Hierarchy of nonhomologous end-joining, single-strand annealing and gene conversion at site-directed DNA double-strand breaks

In mammalian cells, DNA double-strand breaks (DSBs) are repaired by three pathways, nonhomologous end-joining (NHEJ), gene conversion (GC) and single-strand annealing (SSA). These pathways are distinct with regard to repair efficiency and mutagenic potential and must be tightly controlled to preserve viability and genomic stability. Here, we employed chromosomal reporter constructs to characterize the hierarchy of NHEJ, GC and SSA at a single I-SceI-induced DSB in Chinese hamster ovary cells. We discovered that the use of GC and SSA was increased by 6- to 8-fold upon loss of Ku80 function, suggesting that NHEJ is dominant over the other two pathways. However, NHEJ efficiency was not altered if GC was impaired by Rad51 knockdown. Interestingly, when SSA was made available as an alternative mode for DSB repair, loss of Rad51 function led to an increase in SSA activity at the expense of NHEJ, implying that Rad51 may indirectly promote NHEJ by limiting SSA. We conclude that a repair hierarchy exists to limit the access of the most mutagenic mechanism, SSA, to the break site. Furthermore, the cellular choice of repair pathways is reversible and can be influenced at the level of effector proteins such as Ku80 or Rad51

Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks

Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4−/− compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs

PARP inhibition prevents escape from a telomere-driven crisis and inhibits cell immortalisation

Telomeric crisis is the final replicative barrier to cell immortalisation; it is characterised by genome instability and cell death and is triggered when telomeres become critically short and are subjected to fusion. Pre-cancerous lesions, or early stage cancers, often show signs of a telomere crisis, suggesting that escape from telomere crisis is a prerequisite for disease progression. Telomeric crisis therefore represents an attractive, and as yet unexplored, opportunity for therapeutic intervention. Here, we show that two clinically approved PARP inhibitors, selectively eliminate human cells undergoing a telomere-driven crisis. Clonal populations of a colorectal cancer cell line (HCT116), or the plasma cell leukaemia cell line (JJN-3), expressing a dominant-negative telomerase, entered a telomere-driven crisis at defined population doubling points and telomere lengths. The addition of the PARP inhibitors, olaparib or rucaparib prevented these cells from escaping crisis. PARP inhibition did not alter cellular proliferation prior to crisis, rates of telomere erosion or the telomere length at which crisis was initiated, but affected repair of eroded telomeres, resulting in an increased in intra-chromosomal telomere fusion. This was accompanied by enhanced DNA damage checkpoint activation and elevated levels of apoptosis. We propose that PARP inhibitors impair the repair of dysfunctional telomeres and/or induce replicative stress at telomeres to inhibit escape from a telomere crisis. This is the first demonstration that a drug can selectively kill cells experiencing telomeric crisis. We propose that this type of drug, which we term ‘crisolytic’, has the potential to eliminate pre-cancerous lesions and tumours exhibiting short dysfunctional telomeres

Genome-wide gene expression profiling suggests distinct radiation susceptibilities in sporadic and post-Chernobyl papillary thyroid cancers

Papillary thyroid cancers (PTCs) incidence dramatically increased in the vicinity of Chernobyl. The cancer-initiating role of radiation elsewhere is debated. Therefore, we searched for a signature distinguishing radio-induced from sporadic cancers. Using microarrays, we compared the expression profiles of PTCs from the Chernobyl Tissue Bank (CTB, n=12) and from French patients with no history of exposure to ionising radiations (n=14). We also compared the transcriptional responses of human lymphocytes to the presumed aetiological agents initiating these tumours, γ-radiation and H2O2. On a global scale, the transcriptomes of CTB and French tumours are indistinguishable, and the transcriptional responses to γ-radiation and H2O2 are similar. On a finer scale, a 118 genes signature discriminated the γ-radiation and H2O2 responses. This signature could be used to classify the tumours as CTB or French with an error of 15–27%. Similar results were obtained with an independent signature of 13 genes involved in homologous recombination. Although sporadic and radio-induced PTCs represent the same disease, they are distinguishable with molecular signatures reflecting specific responses to γ-radiation and H2O2. These signatures in PTCs could reflect the susceptibility profiles of the patients, suggesting the feasibility of a radiation susceptibility test

Induction and processing of the radiation-induced gamma-H2AX signal and Its link to the underlying pattern of DSB: A combined experimental and modelling study

We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy) and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process, experiments performed at high doses are of relevance in the context of radiation therapy, where hypo-fractionated schemes become increasingly popular