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 ...

In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors.

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Genome‐wide studies in tumor cells have indicated that chromatin‐modifying proteins are commonly mutated in human cancers. The lysine‐specific methyltransferase 2C ( KMT 2C/ MLL 3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT 2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT 2C activity are deficient in homologous recombination‐mediated double‐strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP 1/2 for DNA repair, and treatment with the PARP 1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT 2C expression are attractive targets for therapies with PARP 1/2 inhibitors.

Checkpoint abrogation in G 2 compromises repair of DNA double-strand breaks (DSB) and confers enhanced G 2 chromosomal radiosensitivity in ataxia telangiectasia (AT) cells. To directly test this hypothesis, we combined calyculin A-induced premature chromosome condensation with conventional cytogenetics to evaluate chromosome damage before and after the G 2 checkpoint in irradiated primary AT and normal human lymphocytes and their lymphoblastoid derivatives. Direct analysis of radiation damage in G 2 by premature chromosome condensation reveals practically indistinguishable levels of chromosomal breaks in AT and normal cells. Yet a 4-fold increase in metaphase chromosome damage is observed in AT cells as compared with normal cells which, in contrast to AT cells, exhibit a strong G 2 arrest manifest as an abrupt reduction in the mitotic index. Thus, an active checkpoint facilitates repair of chromosomal breaks in normal cells. Treatment with caffeine that abrogates the G 2 checkpoint without significantly affecting DSB rejoining increases metaphase chromosome damage of normal cells to the AT level but leaves unchanged interphase chromosome damage in G 2 . Caffeine has no effect on any of these end points in AT cells. These observations represent the first direct evidence that the G 2 checkpoint facilitates repair of chromosome damage, presumably by supporting repair of DNA DSBs. Failure to arrest will lead to chromatin condensation and conversion of unrepaired DNA DSBs to chromosomal breaks during G 2 -to-M phase transition. (Cancer Res 2005; 65(24): 11292-6)