MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

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It has been believed that DNA polymerase beta functions for DNA repair in mammalian cells. Although other DNA polymerase are well conserved functionally and structurally between yeast and mammalian cells, no beta-like DNA polymerase has been found in the yeast Saccharomyces cervisiae. We have identified and partially purified a new DNA polymerase. activity which is very similar to Drosophila melanogaster DNA polymerase beta. This polymerase is a basic protein, sensitive to dideoxylnucleotides, but not sensitive as the inhibitors which affect the activity of DNA polymerase alpha, delta, or epsilon, and its apparent MW is about 100 kDa. To further study this new DNA polymerase, we have been trying to clone gene for the DNA polymerase. REV3 gene, which is required for mutagenic DNA repair, encodes a polypeptide of ca. MW of 180 kDa and has several conserved regions specific for a-type DNA polymerases. However, it has not been established that the polypeptide is a DNA polymerase. In this year, we have expressed the REV3 gene product in insect cells using a vaccullo virus expression vector and have shown that the REV3 gene product has a DNA polymerase activity by using activity gel technique.