The goal of the proposal is to understand the mechanism by which an origin of DNA replication is converted into a replication fork and how the proteins at the fork are organized to coordinate the synthesis of the leading and lagging strands. The underlying assumption, which, while plausible, remain to be proved, is that there is a super assembly of proteins at the replication fork and that it functions like a machine whose moving parts are proteins. Wha is less certain is whether there is a single complex assembled at origins that changes conformation in a concerted manner to effect each stage of fork progression or if there are a number of smaller machines each assigned a different specific task and only transiently associated with the fork. To attack such a complex problem requires dividing it into partial reactions. In the coming granting period the investigator will study in depth the Dna2 helicase and DNA polymerase epsilon, with a focus on the dynamics of protein/protein interactions of these key components. They would use yeast as model system to allow a combined genetic and biochemical approach. In the past granting period, the investigators described a helicase essential for chromosomal DNA replication. It was discovered that it interacted with FEN-1, a protein involved in Okazaki fragment processing in SV40 in vitro DNA replication. Further studies of Dna2 have the goal of determining whether the Dna2 helicase plays a role in maturation of the lagging strand in yeast cells and if so, does it have a catalytic role or an architectural/structural role. It should go without saying that to understand the assembly, activation, and movement of the replication fork, one must understand the DNA polymerase. This grant has supported studies of the yeast DNA polymerases for many years. Recently the investigators have focused on pol epsilon. They have used site-directed mutagenesis in an analysis of the C-terminal half of the catalytic core polypeptide, which may have a dual role in DNA replication and sensing DNA damage. The investigators will continue to determine the protein/protein contacts in the pol epsilon holoenzyme and to determine the role of individual subunits. They will use chromatin crosslinking to study the dynamics of interactions between the three DNA polymerases at replication origins and forks, ultimately to get at the question of which polymerase copie which strand, taking advantage of all the polymerase mutants and clones produced under this grant.
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