ENZYMOLOGY OF REPLICATION OF YEAST CHROMOSOMAL DNA

说明(改编自申请人的摘要)：建议的主要目标 研究是为了了解人类染色体DNA复制的机制 真核细胞。该项目的重点是重建 酵母DNA复制叉及其几个组分的深入研究 通过遗传和生化手段。我们知道有几次复制 蛋白质也是细胞正常损伤反应所必需的，另一种 感兴趣的领域是在DNA水平上理解这种反应并研究 随后复制叉子的重塑。其中的四个具体目标 这些建议是：(1)复制因子C的X射线结晶学研究 (RF-C)，一个5亚基的复合体，它装载复制钳制的增殖细胞核抗原，以及 Rf-C亚复合体和替代的Rf-C类复合体 细胞内的损伤反应；(2)了解RF-C的机理研究 这种复合体在Okazaki片段合成过程中负载了增殖细胞核抗原并发挥作用； 增殖细胞核抗原在DNA复制和DNA修复中的结构与功能研究 可能具有特异性的类RF-C复合体的重组和研究 Okazaki片段合成、染色体分离和损伤过程中的功能 细胞内的反应。 因为复制机械的组件和损伤响应 机械在真核生物中是保守的，首席研究人员预计 了解酵母菌中这些机制的作用机制和调控 将成为理解人类细胞中这些过程的模型。 这些过程在人类体内的不适当的功能和调节可能导致 突变和癌症的累积。

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.