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Studies of base excision repair in cell mutants deficient. in either DNA polymerase, or flap endonuclease-1 or both, generated from chicken DT40 cells.

细胞突变体碱基切除修复缺陷的研究。

基本信息

批准号：
15570146
负责人：
KOYAMA Hideki
金额：
$ 2.37万
依托单位：
Yokohama City University, Kihara Institute for Biological Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Base excision repair (BER) is the major pathway in repair of DNA base lesions such as apurinic/apyrimidinic (AP) sites, or deaminated, alkylated or oxidative bases. The BER pathway is divided into DNA polymerase β(Polβ)-dependent short-patch BER and PCNA/flap endonuclease-1 (FEN-1)-dependent long-patch BER. We generated knockout cell lines deficient in either FEN-1 or Polβ, or both from the chicken DT40 cell line and studied differential roles of the proteins in the two subpathways. Surprisingly, double mutant cells could survive nevertheless of deficiency in the subpathways, implying the redundancy of both proteins or the existence of alternative pathways to backup the defects. Three mutant cell lines were all hypersensitive to methyl methanesulfonate, but FEN1-null and double mutants were hypersensitive to hydrogen peroxide compared with wild-type cells. In vitro BER assay, using cell-free extracts and a double-stranded DNA substrate containing one uracil, revealed that while wild-type and Polβ-null cells had an activity to repair the defect, FEN1-null and double mutant cells showed almost no activity. Importantly, this result indicates that FEN1, but not Polβ, is essential for repairing one base defect. Therefore, we are further studying the reason why cells lacking FEN1 but not Polβ are unable to repair the defect.

碱基切除修复(BER)是DNA碱基损伤修复的主要途径，如脱嘌呤/脱嘧啶(AP)位点或脱氨基、烷基化或氧化碱基。误码率通路分为依赖于DNA聚合酶β(POL-β)的短斑块误码率和依赖于增殖细胞核抗原/翻盖核酸内切酶-1(FEN-1)的长斑块型误码率。我们从鸡β细胞系中建立了缺失FEN-1和POL-1或两者都缺乏的基因敲除细胞系，并研究了这两个亚途径中蛋白质的差异作用。令人惊讶的是，双突变细胞仍然可以在亚途径缺乏的情况下存活，这意味着两种蛋白质的冗余或存在替代途径来支持缺陷。与野生型细胞相比，FEN1缺失突变株和双突变株对过氧化氢均敏感。使用无细胞提取物和含有一个尿嘧啶的双链底物进行的体外误码率分析表明，野生型和POLβ缺失细胞具有修复缺陷的活性，而FEN1缺失和双突变细胞几乎没有活性。重要的是，这一结果表明FEN1，而不是POLβ，对于修复一个碱基缺陷是必不可少的。因此，我们正在进一步研究缺乏FEN1而不是POLβ的细胞无法修复缺陷的原因。