ENZYMOLOGY OF REPLICATION OF YEAST CHROMOSOMAL DNA

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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.