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Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.

ABSTRACT: The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.

SUBMITTER: Chappell C

PROVIDER: S-EPMC126026 | biostudies-literature | 2002 Jun

REPOSITORIES: biostudies-literature

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Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.

Chappell Claire C Hanakahi Les A LA Karimi-Busheri Feridoun F Weinfeld Michael M West Stephen C SC

The EMBO journal 20020601 11

The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently ca ...[more]

PMID: 12032095

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Project description:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Keywords: strain_or_line_design; compound_treatment_design

Project description:Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) enhances tumor cell sensitivity to ionizing radiation. It is not clear how this effect is mediated, nor whether this approach can be applied effectively in the clinic. We previously showed that IGF-1R depletion delays repair of radiation-induced DNA double-strand breaks (DSBs), unlikely to be explained entirely by reduction in homologous recombination (HR) repair. The current study tested the hypothesis that IGF-1R inhibition induces a repair defect that involves non-homologous end joining (NHEJ). IGF-1R inhibitor AZ12253801 blocked cell survival and radiosensitized IGF-1R-overexpressing murine fibroblasts but not isogenic IGF-1R-null cells, supporting specificity for IGF-1R. IGF-1R inhibition enhanced radiosensitivity in DU145, PC3 and 22Rv1 prostate cancer cells, comparable to effects of Ataxia Telangiectasia Mutated inhibition. AZ12253801-treated DU145 cells showed delayed resolution of γH2AX foci, apparent within 1 h of irradiation and persisting for 24 h. In contrast, IGF-1R inhibition did not influence radiosensitivity or γH2AX focus resolution in LNCaP-LN3 cells, suggesting that radiosensitization tracks with the ability of IGF-1R to influence DSB repair. To differentiate effects on repair from growth and cell-survival responses, we tested AZ12253801 in DU145 cells at sub-SF50 concentrations that had no early (⩽48 h) effects on cell cycle distribution or apoptosis induction. Irradiated cultures contained abnormal mitoses, and after 5 days IGF-1R-inhibited cells showed enhanced radiation-induced polyploidy and nuclear fragmentation, consistent with the consequences of entry into mitosis with incompletely repaired DNA. AZ12253801 radiosensitized DNA-dependent protein kinase (DNA-PK)-proficient but not DNA-PK-deficient glioblastoma cells, and did not radiosensitize DNA-PK-inhibited DU145 cells, suggesting that in the context of DSB repair, IGF-1R functions in the same pathway as DNA-PK. Finally, IGF-1R inhibition attenuated repair by both NHEJ and HR in HEK293 reporter assays. These data indicate that IGF-1R influences DSB repair by both major DSB repair pathways, findings that may inform clinical application of this approach.

Dataset Information

Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.

Publications

Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.

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