title: Defective chromatin recruitment and retention of NHEJ core components in human tumor cells expressing a Cyclin E fragment.
abstract: Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.
authors.affiliation: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA, School of Biomedical Sciences, Kent State University, Kent, OH 44234, USA, Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA and Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Payel
authors.initials: P
authors.lastname: Chatterjee
authors.forename: Dragos
authors.initials: D
authors.lastname: Plesca
authors.forename: Suparna
authors.initials: S
authors.lastname: Mazumder
authors.forename: Jean
authors.initials: J
authors.lastname: Boutros
authors.forename: Steven M
authors.initials: SM
authors.lastname: Yannone
authors.forename: Alexandru
authors.initials: A
authors.lastname: Almasan
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000528:radiation effects; D002843:Chromatin / Q000378:metabolism*; D019927:Cyclin E / Q000378:metabolism*; D004249:DNA Damage; D059766:DNA End-Joining Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011839:Radiation, Ionizing; D015534:Trans-Activators / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002843:Chromatin; D019927:Cyclin E; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase
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