Defining the Role of Mcm10 in DNA Replication

描述(申请人摘要)：确保准确复制 核基因组在有丝分裂细胞分裂前，采用一种保守机制 在所有真核生物中限制每个DNA合成的起始 复制起点在一个细胞周期内不超过一次。产品中的缺陷 对DNA复制的调控通常会导致有害的后果，例如 细胞死亡或不受控制的细胞分裂，这在许多人类身上都有表现 癌症。这项提议的长期目标是理解 DNA合成的启动是受调控的。这个问题将被提出。 在酿酒酵母中，使用一种 以新型复制因子Mcml0为探针。Mcm10参与了这两个项目 DNA复制的启动和复制分叉的通过 未被点燃的起源。MCM1O的突变不仅会降低治疗效率 特定于原点的启动，但也会在未启动时停止复制分叉 起源。我们已经证明了Mcm1O的自我作用，并且它是一种 CDC28细胞周期蛋白依赖蛋白激酶的调控靶点。具体的 这一建议的目的是：(1)表征自我互动和 Mcml0的磷酸化。相互作用结构域和磷酸化位点(S) 将会被映射。这两种翻译后的具体功能 调控事件将通过表征突变的有缺陷的等位基因来确定 无论是在自我相互作用中还是在磷酸化中。(2)确定具体情况 McmlO在复制起始处的活动。McmlO所需的ARS1序列 结合将通过体外结合试验和体内交联法来确定。 McmlO与其他复制起始因子之间的相互作用， 包括CdC6和MCM2-7复合体，将进行生化表征。(3) 研究McmlO在复制分叉中的作用并鉴定mcmlO 变种人和抑制者。将进行基因分析以确定 McmlO也在DNA合成启动后发挥作用。化验结果将会是 使用mcmlO突变体开发来监控复制之间的相互作用 叉子和未烧制的起源。最后，将进行突变筛选，以 鉴定新的mcmlO突变等位基因和mcm10相互作用蛋白。这些 研究将为理解起源提供一个独特的视角 活动以及起源和复制分叉之间的相互作用。

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.