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

旁路DNA合成(跨损伤DNA复制)是恢复被DNA损伤阻止的复制分叉进展的机制之一。在真核生物中，DNA聚合酶Zeta(ζ)在容易出错的旁路DNA合成中发挥作用。为了阐明容易出错的旁路DNA合成的分子机制，我提出在非洲爪哇卵母细胞提取液中鉴定DNA聚合酶Zeta，并对其进行纯化以进行生化分析。虽然DNA聚合酶Zeta尚未得到纯化，但以下结果将使利用免疫学和生物化学方法纯化该酶成为可能。(I)已从鸡蛋中分离到编码非洲爪蛙Rev3的CDNA。(2)利用固定密码子偏向的pET系统对该基因的两部分进行了细菌表达。分别针对这两种蛋白制备了两种抗Rev3抗体。通过一种抗体对非洲爪哇卵母细胞提取物进行免疫沉淀，另一种抗体对沉淀物进行探测，在提取物中鉴定出一种350 kDa的蛋白质。该蛋白的相对分子质量与Xeflqpus Rev3的预期大小一致。(Iii)建立了体外检测旁路DNA合成的检测体系，在非洲爪哇卵母细胞提取液中至少发现了三种不同的旁路活性。检查免疫检测到的Rev3蛋白是否与其中一种旁路活动有关是一件有趣的事情。

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.