Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining.

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)/Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.

Moscariello M ，Wieloch R ，Kurosawa A ，Li F ，Adachi N ，Mladenov E ，Iliakis G ... -  《-》

Marked contribution of alternative end-joining to chromosome-translocation-formation by stochastically induced DNA double-strand-breaks in G2-phase human cells.

Ionizing radiation (IR) induces double strand breaks (DSBs) in cellular DNA, which if not repaired correctly can cause chromosome translocations leading to cell death or cancer. Incorrect joining of DNA ends generating chromosome translocations can be catalyzed either by the dominant DNA-PKcs-dependent, classical non-homologous end-joining (c-NHEJ), or by an alternative end-joining (alt-EJ) process, functioning as backup to abrogated c-NHEJ, or homologous recombination repair. Alt-EJ operates with slower kinetics as compared to c-NHEJ and generates larger alterations at the junctions; it is also considered crucial to chromosome translocation-formation. A recent report posits that this view only holds for rodent cells and that in human cells c-NHEJ is the main mechanism of chromosome translocation formation. Since this report uses designer nucleases that induce DSBs with unique characteristics in specific genomic locations and PCR to detect translocations, we revisit the issue using stochastically distributed DSBs induced in the human genome by IR during the G2-phase of the cell cycle. For visualization and analysis of chromosome translocations, which manifest as chromatid translocations in cells irradiated in G2, we employ classical cytogenetics. In wild-type cells, we observe a significant contribution of alt-EJ to translocation formation, as demonstrated by a yield-reduction after treatment with inhibitors of Parp, or of DNA ligases 1 and 3 (Lig1, Lig3). Notably, a nearly fourfold increase in translocation formation is seen in c-NHEJ mutants with defects in DNA ligase 4 (Lig4) that remain largely sensitive to inhibitors of Parp, and of Lig1/Lig3. We conclude that similar to rodent cells, chromosome translocation formation from randomly induced DSBs in human cells largely relies on alt-EJ. We discuss DSB localization in the genome, characteristics of the DSB and the cell cycle as potential causes of the divergent results generated with IR and designer nucleases.

Soni A ，Siemann M ，Pantelias GE ，Iliakis G ... -  《-》

Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.

DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches.

Iliakis G ，Murmann T ，Soni A 《-》

Mechanisms of DNA double strand break repair and chromosome aberration formation.

It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10-30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2-10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for exchanges through the joining of incorrect ends and cause the formation of chromosome aberrations in wild type and D-NHEJ mutant cells.

Iliakis G ，Wang H ，Perrault AR ，Boecker W ，Rosidi B ，Windhofer F ，Wu W ，Guan J ，Terzoudi G ，Pantelias G ... -  《-》

Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

Murmann-Konda T ，Soni A ，Stuschke M ，Iliakis G ... -  《-》