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的关键步骤。S期早期复制起始点的激活需要复制前复合体的组装，该复合体随后被调制为起始前复合体，并最终成为完全活跃的起始复合体。这一连串的事件在细胞周期的Gl期开始，并随着2个双向移动的进程复制分叉的建立而终止。进化上保守的蛋白Mcm10参与了DNA复制的起始和延伸步骤。然而，Mcm10在这些过程中的确切作用仍然难以捉摸。我们对酿酒葡萄球菌Mcm10的研究表明，Mcm10是细胞周期调控的，是复制叉的重要组成部分。此外，我们已经证明Mcm10控制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（PCNA）相互作用。因此，Mcm10似乎是复制叉组装的关键协调者，因此是抗癌治疗的一个假定靶点。本资助申请中提出的实验旨在更好地了解Mcm10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：

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.