Understanding the biological function of Mcm10 in yeast

描述(申请人提供)：DNA复制是真核细胞周期中的关键步骤。在S早期阶段激活复制起始点需要组装复制前复合体，然后组装成预起始复合体，最后调节成完全活性的起始复合体。这一系列事件在细胞周期的G1阶段开始，并随着2个双向移动的、进行性复制分叉的建立而终止。进化上保守的Mcm10蛋白参与了DNA复制的起始和延伸步骤。然而，Mcm10的S在这些过程中的确切作用仍然难以捉摸。我们对酿酒酵母Mcm10的研究表明，Mcm10受细胞周期调控，是复制分叉的重要组成部分。此外，我们还发现Mcm10控制着DNA聚合酶-α催化亚基的稳定性。我们的初步结果表明，Mcm10还与DNA聚合酶-epsilon和增殖细胞核抗原(增殖细胞核抗原)相互作用。因此，Mcm10似乎是复制分叉组装中的关键协调者，因此也是抗癌治疗的假定靶点。这项拨款申请中提出的实验旨在更好地了解Mcm10及其与DNA聚合酶-α催化亚单位和其他复制蛋白质的相互作用。这项建议的具体目标是： 1.Mcm10‘S在启动复合体组装中的作用是什么？我们推测，Mcm10除了调节ol-α的稳定性外，还参与预引发复合体的组装。 2.Mcm10的S在复制叉子公司扮演什么角色？我们假设Mcm10与增殖细胞核抗原的相互作用是DNA延长所必需的。3.Mcm10如何稳定DNA聚合酶-α？我们的假设是，Mcm10中的特定结构域是稳定CDC17所必需的。4.在没有Mcm10的情况下，CDC17的降解机制是什么？Mcm10的缺失触发了CDc17的降解。然而，目前还不清楚CDC17是如何被降解的。CDC17有2个与PEST相似的序列，它们被认为是泛素介导的蛋白降解的信号序列。我们的工作假设是，与Mcm10的结合使这些害虫序列无法被降解机制访问。

DESCRIPTION (provided by applicant): DNA replication is a crucial step in the eukaryotic cell cycle. Activation of replication origins in early S phase requires the assembly of a pre-replication complex that is subsequently modulated into a pre-initiation complex and finally into a fully active initiation complex. This cascade of events is initiated in Gl phase of the cell cycle and terminates with the establishment of 2 bidirectionally moving, processive replication forks. The evolutionarily conserved protein Mcm10 has been implicated in both the initiation and elongation steps of DNA replication. However, Mcm10's precise role in these processes has remained elusive. Our studies on Mcm10 in S. cerevisiae suggest that Mcm10 is cell cycle regulated and an essential component of the replication fork. Furthermore, we have shown that Mcm10 controls the stability of the catalytic subunit of DNA polymerase-alpha. Our preliminary results suggest that Mcm10 also interacts with DNA polymerase-epsilon and PCNA (proliferating cell nuclear antigen). Thus, Mcm10 appears to be a key coordinator in replication fork assembly and is therefore, a putative target for anti-cancer treatment. The experiments proposed in this grant application are targeted to achieve a better understanding of Mcm10 and its interactions with the catalytic subunit of DNA polymerase-alpha and other replication proteins. The specific aims of this proposal are: 1. What is Mcm10's role in initiation complex assembly? We hypothesize that in addition to regulating the stability of pol-alpha Mcm10 participates in preinitiation complex assembly. 2. What is Mcm10's role at replication forks? We hypothesize that Mcm10 interaction with PCNA is required for DNA elongation. 3. How does Mcm10 stabilize DNA polymerase-alpha? Our hypothesis is that specific domains in Mcm10 are required to stabilize Cdc17. 4. What is the mechanism of Cdc17 degradation in the absence of Mcm10? Loss of Mcm10 triggers the degradation of Cdc17. However, it remains unclear how Cdc17 is degraded. Cdc17 has 2 matches to PEST-like sequences, which have been implicated as signal sequences for ubiquitin-mediated proteolysis. Our working hypothesis is that binding to Mcm10 renders these PEST sequences inaccessible to the degradation machinery.