Vispé Stephane, Ho Erick L Y, Yung Tetsu M C, Satoh Masahiko 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.
J Biol Chem. 2003 Sep 12;278(37):35279-85. doi: 10.1074/jbc.M303448200. Epub 2003 Jun 27.
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.
双链DNA断裂是电离辐射诱导产生的最具致死性的DNA损伤类型。此前，我们报道过在无细胞双链DNA断裂形成实验中，双链DNA断裂可由位于相反DNA链上相隔18或30个碱基对的两处DNA损伤经酶促产生（维斯佩，S.，和佐藤，M. S.（2000年）《生物化学杂志》275卷，27386 - 27392页）。在我们所开展的实验中，这两处DNA损伤被参与碱基切除修复的酶转化为单链缺口。我们发现这些单链缺口被转化为双链DNA断裂；然而，这并非是由于DNA的自发变性。因此，我们提出了一个模型，即DNA聚合酶δ/ε通过在单链缺口处产生修复补丁而发生碰撞，从而导致双链DNA断裂的形成。我们对该模型进行了测试，并研究了是否有其他酶/因子参与双链DNA断裂的形成。在此我们报告，在该实验中，参与碱基切除修复的酶——瓣状核酸内切酶-1（FEN-1），而非DNA聚合酶δ/ε，负责双链DNA断裂的形成。此外，通过将瓣状核酸内切酶-1表达构建体转染到细胞中，从而改变它们的瓣状核酸内切酶-1含量，我们发现在对这些细胞进行γ射线照射后，双链DNA断裂的数量增加。这些结果表明瓣状核酸内切酶-1作为双链DNA断裂形成因子发挥作用。由于FEN-1是一种在DNA修复和DNA复制中发挥作用的必需酶，双链断裂可能作为FEN-1活性的副产物在细胞中产生。
