ENZYMOLOGY OF REPLICATION OF YEAST CHROMOSOMAL DNA

描述（改编自申请人的摘要）：拟议的主要目标 研究的目的是了解染色体DNA复制的机制， 真核细胞该项目的重点是重建 酵母DNA复制叉及其几种组分的深入研究 通过遗传和生化手段。在知道几个复制品 蛋白质也是细胞正常损伤反应所必需的，另一个 感兴趣的领域是在DNA水平上理解这种反应， 复制叉的后续改造。四个具体目标 本文主要内容如下：（1）复制因子C X射线晶体学研究 （RF-C），一种装载复制钳PCNA的5亚基复合物，以及 RF-C的亚复合物和替代的RF-C样复合物，其参与 细胞损伤反应;（2）RF-C的机制研究，以了解如何 该复合体装载PCNA并在冈崎片段合成期间起作用;（3） PCNA在DNA复制和DNA修复中的结构与功能研究;（4） 可能具有特异性的类RF-C复合物的构建及研究 在冈崎片段合成、染色体分离和损伤过程中发挥作用 细胞内的反应。 因为复制机器的组成部分和损伤反应 机器在真核生物中是保守的，首席研究员预计， 了解酵母中这些机器的机制和调节 将作为理解人类细胞中这些过程的模型。 这些过程在人类中的不适当的功能和调节可能导致 突变和癌症的积累。

DESCRIPTION (adapted from applicant's abstract): The main goal of the proposed research is to understand the mechanism of chromosomal DNA replication in eukaryotic cells. The emphasis of the project is on the reconstitution of the yeast DNA replication fork and an intensive study of several of its components by genetic and biochemical means. With the knowledge that several replication proteins are also required for a normal damage response in the cell, another area of interest is to understand this response at the DNA level and to study the subsequent remodeling of the replication fork. The four specific aims in the proposal are: (1) X-ray crystallographic studies of Replication Factor C (RF-C), a 5-subunit complex which loads the replication clamp PCNA, and of sub-complexes of RF-C and alternative RF-C-like complexes which are involved in damage response in the cell; (2) Mechanistic studies of RF-C to understand how this complex loads PCNA and functions during Okazaki fragment synthesis; (3) Structure-function studies of PCNA in DNA replication and DNA repair; (4) Reconstitution and study of RF-C-like complexes which may have specific functions during Okazaki fragment synthesis, chromosome segregation, and damage response in the cell. Because the components of the replication machinery and the damage response machinery are conserved in eukaryotes the principal investigator expects that an understanding of the mechanism and regulation of these machineries in yeast will serve as a model for understanding these processes in human cells. Improper function and regulation of these processes in humans may lead to the accumulation of mutations and cancer.