Enzymatic Mechanisms of DNA Replication

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DESCRIPTION (provided by applicant): Genomic approaches and the discovery that E. coli UmuC and DinB, yeast RAD30 and Xeroderma pigmentosum variant (XPV) encode DNA polymerases have led to an explosion in the number of known DNA polymerases. We have discovered, originally via a two hybrid screen, that the essential but non-catalytic C terminal domain of yeast DNA polymerase (pol) epsilon, a polymerase required for DNA replication, repair, and the DNA replication checkpoint, interacts both physically and functionally with one of the most novel of the new polymerases, pol sigma. Yeast pol sigma mutants are defective in completion of S phase, in DNA repair, in chromosome condensation, and in sister chromatid cohesion. The discovery of this interaction lends a new dimension to our continuing search for the precise role of pot epsilon in chromosome dynamics during S phase. We find that pol epsilon mutants are defective in cohesion. The overall goat of our studies is to determine how pol epsilon and pol sigma interact in these various cellular processes. First, to define the in vivo role of the interaction, structure/function analysis will be carried out by using site directed mutations and correlating effects on interaction between the two proteins with phenotypes of resulting mutants, especially with respect to sister chromatid cohesion. Second, we will characterize pol sigma biochemically- definition of substrates, fidelity, processivity, and stimulation by PCNA. A defining aspect of our studies is focus on the interaction between pol sigma and pot epsilon. We will also address pol epsilon's interaction with another cohesion protein,Ctf18, a component of an alternative polymerase clamp loader. The key questions in the nascent field of sister chromatid cohesion are what is the nature of the links between the chromosomes and what is the mechanism by which DNA replication contributes to forming these links. With this in mind, we will determine if pol epsilon is required for establishing cohesion and/or maintenance. We will study the fate of replication forks at sites of cohesion in vivo and the association of polymerases with these sites. We will use chromatin immunoprecipitation, 2D gel electrophoresis of DNA replication intermediates, and microscopic analysis of chromosome dynamics using fluorescence microscopy and the GFP-repressor/operator tagging strategy, techniques set up by us during the previous granting period. Several collaborations are in place to insure progress.