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

碱基切除修复（BER）是修复DNA碱基损伤的主要途径，如无尿嘧啶/无嘧啶（AP）位点，或脱胺、烷基化或氧化碱基。BER途径分为DNA聚合酶β(Polβ)依赖的短补丁BER和PCNA/flap内切酶-1 （FEN-1）依赖的长补丁BER。我们从鸡DT40细胞系中获得了FEN-1或pol - β缺失的敲除细胞系，并研究了这两种蛋白在两种亚通路中的差异作用。令人惊讶的是，双突变细胞可以在亚通路缺乏的情况下存活，这意味着两种蛋白质的冗余或存在替代通路来支持缺陷。与野生型细胞相比，3个突变细胞系均对甲基磺酸敏感，但FEN1-null和双突变细胞系对过氧化氢敏感。体外BER测定，使用无细胞提取物和含有一个尿嘧啶的双链DNA底物，显示野生型和Polβ-null细胞具有修复缺陷的活性，而FEN1-null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，是修复一个碱基缺陷所必需的。因此，我们正在进一步研究缺乏FEN1而没有Polβ的细胞无法修复缺陷的原因。

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.