Our long term goals are two-fold: to understand what factors maintain or perturb the fidelity of mammalian DNA polymerases and to understand how these enzymes along with associated DNases take part in DNA excision repair. In the immediate future we propose to study changes which occur in DNA polymerase Alpha as the cell enters a non-cycling state due to population senescence, differentiation or arrest in G-o due to medium exhaustion. We also propose to study how these changes as well as phosphorylation by protein kinase C affect the fidelity and activity levels of DNA polymerase Alpha. Finally, several complex forms of DNA polymerases Alpha, Beta and Delta would be studied in order to learn about factors which affect fidelity. Using very defined damaged DNA and chromatin substrates we propose also to study excision and repair synthesis mediated by several forms of Alpha, Beta and Delta polymerase and by Gamma polymerase, each alone and in conjunction with DNases found to be associated with these enzymes. Finally, we have purified to homogeneity a 220 kilodalton factor which is required for DNA repair synthesis in permeabilized cells and we propose to study the catalytic properties of this factor and its possible relation to DNA polymerase Delta.
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