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

点击翻译按钮获取中文摘要

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.