Defining the Role of Mcm10 in DNA Replication

描述（申请人摘要）：为了确保精确复制 有丝分裂细胞分裂之前的核基因组，采用保守的机制 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中不超过一次。缺陷在 DNA复制的调节常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，这在许多人类中都有体现 癌症。该提案的长期目标是了解如何 DNA 合成的起始受到调节。问题将会被解决 使用遗传和生物化学方法在酿酒酵母中进行 新型复制因子 Mcml0 作为探针。 Mcm10 参与了这两个方面 DNA复制的起始和复制叉的通过 未烧制的起源。 MCM1O 的突变不仅降低了效率 特定于起源的启动，但也会在未激发时停止复制叉 起源。我们已经证明Mcm1O可以自相互作用，并且它是一个 Cdc28 细胞周期蛋白依赖性蛋白激酶的调节目标。具体的 该提案的目标是：（1）描述自交互和 Mcml0 的磷酸化。相互作用结构域和磷酸化位点 将被映射。这两个翻译后的具体功能 调控事件将通过表征突变等位基因缺陷来确定 无论是自相互作用还是磷酸化。 (2)确定具体 McmlO 在复制起点的活性。 McmlO 所需的 ARS1 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 Cdc6 和 MCM2-7 复合体，将进行生化表征。 (3) 研究 McmlO 在复制叉上的作用并表征 mcmlO 突变体和抑制子。将进行遗传分析以确定是否 McmlO也在DNA合成开始后发挥作用。化验将是 使用 mcmlO 突变体开发来监测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 鉴定新的 mcmlO 突变等位基因和 Mcm 10 相互作用蛋白。这些 研究将为理解起源提供独特的视角 活性以及起点和复制叉之间的相互作用。

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.