Double-strand DNA Break Formation Mediated by Flap Endonuclease-1

Next Section Abstract 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 (Vispe, 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 δ/ϵ, 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 δ/ϵ, 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 γ-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.