Understanding the biological function of Mcm10 in yeast

描述（由申请人提供）：DNA复制是真核细胞周期中的关键步骤。在早期S期激活复制起点需要组装复制前复合物，其随后被调节成前起始复合物，并最终成为完全活性的起始复合物。这种级联事件在细胞周期的G1期开始，并以2个双向移动的进行性复制叉的建立而终止。进化上保守的蛋白质Mcm 10与DNA复制的起始和延伸步骤都有牵连。然而，Mcm 10在这些过程中的确切作用仍然难以捉摸。本研究对S.酿酒酵母表明，Mcm 10是细胞周期调节和复制叉的重要组成部分。此外，我们已经表明，Mcm 10控制DNA聚合酶-α的催化亚基的稳定性。我们的初步研究结果表明，Mcm 10也与DNA聚合酶和增殖细胞核抗原（PCNA）相互作用。因此，Mcm 10似乎是复制叉组装中的关键协调者，因此是抗癌治疗的假定靶点。在这项资助申请中提出的实验旨在更好地了解Mcm 10及其与DNA聚合酶α和其他复制蛋白的催化亚基的相互作用。这项建议的具体目标是： 1. Mcm 10在起始复合物组装中的作用是什么？ 我们推测，除了调节pol-alpha Mcm 10的稳定性参与preinitiation复杂的组装。 2. Mcm 10在复制叉中的作用是什么？ 我们推测Mcm 10与PCNA的相互作用是DNA延伸所必需的。3. Mcm 10如何稳定DNA聚合酶-α？ 我们的假设是Mcm 10中的特定结构域是稳定Cdc 17所必需的。4.在没有Mcm 10的情况下，Cdc 17降解的机制是什么？ Mcm 10的丢失触发Cdc 17的降解。然而，目前还不清楚Cdc 17是如何降解的。Cdc 17与PEST样序列有2个匹配，PEST样序列被认为是泛素介导的蛋白水解的信号序列。我们的工作假设是，结合Mcm 10使这些PEST序列无法进入降解机制。

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.