Understanding the biological function of Mcm10 in yeast

了解 Mcm10 在酵母中的生物学功能

• 批准号: 7455040
• 负责人: Anja-Katrin Bielinsky
• 金额: $ 21.6万
• 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/7455040
• 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。早期S期复制起点的激活需要组装预复制复合体，随后将其调节为预起始复合体，最后调节为完全活性的起始复合体。该级联事件在细胞周期的G1期开始，并随着2个双向移动、进行性复制叉的建立而终止。进化上保守的蛋白质 Mcm10 与 DNA 复制的起始和延伸步骤有关。然而，Mcm10 在这些过程中的确切作用仍然难以捉摸。我们对酿酒酵母中 Mcm10 的研究表明，Mcm10 受细胞周期调节，并且是复制叉的重要组成部分。此外，我们还发现 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 PCNA（增殖细胞核抗原）相互作用。因此，Mcm10 似乎是复制叉组装中的关键协调子，因此是抗癌治疗的推定靶标。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。该提案的具体目标是： 1. Mcm10 在启动复合物组装中起什么作用？ 我们假设除了调节 pol-α 的稳定性外，Mcm10 还参与启动前复合物的组装。 2. Mcm10 在复制分叉中的作用是什么？ 我们假设 Mcm10 与 PCNA 相互作用是 DNA 延伸所必需的。 3. Mcm10如何稳定DNA聚合酶-α？ 我们的假设是 Mcm10 中的特定结构域需要稳定 Cdc17。 4. 在没有Mcm10的情况下，Cdc17的降解机制是什么？ Mcm10 的丢失会触发 Cdc17 的降解。然而，目前尚不清楚 Cdc17 是如何降解的。 Cdc17 与 PEST 样序列有 2 个匹配，这些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，与 Mcm10 的结合使得降解机制无法访问这些 PEST 序列。