GENETIC ANALYSIS OF MCM PROTEIN FUNCTION IN S POMBE

这项研究计划的广泛目标是描述控制DNA复制启动的调控元件网络，并将其与细胞周期相耦合，特别关注一种被称为MCM蛋白质的保守分子家族。这些蛋白质存在于从酵母到人类的各种真核生物中。MCM蛋白的突变会损害DNA复制，导致染色体损伤和遗传不稳定。这种基因组完整性缺陷可能导致或促成癌症的发展，也可能导致发育缺陷。因此，了解DNA复制的调控直接关系到人类的健康。这项建议利用一个模型真核系统的遗传学，分裂酵母S.pombe，来研究MCM蛋白在DNA复制中的作用。真核细胞包含六种相关的MCM蛋白，它们组装在一个大的复合体中，每一种蛋白都是生存所必需的。这六种蛋白质有什么不同之处？每一个都为MCM复合体的组装、本地化和活动提供了什么功能？裂解酵母系统特别适合于解决这些问题，因为它的复制起点的结构和MCM蛋白的行为与高等真核生物中的非常相似，而且它很容易利用遗传学和分子生物学来操纵。为了确定每个MCM蛋白在S.pombe中复杂组装和定位中的贡献，将对单个MCM进行突变分析，并使用已建立的方案检查这些突变对体内复杂行为的影响。Mcm突变对DNA复制和细胞周期进程的影响将被确定。将调查与已知基因的遗传和物理相互作用，并寻找MCM蛋白和已知细胞周期调节因子之间相互作用的证据。最后，将使用遗传筛选来识别MCM蛋白与之相互作用的其他基因。在整个研究过程中，单个MCM的行为、它们对复制的影响以及它们在MCM复合体中的位置将是相关的。这项研究将有助于全面描述这些保守的蛋白质是如何在活细胞内的蛋白质网络中组装和发挥作用的。

The broad goal of this research program is to describe the network of regulatory elements that control the initiation of DNA replication and couple it to the cell cycle, with particular focus on a conserved family of molecules called MCM proteins. These proteins are found in eukaryotes ranging from yeast to man. Mutations in MCM proteins impair DNA replication, resulting in chromosome damage and genetic instability. Such defects in genome integrity can cause or contribute to development of cancer, and can also cause defects in development. Thus, understanding the regulation of DNA replication has direct relevance to human health. This proposal exploits the genetics of a model eukaryotic system, the fission yeast S. pombe, to investigate the role of the MCM proteins in DNA replication. Eukaryotic cells contain six related MCM proteins assembled in a large complex, and each protein is essential for viability. What distinguishes these six proteins from one another? What functions does each provide to the assembly, localization, and activity of the MCM complex? The fission yeast system is particularly well suited to address these questions, because the structure of its replication origins and behavior of its MCM proteins closely resemble those in higher eukaryotes, and it is easily manipulated using genetics and molecular biology. In order to identify the contribution of each MCM protein to complex assembly and localization in s. pombe, individual MCMs will be subjected to mutational analysis and the effects of these mutations upon complex behavior in vivo will be examined using established protocols. Effects of mcm mutations on DNA replication and cell cycle progression will be determined. Genetic and physical interactions with known genes will be investigated, and evidence for interactions between MCM proteins and known cell cycle regulators will be sought. Finally, a genetic screen will be employed to identify additional genes with which the MCM proteins interact. Throughout the study, the behavior of individual MCMs, their effect on replication, and their position within the MCM complex will be correlated. This study will contribute to a complete description of how these conserved proteins are assembled and function within the network of proteins inside the living cell.