Double-strand DNA break formation mediated by flap endonuclease-1.
Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.
Vispé
Stephane
S
Laboratory of DNA Repair, Health and Environment Unit, Laval University Medical Center, CHUQ, Faculty of Medicine, Laval University, 2705 Boulevard Laurier, Sainte-Foy, Québec G1V 4G2, Canada.
Ho
Erick L Y
EL
Yung
Tetsu M C
TM
Satoh
Masahiko S
MS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D045585:Flap Endonucleases; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human