Biochemical studies of eukaryotic DNA polymerase zeta functioning in bypass DNA synthesis

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Bypass DNA synthesis (translesion DNA replication) is one of the mechanisms that retrieve progression of replication fork halted by DNA damages. In eukaryote, DNA polymerase zeta (ζ) functions in error-prone bypass DNA synthesis. To elucidate molecular mechanisms of error-prone bypass DNA synthesis, I proposed to identify DNA polymerase zeta in Xenopus oocyte extract and purify the enzyme to analyze it biochemically. Although DNA polymerase zeta has not been purified yet, the following results will make feasible to purify the enzyme using immunological and biochemical methods.(i) The CDNA encoding Xenopus REV3 has been isolated from egg. The predicted size of Xenopus KEV3 was about 350kDa.(ii) Two parts of the cDNA were bacterially expressed using the pET system in which a codon bias is fixed to express eukaryotic gene. Two kinds of anti-REV3 antibodies were raised against those two proteins, respectively. By immunoprecipitating Xenopus oocyte extract by one antibody and probing the precipitate by the other antibody, a 350kDa protein has been identified in the extract. The molecular weight of the protein coincided to the expected size of the Xeflqpus REV3.(iii) An assay system to detect bypass DNA synthesis in vitro has been constructed Using the assay, at least three distinct bypass activities have been found in Xenopus oocyte extract. It is interesting to examine if the immunologically detected REV3 protein is responsible to one of the bypass activities.