Since changes of DNA sequence have profound genetic consequences, it is important to understand the mechanisms for maintaining and replicating these sequences if we are to understand fully the basis of genetic diseases and possibly degenerative diseases such as cancer and aging. The DNA polymerases are obviously key players in DNA metabolism, and it is our goal to understand how these enzymes contribute to maintaining genetic integrity. Several years ago we identified and purified a DNA polymerase, pol epsilon, that served to mediate DNA repair synthesis in permeabilized diploid human fibroblasts. However, Akio Sugino and coworkers have now suggested that in yeast, pol epsilon participates in DNA replication. the proposal is to continue to clone and to sequence a cDNA from a HeLa library with strong homology to the yeast pol epsilon gene. Sequence information will be used to make antibodies to the cDNA protein and antibodies will be utilized to confirm that the cDNA is that of pol epsilon, to purify the enzyme, and to identify other proteins bound to the enzyme. mRNA, protein, and activity of pol epsilon will be monitored through the cell cycle and also monitored in non-cycling HeLa cells and in differentiated neuroblastoma cells as well as in cells exposed to DNA damaging agents. Finally, catalytic properties of pol epsilon and a form(s) of the enzyme with a smaller catalytic subunit will be compared as will their subunit structures. Ultimately we wish to understand what role(s) pol epsilon might have in DNA repair and elongation during DNA replication. Meanwhile, the utilization of RNA primers by pols alpha, beta, delta and epsilon will be methodically studied and removal of RNA primers and replacement with DNA by a complex of polbeta, DNase V, and an RNase H will be studied in model systems. Our previous experience with HeLa DNA polymerases alpha, beta and epsilon, and an accumulation of pol delta as a byproduct has placed us in a unique situation of having stocks of each of these enzymes and substrates so that comparisons of catalytic properties can be carried out in single experiments. In this manner, we hope to contribute to the knowledge of the roles of the individual DNA polymerases in DNA metabolism.
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