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Defining the Role of Mcm10 in DNA Replication

定义 Mcm10 在 DNA 复制中的作用

基本信息

批准号：
6894255
负责人：
MING LEI
金额：
$ 25.5万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-06-01 至 2007-05-31
项目状态：
已结题

项目摘要

DESCRIPTION (APPLICANT'S ABSTRACT): To ensure precise duplication of the nuclear genome befOre mitotic cell division, a conserved mechanism is employed in all eukaryotes to limit the initiation of DNA synthesis from each replication origin to no more than once in one cell cycle. Defects in the regulation of DNA replication often lead to detrimental consequences such as cell death or uncontrolled cell division, which is manifested in many human cancers. The long-term objective of this proposal is to understand how the initiation of DNA synthesis is regulated. The question will be approached genetically and biochemically in the yeast Saccharomyces cerevisiae, using a novel replication factor, Mcml0, as the probe. Mcm10 is involved in both initiation of DNA replication and the passage of replication forks through unfired origins. Mutations in MCM1O not only reduce the efficiency of origin-specific initiation, but also stall replication forks at unfired origins. We have demonstrated that Mcm1O self-interacts, and that it is a target of regulation by the Cdc28 cyclin-dependent protein kinase. The specific aims of this proposal are: (1) Characterize the self-interaction and phosphorylation ofMcml0. The interaction domain and the phosphorylation site(s) will be mapped. The specific functions of these two post-translational regulatory events will be determined by characterizing mutant alleles defective either in the self-interaction or phosphorylation. (2) Determine specific activities of McmlO at replication origins. ARS1 sequences required for McmlO binding will be determined by in vitro binding assays and in vivo crosslinking. The interaction between McmlO and other replication initiation factors, including Cdc6 and the MCM2-7 complex, will be characterized biochemically. (3) Investigate the roles of McmlO on the replication fork and characterize mcmlO mutants and suppressors. Genetic analysis will be carried out to determine if McmlO also plays a role after the initiation of DNA synthesis. An assay will be developed using mcmlO mutants to monitor the interaction between replication forks and unfired origins. Finally, mutant screens will be carried out to identify new mcmlO mutant alleles and Mcm 10-interacting proteins. These studies will provide a unique perspective to the understanding of origin activity and the interactions between the origin and the replication fork.

描述（申请人摘要）：为确保准确复制核基因组在有丝分裂细胞分裂之前，采用保守机制在所有的真核生物中，复制起点在一个细胞周期中不超过一次。缺陷 DNA复制的调节通常会导致有害的后果，细胞死亡或不受控制的细胞分裂，这表现在许多人类癌的本提案的长期目标是了解 DNA合成的起始受到调节。这个问题将被处理基因和生化在酵母酿酒酵母，使用新的复制因子Mcml 0作为探针。Mcm 10参与了这两项工作 DNA复制的起始和复制叉的通过无火的起源MCM 10的突变不仅降低了源特定的启动，但也会在未激发时停止复制分叉起源.我们已经证明了Mcm 1 O的自我相互作用，它是一个 Cdc 28细胞周期蛋白依赖性蛋白激酶的调节靶点。具体该建议的目的是：（1）描述自交互，磷酸化Mcml 0.相互作用结构域和磷酸化位点将被映射。这两种翻译后蛋白的具体功能调节事件将通过表征突变等位基因缺陷来确定无论是自我作用还是磷酸化。(2)确定具体 McmlO在复制起点的活性。McmlO所需的ARS 1序列结合将通过体外结合测定和体内交联来确定。 McmlO与其他复制起始因子之间的相互作用，包括Cdc 6和MCM 2 -7复合物，将进行生物化学表征。（三）调查McmlO在复制分叉上的角色并描述mcmlO的特征突变体和抑制子。将进行基因分析，以确定是否 McmlO在DNA合成起始后也起作用。将进行测定，使用mcmlO突变体开发以监测复制之间的相互作用叉子和未燃烧的起源。最后，将进行突变体筛选，鉴定新的mcm 10突变等位基因和Mcm 10相互作用蛋白。这些研究将提供一个独特的视角来了解起源活动以及起点和复制叉之间的相互作用。