SUMMARY The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair—and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

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.

Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.

In the above article, we inadvertently provided the wrong catalog numbers for two monoclonal antibodies generated by Sigma Aldrich against the proteins RNF20 and RNF40. The correct catalog numbers are XYZ for the RNF20 antibody and ABC for the RNF40 antibody. The Supplemental Information file has been updated online and now includes these correct catalog numbers. We regret any inconvenience this may have caused.

The mechanisms by which inhibition of the epidermal growth factor receptor (EGFR) sensitizes non-small cell lung cancer (NSCLC) cells to ionizing radiation remain poorly understood. We set out to characterize the radiosensitizing effects of the tyrosine kinase inhibitor erlotinib and the monoclonal antibody cetuximab in NSCLC cells that contain wild-type p53. Unexpectedly, EGFR inhibition led to pronounced cellular senescence but not apoptosis of irradiated cells both in-vitro and in-vivo. Senescence was completely dependent on wild-type p53 and associated with a reduction in cell number as well as impaired clonogenic radiation survival. Study of ten additional NSCLC cell lines revealed that senescence is a prominent mechanism of radiosensitization in 45% of cell lines and occurs not only in cells with wild-type p53 but also in cells with mutant p53 where it is associated with an induction of p16. Interestingly, senescence and radiosensitization were linked to an increase in residual radiation-induced DNA double-strand breaks irrespective of p53/p16 status. This effect of EGFR inhibition was at least partially mediated by disruption of the MEK-ERK pathway. Thus, our data indicate a common mechanism of radiosensitization by erlotinib or cetuximab across diverse genetic backgrounds. Our findings also suggest that assays that are able to capture the initial proliferative delay that is associated with senescence should be useful for screening large cell line panels to identify genomic biomarkers of EGFR inhibitor-mediated radiosensitization.

DNA double-strand breaks constitute the most dangerous type of DNA damage induced by ionising radiation (IR). Accordingly, the resistance of cells to IR is modulated by three intimately related cellular processes: DNA repair, recombination, and replication. Significant discoveries in this field of research have been made over the last few years. A picture seems to be emerging in which perturbations of recombination in cancer cells are a more widespread cause of genomic instability than previously appreciated. Conversely, such cells may also be more sensitive to certain chemotherapeutic drugs and to IR. Thus, the alterations in recombination that promote carcinogenesis by causing genomic instability may also be the weakness of the tumours that arise in this setting, a concept which could hold great promise for the advancement of cancer treatment in the not too distant future. British Journal of Cancer (2004) (Steel, 1996). It is established that the creation of a DNA double-strand break (DSB) represents the principal lesion that, if not adequately repaired, can lead to cell death via the generation of lethal chromosomal aberrations or the direct induction of apoptosis. Alternatively, an inaccurately repaired or unrepaired DSB may result in mutations or genomic rearrangements in a surviving cell, which in turn can lead to genomic instability and subsequently result in malignant cell transformation. Complex damage response pathways have evolved, and are evolutionary conserved, to protect the cell from the potentially deleterious effects of a DSB. Two principal recombinational repair pathways have been recognised, homologous recombination (HR) and nonhomologous end-joining (NHEJ), that employ entirely separate protein complexes. Briefly, DSB repair by HR requires an undamaged template molecule that contains a homologous DNA sequence, typically on the sister chromatid in the S and G2 phases of the cell cycle. In contrast, nonhomologous re-joining of two double-stranded DNA ends, which may occur in all cell-cycle phases, does not require an undamaged partner and does not rely on extensive homologies between the recombining ends. The study of these pathways has proved to be a rapidly evolving field of research over the past few years. Considerable interest has been generated by the realisation that defective HR and, in some cases, NHEJ can be causally linked to impaired DNA replication, genomic instability, human chromosomal instability syndromes, cancer development, or cellular hypersensitivity to DNA-damaging agents.Here, we discuss some of the recent advancements in basic research on DNA repair, recombination, and replication, which could hold great promise for the advancement of cancer treatment in the not too distant future. To this end, the prevailing theme is that the genetic alterations in recombination that lead to genomic instability and malignant transformation may also determine how tumour cells respond to IR and certain chemotherapeutic drugs (Venkitaraman, 2003). We are only able to consider a small numbe...

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.

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