GENETIC ANALYSIS OF MCM PROTEIN FUNCTION IN S POMBE

粟酒裂殖酵母MCM蛋白功能的遗传分析

• 批准号: 6181462
• 负责人: SUSAN L FORSBURG
• 金额: $ 41.45万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181462
• 关键词: DNA binding protein; DNA replication; Schizosaccharomyces pombe; cell cycle; cyclin dependent kinase; fungal genetics; fungal proteins; gene mutation; laboratory rabbit; phosphorylation; polymerase chain reaction; protein biosynthesis; protein localization; protein sequence; protein structure function

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.

这项研究计划的广泛目标是描述控制DNA复制启动并将其与细胞周期偶联的调控元件网络，特别关注称为MCM蛋白的保守分子家族。 这些蛋白质存在于从酵母到人类的真核生物中。MCM蛋白质的突变会损害DNA复制，导致染色体损伤和遗传不稳定。基因组完整性的这种缺陷可导致或促成癌症的发展，并且还可导致发育缺陷。因此，了解DNA复制的调控与人类健康直接相关。这个建议利用了真核系统的模型，分裂酵母S。pombe的，以研究MCM蛋白在DNA复制中的作用。真核细胞含有六个相关的MCM蛋白组装在一个大的复合物，每个蛋白质是必不可少的生存能力。这六种蛋白质之间有什么区别？它们各自对MCM复合体的组装、定位和活性提供了什么功能？裂变酵母系统特别适合解决这些问题，因为其复制起点的结构和MCM蛋白的行为与高等真核生物中的非常相似，并且很容易使用遗传学和分子生物学进行操作。为了确定每个MCM蛋白对复合物组装和定位的贡献。对于粟酒裂殖酵母，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。 将确定mcm突变对DNA复制和细胞周期进程的影响。遗传和物理与已知基因的相互作用将被调查，并将寻求MCM蛋白和已知的细胞周期调节因子之间的相互作用的证据。最后，将采用遗传筛选来鉴定与MCM蛋白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复合物中的位置将相互关联。这项研究将有助于完整地描述这些保守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。