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: 6901915
• 负责人: SUSAN L FORSBURG
• 金额: $ 41.15万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6901915
• 关键词: DNA binding protein; DNA replication; Schizosaccharomyces pombe; cell cycle; chromatin; chromatin immunoprecipitation; cyclin dependent kinase; fungal genetics; fungal proteins; gene mutation; laboratory rabbit; phosphorylation; polymerase chain reaction; protein biosynthesis; protein localization; protein sequence; protein structure function

DESCRIPTION (provided by applicant): The broad goal of this research program is to understand the link between DNA replication and genome integrity in a model organism, Schizosaccharomyces pombe. The processes of chromosome replication and chromatin assembly are intimately coupled with genome stability and malignancy. Progressive loss of genome integrity can cause or exacerbate cancer. Fission yeast offers a powerful model system with facile genetics, and large chromosomes and replication origins reminiscent of those in metazoan cells. Recent completion of the genome sequence of S. pombe provides new tools allowing rapid identification and characterization of protein function in vivo, making predictions that can then be tested in human cells. The conserved MCM proteins are required both for initiation and elongation stages of DNA synthesis in all eukaryotes. Analysis of MCM function in vivo provides the essential cellular context for their role during S phase. This proposal provides evidence linking MCMs to proteins with multiple roles in DNA metabolism. The first aim will investigate evidence that loss of MCM function leads to double strand breaks in the DNA, and determine how the cell uses proteins of the homologous recombination machinery to prevent this. Interactions between MCMs and recombination machinery will also be examined in human cells. The second aim will characterize MCM function by mutational analysis of residues predicted to play important structural roles, and will characterize the localization of wild type and mutant proteins on replicating chromatin using new methods of visualization. This aim will also analyze modifications of MCM proteins during normal cell cycle and perturbed S phase. The third aim will investigate how chromatin structure is linked to MCM function by examining the links between origin timing and histone modifications, investigating previously identified interactions between histone modifying enzymes and MCMs, and determine how MCMs gain access to DNA within a chromatinized template. All these proteins exist in humans and both have links to cancer, but the in vivo, genetically oriented approach is not possible in tissue culture. However, predictions generated in the yeast system can be tested in human cells. Thus, studies in simple yeast cells have direct relevance to understanding the role of these factors in human cell division.

描述（由申请人提供）：本研究计划的主要目标是了解模式生物粟酒裂殖酵母中DNA复制和基因组完整性之间的联系。染色体复制和染色质装配的过程与基因组的稳定性和恶性程度密切相关。基因组完整性的逐渐丧失可导致或加重癌症。裂变酵母提供了一个强大的模型系统，具有简单的遗传学，大染色体和复制起点，让人想起后生动物细胞中的那些。最近完成的S. pombe提供了新的工具，允许在体内快速鉴定和表征蛋白质功能，进行预测，然后可以在人类细胞中进行测试。在所有真核生物中，保守的MCM蛋白在DNA合成的起始和延伸阶段都是必需的。对MCM体内功能的分析为其在S期的作用提供了必要的细胞背景。这一提议提供了将MCMs与在DNA代谢中具有多种作用的蛋白质联系起来的证据。第一个目标是研究MCM功能丧失导致DNA双链断裂的证据，并确定细胞如何使用同源重组机制的蛋白质来防止这种情况。MCMs和重组机制之间的相互作用也将在人类细胞中进行研究。第二个目标将表征MCM功能的预测发挥重要的结构作用的残基的突变分析，并将表征本地化的野生型和突变体蛋白质复制染色质使用新的可视化方法。这一目标也将分析MCM蛋白在正常细胞周期和扰动S期的修饰。第三个目标将研究染色质结构是如何连接到MCM功能，通过检查起源时间和组蛋白修饰之间的联系，研究以前确定的组蛋白修饰酶和MCMs之间的相互作用，并确定MCMs如何获得染色质化模板内的DNA。所有这些蛋白质都存在于人类中，并且都与癌症有关，但体内遗传导向的方法在组织培养中是不可能的。然而，在酵母系统中产生的预测可以在人类细胞中进行测试。因此，在简单酵母细胞中的研究与理解这些因子在人类细胞分裂中的作用有直接关系。