title: Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.
abstract: Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
authors.forename: Agnes
authors.initials: A
authors.lastname: Schipler
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Mladenova
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
authors.forename: Aashish
authors.initials: A
authors.lastname: Soni
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Nikolov
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
authors.forename: Janapriya
authors.initials: J
authors.lastname: Saha
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany.
authors.forename: Emil
authors.initials: E
authors.lastname: Mladenov
authors.affiliation: Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, 45122 Essen, Germany georg.iliakis@uk-essen.de.
authors.forename: George
authors.initials: G
authors.lastname: Iliakis
mesh_terms: D000818:Animals; D016466:CHO Cells; D016923:Cell Death; D033481:Chromosomes, Mammalian / Q000378:metabolism*; D000072837:Chromothripsis; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016678:Genome; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008677:Metaphase; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction; D014178:Translocation, Genetic; D000071857:Tumor Suppressor p53-Binding Protein 1 / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; D049452:Green Fluorescent Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific