Roles of DNA Polymerase Epsilon in Yeast

描述(由申请人提供)：我们建议鉴定酵母DNA聚合酶epsilon全酶第二大亚基Dpb2中的突变。POL epsilon对于复制叉的启动和延长是必不可少的，而Dpb2可以在调节聚合酶活性方面发挥重要作用。我们将利用这些突变体来研究Dpb2在以下方面的生理作用：复制的启动、保真度、与epsilon全酶的其他亚单位的相互作用以及检查点。近几年在美国家长基金的资助下产生的资源构成了申请的基础。虽然已经对polE的催化亚基进行了彻底的研究，但Dpb2亚基的特性仍然很差。我们将使用质粒改组来鉴定新的dpb2突变体。我们将在其中筛选突变子表型、起始缺陷和延伸缺陷。生物体如何高精度地复制它们的DNA是一个具有根本意义的问题。复制的保真度有三个主要组成部分：DNA聚合酶的碱基选择，3‘-5’核酸外切酶的校对和错配修复。这三个过程的连续作用是基因组复制高保真的原因。人们对DNA聚合酶辅助蛋白在该序列中的可能作用知之甚少。波兰实验室在研究细菌中DNA复制的保真度方面有一个完善的计划。现在，它希望通过与美国实验室的合作，将这些研究扩展到真核生物模型酿酒酵母。如果发现突变体，将在波兰作为单突变体和双突变体进行研究，这些突变体在错配修复方面存在缺陷，其他突变体将在美国研究。如果没有发现突变等位基因，波兰小组将把兴趣扩展到聚合酶功能的其他方面，包括但不限于启动，需要Dpb2。这些突变体将被用来确定Dpb2在全酶启动和/或延伸中的特定功能(S)。Dpb2可能通过蛋白质/蛋白质相互作用来调节其效应。我们将研究突变的Dpb2蛋白与其他Polepsilon亚基和辅助蛋白如Dpb1、SLD5、Trf4、5的相互作用。

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.