STRUCTURE/FUNCTION OF YEAST DNA POLYMERASE DELTA

该研究的主要目标是研究DNA聚合酶δ（Pol-delta）的结构和功能，Pol-delta是细胞中用于DNA复制和DNA修复的主要DNA聚合酶。研究将在酵母酿酒酵母中进行，这是DNA代谢基础研究的一个极好的模型系统。该实验室最近的研究为Pol-Delta的结构提供了新的线索。最重要和最有趣的是，这种酶具有整体二聚体结构。这些新发现最明显的暗示是Pol-delta可能在DNA复制叉处起二聚体的作用。Pol-delta的两个小亚基POL 31和POL 32的基因以及酵母和大肠杆菌中的过表达系统的可用性促进了这些最近的研究。杆菌拟议的研究将侧重于了解Pol-delta在复制叉处的二聚体结构的功能意义以及Pol-delta的小亚基在建立这种结构中的作用。主要问题是：Pol 32 p亚基的二聚化结构域和增殖细胞核抗原（PCNA）结合结构域如何影响酵母细胞的生长和损伤反应？最近的研究表明，二聚化和PCNA结合驻留在这个亚基。将进行随机和定点诱变以消除任一功能，并在体内和体外测试结果。Pol-delta是体外功能性二聚体吗？将设计特定的底物来测试这个问题。单体Pol-delta突变体的可用性将促进这些研究。Pol-delta复制哪条链，Pol-delta的作用是什么？研究将在具有特定核苷酸类似物的可渗透细胞中进行。Pol-delta是否有额外的亚基？将对POL 31或POL 32中的菌株突变体进行抑制子分析，以分离推定的额外亚基。这些研究对于进一步了解真核细胞中DNA复制的机制具有重要意义。由于一些DNA复制因子也在DNA修复中起作用，因此了解酵母中这两种途径的调节和相互作用对于理解人类中的类似过程非常重要。人类对这些过程的不当调节可能导致癌症。

The main goal of the research is to study the structure and function of DNA polymerase delta (Pol-delta), the principal DNA polymerase in the cell for DNA replication and for DNA repair. Studies will be carried out in the yeast Saccharomyces cerevisiae, an excellent model system for basic studies in DNA metabolism. Recent studies from this laboratory have shed new light on the structure of Pol-delta. Most significantly and intriguingly, the enzyme has an overall dimeric structure. The most obvious implication of these new findings is that Pol-delta may function as a dimer at the DNA replication fork. These recent studies have been facilitated by the availability of the genes for the two small subunits of Pol-delta, POL31 and POL32, and of overexpression systems in yeast and in E. coli. The proposed studies will focus on gaining an understanding of the functional significance of the dimeric structure of Pol-delta at the replication fork and the role of the small subunits of Pol-delta in establishing this structure. Major questions addressed in this proposal are: How do the dimerization domain and the Proliferating Cell Nuclear Antigen (PCNA)- binding domain of the Pol32p subunit affect yeast cell growth and damage-response? Recent studies indicate that dimerization and PCNA- binding reside in this subunit. Random and site-directed mutagenesis will be carried out to eliminated either function and test the consequences in vivo and in vitro. Is Pol-delta a functional dimer in vitro? Specific substrates will be designed to test this question. These studies will be facilitated by the availability of monomeric Pol-delta mutants. Which strand(s) does Pol-delta replicate and what is the role of Pol-epsilon? Studies will be carried out in permeable cells with specific nucleotide analogs. Does Pol-delta have additional subunit(s)? A suppressor analysis of strains mutant in POL31 or POL32 will be carried out to isolate a putative additional subunit. These studies are important for a further understanding of the mechanism of DNA replication in the eukaryotic cell. As some DNA replication factors also function in DNA repair, an understanding of the regulation and the interaction of these two pathways in yeast is important for understanding analogous processes in humans. Improper regulation of these processes in humans may lead to cancer.