MECHANISM OF DNA RECOMBINATION AND REPAIR IN YEAST SACCHAROMYCES CEREVISIAE

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It has been believed that DNA polymerase beta is required for DNA repair reaction in mammalian cells. Although other DNA polymerases are well conserved functionally and structurally between yeast and mammalian cells, so far no DNA polymerase beta-like DNA polymerase activity has not been identified in yeast cells. Thus, we have tried to identify a new DNA polymerase activity that resemble to mammalian DNA polymerase beta. We successfully identified in the crude extracts of S. cerevisiae cells and partially purified such activity. Although its apparent molecular weight of the DNA polymerase polypeptide was considerably larger than that of DNA polymerase beta, its biochemical properties were very similar to those of DNA polymerase beta. To study this new DNA polymerase's function, we have been trying to clone a gene encoding the DNA polymerase using antibody against the purified DNA polymerase and the PCR technique. DNA helicase is believed to be required not only for DNA replication, but also for DNA repair and recombination. We have identified and purfied a new DNA helicase activity. The apparent molecular weight and biochemical properties of the purified polypeptide, and genetical studies indicated that this DNA helicase is distinguished from the previously identified DNA helicases in yeast, suggesting that it is encoded by a new gene. Using the cloned S. cerevisiae DST2 which encodes DNA strand transfer protein beta as a hybridization probe, we have identified two homologs of DST2 in S. pombe. One of them which encodes a 120-kDa polypeptide was found to be an essential gene for S. pombe cell growth, suggesting that it plays an important role in S. pombe. Using the 120-kDa polypeptide gene as a probe, we have also identified and cloned a S. cerevisiae homolog which encodes a 120-kDa polypeptide. Like S. pombe, the S. cerevisiae 120-kDa polypeptide gene is essential for cell growth. To find the function of the 120-kDa polypeptide in the cell, we have isolated various temperature- sensitive mutants.