ENZYMOLOGY OF REPLICATION OF YEAST CHROMOSOMAL DNA

建议的研究的主要目的是了解心绞痛的机制 真核细胞中的染色体DNA复制。的重点是 该项目是关于酵母DNA复制叉和 通过遗传和生物化学手段对其成分进行深入研究。 这些研究不仅形成了对心脏解剖结构的全面分析 复制的分叉，也是长期发展的必要前奏 我们研究的目标，这是具体的启蒙和 从酵母复制起点延长DNA复制。这个 复制型DNA聚合酶增量全酶由DNA组成 复制钳--增殖细胞核抗原聚合酶 (增殖细胞核抗原)和钳位加载器复制因子C(RF-C)。结构和 五亚单位RF-C复合体的机理研究将集中在 Rf-C各亚基的活性需求及其作用机制 RF-C通过其将增殖细胞核抗原装载在模板-引物处。令人敬爱的机械师 对负荷途径的洞察将有助于理解这一途径 冈崎片断合成过程中的全酶循环。一种结构- 增殖细胞核抗原的功能分析及其与新的增殖细胞核抗原相互作用的鉴定 蛋白质将进一步定义增殖细胞核抗原在DNA复制、细胞中的作用 周期进程和DNA修复。相互作用的性质和 特定的增殖细胞核抗原突变体的影响将被调查。叉子伸长 研究和Okazaki片段成熟研究专门调查 细胞DNA解旋酶在这些过程中的作用。 这些研究对于进一步了解这一机制具有重要意义。 真核细胞中的DNA复制。因为一些DNA复制 因子也在DNA修复中发挥作用，这是对调节的理解 而这两条途径在酵母中的相互作用对于 理解人类的相似过程。对这些不适当的监管 人类的过程可能会导致癌症。

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 its components by genetic and biochemical means. These studies not only form a comprehensive analysis of the anatomy of the replication fork, but are also a necessary prelude to the long-term objective of our research, which is the specific initiation and elongation of DNA replication from a yeast replication origin. The replicative DNA polymerase delta holoenzyme consists of the DNA polymerase, the replication clamp Proliferating Cell Nuclear Antigen (PCNA), and the clamp loader Replication Factor C (RF-C). Structural and mechanistic studies of the five-subunit RF-C complex will focus on the requirement of each of the RF-C subunits for activity and the mechanism by which RF-C loads PCNA at the template-primer. A dear mechanistic insight in the pathway of loading will aid in understanding the pathway of holoenzyme recycling during Okazaki fragment synthesis. A structure- function analysis of PCNA and the identification of new PCNA-interacting proteins will further define a role for PCNA in DNA replication, cell cycle progression and DNA repair. The nature of the interactions and the effect of specific PCNA mutants will be investigated. Fork elongation studies and Okazaki fragment maturation studies specifically investigate the roles of cellular DNA helicases in these processes. 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.