GENETIC ANALYSIS OF CHROMOSOME SEGREGATION IN YEAST

酵母染色体分离的遗传分析

• 批准号: 2178396
• 负责人: CONNIE A HOLM
• 金额: $ 22.57万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178396
• 关键词: DNA repair; DNA replication; alleles; cell cycle; cell cycle proteins; cell growth regulation; chromosome movement; fungal genetics; gene interaction; genetic techniques; guanosinetriphosphatases; intermolecular interaction; mutant; phenotype; protein structure function; site directed mutagenesis; telomere; yeasts

Our ultimate goal is to understand the molecular mechanisms of chromosome segregation. Specifically, we want to understand how chromosomes are replicated precisely, how their structural integrity is maintained, and how their separation is coordinated with other events in the cell cycle. To probe these issues, we will study mutations in two genes that encode biochemically well-characterized proteins that may play essential roles in both the mechanics of DNA replication and its coordination with mitosis. CDC44 (the large subunit of replication factor C) and PCNA (proliferating cell nuclear antigen) have been shown to interact both biochemically and genetically, and each has characteristics that suggest a dual role in the cell cycle. Thorough biochemical analysis reveals that replication factor C and PCNA act together to promote the processivity of DNA polymerase delta in vitro. Nonetheless, cell-cycle analysis of a cdc44 mutant suggests that a critical mitotic (post-replicative) function is perturbed. Correspondingly, the association of PCNA with most cyclin-dependent-kinase complexes in vivo suggests that PCNA may play an important role in the coordination of DNA synthesis with other cellular events. To analyze the roles of CDC44 and PCNA in living cells we will create random and site-directed mutations in vitro and analyze the phenotypes they confer. Perturbations of DNA replication will be signaled by elevated mutation rates, disrupted DNA synthesis, and an appropriate cell-cycle arrest. Perturbations of mitotic functions will be revealed by a post- replicative arrest or phenotypes characteristic of a regulatory defect. Because CDC44 and PCNA appear to be components of a large intracellular complex, complete understanding of their roles in the cell will require an understanding of the components with which they interact. Thus, pseudoreversion and unlinked non-complementation will be used to identify functional interactions that take place in vivo. Mutagenesis and phenotypic analysis will then be used to examine the roles of the newly identified gene products in the vital processes of the cell cycle. A related project will examine the effect on telomere metabolism of perturbing DNA replication. The allele-specific elongation of telomeres in cdc44 and cdcl7 (DNA polymerase delta) mutants suggests that specific perturbations of DNA replication can unbalance the processes that maintain telomeres. To determine the characteristics that lead to this imbalance, telomere elongation will be assessed in cdc44 and cdcl7 mutants with different phenotypes, and the effects of cell-cycle delays will be examined. Taken together, our studies of CDC44 and PCNA will lead to an improved understanding of DNA replication and the cell cycle, and ultimately of genetic diseases such as cancer.

我们的最终目标是了解染色体的分子机制 隔离。具体来说，我们想了解染色体的 精确地复制了它们的结构完整性如何维持，以及 它们的分离如何与细胞周期中的其他事件协调。 为了探究这些问题，我们将研究两个编码基因的突变 特征良好的蛋白质可能在 DNA复制的力学及其与有丝分裂的协调。 Cdc44（复制因子C的大亚基）和PCNA（增殖 细胞核抗原）已显示出生化和 从遗传上讲，每个特征都表明在 细胞周期。彻底的生化分析表明，复制因子 C和PCNA一起起作用以促进DNA聚合酶的加工性 体外三角洲。但是，CDC44突变体的细胞周期分析 表明关键有丝分裂（复制后）功能受到干扰。 相应地，PCNA与大多数细胞周期蛋白依赖性激酶的关联 体内复合物表明PCNA可能在 DNA合成与其他细胞事件的协调。 为了分析CDC44和PCNA在活细胞中的作用，我们将创建 体外随机和位置定向突变并分析表型 他们会议。 DNA复制的扰动将通过升高发出信号 突变速率，DNA合成的破坏和适当的细胞周期 逮捕。有丝分裂功能的扰动将通过后揭示 调节缺陷的复制停滞或表型的特征。 因为cdc44和pCNA似乎是大细胞内的组成部分 复杂，完全了解其在牢房中的角色将需要 了解它们相互作用的组件。因此， 伪version和未链接的不集成将用于识别 在体内发生的功能相互作用。诱变和 然后，将使用表型分析来检查新的角色 在细胞周期的重要过程中确定了基因产物。 一个相关项目将研究对端粒代谢的影响 干扰DNA复制。端粒的等位基因特定延伸 CDC44和CDCL7（DNA聚合酶三角洲）突变体表明特定 DNA复制的扰动会使维持的过程不平衡 端粒。为了确定导致这种不平衡的特征， 端粒伸长率将在CDC44和CDCL7突变体中进行评估 不同的表型，细胞周期延迟的影响将是 检查。 综上所述，我们对CDC44和PCNA的研究将导致改进 了解DNA复制和细胞周期，并最终 遗传疾病，例如癌症。