title: Marked contribution of alternative end-joining to chromosome-translocation-formation by stochastically induced DNA double-strand-breaks in G2-phase human cells.
abstract: Ionizing radiation (IR) induces double strand breaks (DSBs) in cellular DNA, which if not repaired correctly can cause chromosome translocations leading to cell death or cancer. Incorrect joining of DNA ends generating chromosome translocations can be catalyzed either by the dominant DNA-PKcs-dependent, classical non-homologous end-joining (c-NHEJ), or by an alternative end-joining (alt-EJ) process, functioning as backup to abrogated c-NHEJ, or homologous recombination repair. Alt-EJ operates with slower kinetics as compared to c-NHEJ and generates larger alterations at the junctions; it is also considered crucial to chromosome translocation-formation. A recent report posits that this view only holds for rodent cells and that in human cells c-NHEJ is the main mechanism of chromosome translocation formation. Since this report uses designer nucleases that induce DSBs with unique characteristics in specific genomic locations and PCR to detect translocations, we revisit the issue using stochastically distributed DSBs induced in the human genome by IR during the G2-phase of the cell cycle. For visualization and analysis of chromosome translocations, which manifest as chromatid translocations in cells irradiated in G2, we employ classical cytogenetics. In wild-type cells, we observe a significant contribution of alt-EJ to translocation formation, as demonstrated by a yield-reduction after treatment with inhibitors of Parp, or of DNA ligases 1 and 3 (Lig1, Lig3). Notably, a nearly fourfold increase in translocation formation is seen in c-NHEJ mutants with defects in DNA ligase 4 (Lig4) that remain largely sensitive to inhibitors of Parp, and of Lig1/Lig3. We conclude that similar to rodent cells, chromosome translocation formation from randomly induced DSBs in human cells largely relies on alt-EJ. We discuss DSB localization in the genome, characteristics of the DSB and the cell cycle as potential causes of the divergent results generated with IR and designer nucleases.
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.
authors.forename: Aashish
authors.initials: A
authors.lastname: Soni
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Siemann
authors.affiliation: Institute of Nuclear Technology and Radiation Protection, National Centre for Scientific Research "Demokritos,"Aghia Paraskevi Attikis, Athens, Greece.
authors.forename: Gabriel E
authors.initials: GE
authors.lastname: Pantelias
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany. Electronic address: Georg.Iliakis@uk-essen.de.
authors.forename: George
authors.initials: G
authors.lastname: Iliakis
mesh_terms: D002460:Cell Line; D002877:Chromosomes, Human / Q000528:radiation effects*; D020732:Cytogenetic Analysis; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000187:drug effects; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016195:G2 Phase / Q000187:drug effects / Q000528:radiation effects*; D045325:HCT116 Cells; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D012545:Schiff Bases / Q000494:pharmacology; D014178:Translocation, Genetic / Q000187:drug effects
chemical_list: C578537:5,6-bis(benzylideneamino)-2-mercaptopyrimidin-4-ol; C000607892:LIG1 protein, human; C000607893:LIG4 protein, human; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011743:Pyrimidines; D012545:Schiff Bases; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011088:DNA Ligases; D000072481:DNA Ligase ATP
keywords: Alternative end joining (alt-EJ); Chromatid translocations; Chromosomal translocations; Classical non homologous end joining (c-NHEJ); Ionizing radiation (IR)