Understanding the biological function of Mcm10 in yeast

了解 Mcm10 在酵母中的生物学功能

• 批准号: 7254157
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 21.61万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7254157
• 关键词: Affect; Applications Grants; Binding; Biological Assay; Biological Process; Catalytic Domain; Cell Cycle; Chromatin; Complex; DNA; DNA Polymerase I; DNA Polymerase II; DNA biosynthesis; DNA polymerase alpha-primase; Eukaryotic Cell; Event; Half-Life; Molecular Chaperones; Mutation; Nature; Nuclear; Peptide Signal Sequences; Phase; Polymerase; Post-Translational Protein Processing; Pre-Replication Complex; Process; Proliferating Cell Nuclear Antigen; Proteins; Recruitment Activity; Replication Origin; Research Personnel; Role; Saccharomyces cerevisiae; Temperature; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Work; Yeasts; cancer therapy; multicatalytic endopeptidase complex; mutant; research study

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.

描述（由申请人提供）：DNA复制是真核细胞周期中的关键步骤。复制起源在早期阶段的激活需要组装一个预复合复合物，该复制复合物随后被调节为发射络合物，并最终变成完全活跃的启动复合物。这种级联的事件是在细胞周期的GL阶段启动的，并通过建立2个双向移动的，可进行的复制叉来终止。进化保守的蛋白MCM10已与DNA复制的起始和伸长步骤有关。但是，MCM10在这些过程中的确切作用仍然难以捉摸。我们对酿酒酵母中MCM10的研究表明，MCM10是细胞周期的调节，也是复制叉的重要组成部分。此外，我们已经表明MCM10控制DNA聚合酶-Alpha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PCNA（增殖细胞核抗原）相互作用。因此，MCM10似乎是复制叉组件中的关键协调员，因此是抗癌治疗的推定目标。本赠款应用中提出的实验旨在更好地了解MCM10及其与DNA聚合酶-Alpha和其他复制蛋白的催化亚基的相互作用。该提案的具体目的是： 1。MCM10在启动复合组装中的作用是什么？ 我们假设，除了调节pol-Alpha MCM10的稳定性外，还参与了预启用复合体组件。 2。MCM10在复制叉中的作用是什么？ 我们假设MCM10与PCNA的相互作用是DNA伸长所必需的。 3。MCM10如何稳定DNA聚合酶-Alpha？ 我们的假设是，MCM10中的特定域是稳定Cdc17所需的。 4。在没有MCM10的情况下，CDC17降解的机理是什么？ MCM10的损失会触发CDC17的降解。但是，目前尚不清楚CDC17是如何降解的。 CDC17与虫害样序列有2种匹配，这些序列已被视为泛素介导的蛋白水解的信号序列。我们的工作假设是，与MCM10的结合使这些害虫序列无法访问降解机制。