ENZYMOLOGY OF REPLICATION OF YEAST CHROMOSOMAL DNA

这项研究的主要目的是了解 真核细胞中的染色体DNA复制。的重点 项目是关于酵母DNA复制叉的重建， 通过遗传学和生物化学方法对其成分进行深入研究。 这些研究不仅形成了一个全面的解剖分析， 复制分叉，但也是长期的必要前奏 我们的研究目标，这是具体的启动和 从酵母复制起点延伸DNA复制。的 复制型DNA聚合酶δ全酶由DNA 聚合酶，复制钳，扩增细胞核抗原 （PCNA）和钳夹装载复制因子C（RF-C）。结构和 五亚基RF-C复合物的机制研究将集中在 每个RF-C子单元对活动和机制的要求 RF-C将PCNA负载在模板引物上。亲爱的机械 对负荷途径的洞察将有助于理解 在冈崎片段合成过程中全酶循环的过程。一个结构- PCNA的功能分析及新的PCNA相互作用蛋白的鉴定 蛋白质将进一步确定PCNA在DNA复制、细胞增殖和细胞凋亡中的作用。 周期进展和DNA修复。相互作用的性质和 将研究特异性PCNA突变体的作用。叉形伸长 研究和冈崎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该机制具有重要意义 真核细胞中的DNA复制。一些DNA复制 因子也在DNA修复中起作用， 酵母中这两种途径的相互作用对于 理解人类的类似过程。对这些问题的不当监管 可能导致癌症。

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.