Defective flap endonuclease 1 activity in mammalian cells is associated with impaired DNA repair and prolonged S phase delay.
Flap endonuclease 1 (FEN-1) is a 5'-3' flap exo-/endonuclease that plays an important role in Okazaki fragment maturation, nonhomologous end joining of double-stranded DNA breaks, and long patch base excision repair. Here, we demonstrate that the wild type FEN-1 binds tightly to chromatin in conjunction with proliferating cell nuclear antigen (PCNA) recruitment after MMS treatment, and the nuclease-defective FEN-1 increased the sensitivity of the cells to methylmethane sulfonate (MMS) and to UV light but not to ionizing radiation. In contrast, the cells expressing the nuclease-defective and PCNA binding-defective double mutant FEN-1 exhibited sensitivities similar to those in the cells expressing the wild type FEN-1. MMS treatment caused a prolonged delay of S phase progression and impairment in colony-forming activity of cells expressing nuclease-defective FEN-1. A comet assay demonstrated that DNA repair after MMS or UV treatment was impaired in the cells expressing nuclease-deficient FEN-1 but not in the cells with double-mutated FEN-1. Taken together, these findings suggest that FEN-1 plays an essential role in the DNA repair processes in mammalian cells and that this activity of FEN-1 is PCNA-dependent.
Shibata
Yoshiyuki
Y
Department of Radiology and Cancer Biology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.
Nakamura
Takashi
T
D021581:Active Transport, Cell Nucleus; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000032:analysis / Q000502:physiology*; D045585:Flap Endonucleases; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D016196:S Phase; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D002843:Chromatin; D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D008741:Methyl Methanesulfonate; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human