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

真核 DNA 聚合酶 zeta 在旁路 DNA 合成中发挥作用的生化研究

• 批准号: 12680676
• 负责人: AKIYAMA Masahiro
• 金额: $ 2.37万
• 依托单位: NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680676/
• 关键词: bypass DNA synthesis; translesion DNA replication; Xenopus; DNA polymerase zeta; REV3 gene; DNA damages; mutation; replication fork; DNAポリメラーゼ; アフリカツメガエル卵母細胞; スライディングクランプ

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.

旁路DNA合成（translesion DNA replication）是恢复因DNA损伤而停止的复制叉进展的机制之一。在真核生物中，DNA聚合酶zeta（zeta）在易错旁路DNA合成中起作用。为了阐明错误倾向旁路DNA合成的分子机制，我建议在非洲爪蟾卵母细胞提取物中鉴定DNA聚合酶zeta，并纯化酶以进行生化分析。虽然DNA聚合酶zeta尚未纯化，但以下结果将使使用免疫学和生物化学方法纯化该酶变得可行。(i)编码非洲爪蟾REV3的cDNA已从卵中分离出来。预测的Xenopus KEV 3的大小约为350kDa。(ii)将两部分cDNA用pET系统进行细菌表达，其中密码子偏好性被固定以表达真核基因。分别针对这两种蛋白质产生两种抗REV3抗体。通过用一种抗体免疫沉淀爪蟾卵母细胞提取物并用另一种抗体探测沉淀物，在提取物中鉴定出一种350kDa的蛋白。蛋白质的分子量与Xeflqpus REV3的预期大小一致。(iii)本文建立了一个体外检测旁路DNA合成的实验系统。利用该实验，在非洲爪蟾卵母细胞提取物中发现了至少三种不同的旁路活性。有趣的是检查免疫学检测到的REV3蛋白是否负责旁路活性之一。