Roles of DNA Polymerase Epsilon in Yeast

DNA 聚合酶 Epsilon 在酵母中的作用

• 批准号: 6834362
• 负责人: Judith L CAMPBELL
• 金额: $ 4.03万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6834362
• 关键词: DNA directed DNA polymerase; DNA repair; DNA replication; Poland; Saccharomyces cerevisiae; alleles; enzyme activity; enzyme structure; genetic screening; mutant; phenotype; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): We propose to identify mutants in the second largest subunit, Dpb2, of the DNA polymerase epsilon holoenzyme of yeast. Pol epsilon is essential for initiation and for elongation of replication forks, and Dpb2 can be expected to play an important role in modulating the polymerase activity. We will use the mutants to investigate the physiological role of Dpb2 in: initiation of replication, fidelity, interactions with other subunits of the epsilon holoenzyme, and checkpoints. The resources produced over recent years under funding from the American parent grant form the foundation for the application. While the catalytic subunit of pol E has been thoroughly investigated, the Dpb2 subunit remains poorly characterized. We will identify new dpb2 mutants using plasmid shuffling. We will screen among them for mutator phenotype, initiation defects, and elongation defects. The question of how organisms duplicate their DNA with high accuracy is of fundamental interest. Three major components contribute to the fidelity of replication: base selection by DNA polymerase, proofreading by 3'-5' exonuclease, and mismatch repair. The sequential action of these three processes is responsible for the high fidelity of genome duplication. Little is known about the possible role of DNA polymerase accessory proteins in this sequence. The Polish laboratory has a well-established program in studying DNA replication fidelity in bacteria. It would now like to extend these studies to the model eukaryote, Saccharomyces cerevisiae by collaborating with the American labortory. If mutator mutants are found, they will be studied in Poland, both as single mutants and as double mutants having defects in mismatch repair, with other mutants studied in America. If mutator alleles are not found, the Polish group will extend its interests to other aspects of polymerase function, including but not limited to initiation, that require Dpb2. The mutants will be used to determine the specific function(s) of Dpb2 within the holoenzyme in initiation and/or elongation. A likely mechanism by which Dpb2 might mediate its effect is through protein/protein interactions. We will investigate interaction of the mutant dpb2 proteins with other pol epsilon subunits and accessory proteins such as Dpbl 1,Sld5,Trf4,5.

描述（由申请人提供）：我们建议在酵母的DNA聚合酶Epsilon Holoenzyme的第二大亚基DPB2中识别突变体。 Pol Epsilon对于启动和伸长复制叉是必不可少的，并且可以预期DPB2在调节聚合酶活性中起重要作用。我们将使用突变体研究DPB2在：复制的启动，忠诚度，与Epsilon Holoenzyme的其他亚基的相互作用和检查点的生理作用。近年来，美国父母赠款的资助构成了该申请的基础。虽然对pol e的催化亚基进行了彻底的研究，但DPB2亚基的特征仍然很差。我们将使用质粒改组确定新的DPB2突变体。我们将在其中筛选出突变器表型，启动缺陷和伸长缺陷。有机体如何以高精度复制其DNA的问题具有基本的兴趣。 三个主要组成部分有助于复制的保真度：通过DNA聚合酶选择碱基选择，通过3'-5'核酸酶进行校对和不匹配修复。这三个过程的顺序作用负责基因组重复的高保真度。关于DNA聚合酶辅助蛋白在此序列中的可能作用知之甚少。波兰实验室在研究细菌中的DNA复制保真度方面拥有良好的计划。现在，它希望通过与American Labortory合作，将这些研究扩展到真核生物酿酒酵母的模型。如果发现突变体突变体，则将在波兰进行研究，无论是单个突变体还是具有不匹配修复缺陷的双突变体，在美国研究了其他突变体。如果找不到突变器等位基因，则波兰组将将其兴趣扩展到需要DPB2的聚合酶功能的其他方面，包括但不限于启动。突变体将用于确定全酶在开始和/或伸长率中DPB2的特定函数。 DPB2可能介导其作用的可能机制是通过蛋白质/蛋白质相互作用。我们将研究突变型DPB2蛋白与其他pol Epsilon亚基和辅助蛋白（例如DPBL 1，SLD5，TRF4,5）的相互作用。