最近对维持染色体完整性的因子进行的全基因组筛选发现，Mcm10是少数几个在防止DNA损伤方面非常有效的复制蛋白之一。然而，Mcm10如何促进无扰动的S相进展仍然是难以捉摸的。我们在酿酒酵母中的研究表明，Mcm10是真核生物复制叉的一个组成部分，并通过与其他叉成员的大量相互作用协调DNA解旋和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夹、增殖细胞核抗原（PCNA）。因此，Mcm10可能通过适当调节不同的分叉活动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，Mcm10也可能通过结合异三聚体检查点箝位9-1-1 （S. pombe和哺乳动物中的Rad9/Rad1/Hus1和S. cerevisiae中的Ddc1/Rad17/Mec3）更直接地促进S期检查点应答。虽然PCNA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夹子（PCNA）的结合而不是与另一个夹子（9-1-1）的结合中起着至关重要的作用。在本拨款申请的第一部分，我们将继续对出芽酵母中Mcm10的结构-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PCNA相互作用蛋白（PIP）盒子，并证明了该PIP盒子是酿酒酵母中泛素化Mcm10与PCNA结合所必需的。虽然Mcm10和9-1-1复合物之间的相互作用部分依赖于功能性PIP盒子，但它独立于泛素化。我们的假设是泛素化提供了一个分子开关，将Mcm10靶向到PCNA。在本应用程序的第二部分，我们将开始探索连接MCM10到细胞基因组完整性网络的途径。为此，我们对温度敏感的mcm10-1突变体进行了全基因组合成致死性筛选。将验证并进一步检查此屏幕的顶部命中。该应用程序的总体目标是获得Mcm10如何促进基因组稳定性维持以及细胞如何抵消Mcm10缺陷分叉处的DNA损伤的机制理解。这些研究最终将有助于剖析导致人类肿瘤发生的基因组改变的分子事件。

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.