The Roles of DNA Polymerase Epsilon in Yeast

点击翻译按钮获取中文摘要

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.