Backup Non-homologous End Joining Research Articles

Variants of Mre11 induce genomic instability

DNA double‐strand break (DSB) is the most lethal type of DNA damage and if not properly repaired could destabilize the genome. Cells repair DSBs by three repair pathways‐homology‐directed repair (HDR), non‐homologous end‐joining (NHEJ) and a recent back‐up NHEJ pathway has been reported and is referred to as alternate‐NHEJ (Alt‐NHEJ). Mre11 plays a role in all the three DSB repair pathways. Mre11 has single‐strand endonuclease activity and double‐strand‐specific 3′–5′ exonuclease activity which are essential for DNA end‐processing. Mre11 has been shown to be overexpressed in breast cancer and high Mre11 expression was found to be associated with a more malignant behavior in breast cancer. Mutations in Mre11 genes can have a significant impact on cancer prognosis and treatment. Here we show that cells expressing variants of Mre11 are sensitive to DNA damaging agent etoposide. We also found that the Mre11 variants influence response to various DNA damaging agents and induce genomic instability. Work is on‐going to characterize how the Mre11 variants alter DSB repair mechanisms. This will provide mechanistic insights into the role of the Mre11 variants inducing genomic instability and cancer and has potential to influence cancer therapeutics.Support or Funding InformationThis research was supported by Murdock College Research Program for Natural Sciences ‐ Life Sciences grant (#201812085) and Linfield College Faculty Student Research Collaborative Grant (FSRCG) to SR

MODELLING DSB REPAIR KINETICS FOR DNA DAMAGE INDUCED BY PROTON AND CARBON IONS.

Proton and carbon therapy are the main choices of particle therapy for cancer treatment. Particle dose distribution is superior to conventional photon therapy dose distribution due to Bragg peak. However, the basic biology of cellular damage and cell death is not well understood. The aim of this work is to present a mechanistic model of double strand break (DSB) repair that predicts the repair kinetics of damage induced by particles employed in cancer therapy. Monte Carlo Track Damage Simulation (MCDS) was employed to model DNA damage. The frequency of DSB and SSB was computed for proton and carbon ions. DSBs were subjected to repair model to calculate the repair kinetics. Two distinct DSB repair models dependent on the cell cycle were proposed. The DSB repair model contains non-homologous end joining (NHEJ), homologous recombination (HR) and back up non-homologous end joining (B-NHEJ) repair processes. The DSB complexity results in the switch in the repair pathway from NHEJ to a slower process that starts with DSB end resection. DSB end resection in early S and G1 phases of the cell cycle enhances the B-NHEJ repair pathway, while in late S and G2 phases of the cell cycle promotes HR repair pathway. The repair model was transformed to a set of nonlinear differential equations. The model calculates the overall repair kinetics and protein temporal repair activity at the site of damage. The damage and repair model provides a detailed mechanistic understanding of all processes that are involved in the damage induction and repair. The number of DSB and their complexity increase as the particle energy decreases due to the proximity of particle interactions in water. The repair kinetics show a biphasic behaviour that is due to the NHEJ fast repair of simple type DSB and HR slow repair of complex type DSB.

DNA polymerase beta participates in DNA End-joining.

DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA polymerases lambda (Pol λ) and mu (Pol μ) promote DNA end-joining. Here, we show that DNA polymerase beta (Pol β), another member of the X family of DNA polymerases, plays a role in aNHEJ. In the absence of DNA Pol β, fewer small deletions are observed. In addition, depletion of Pol β results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol β in functions in aNHEJ and provide mechanistic insight into its role in this process.

Abstract 2492: DNA polymerase beta participates in DNA end-joining

Abstract DNA double strand breaks (DSBs) are one of the most deleterious lesions. If left unrepaired, DSBs lead to genomic instability and carcinogenesis. The cells combat DSBs by two classical pathways that include homologous recombination (HR) which requires the sister chromatid for sequence homology, and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred non-canonical NHEJ which has been given many names, such as alternative NHEJ or microhomology-mediated end joining (MMEJ). The enzymatic mechanisms of non-canonical NHEJ are not well defined. NHEJ requires processing enzymes including nucleases and polymerases, although the roles of these enzymes in the pathway are poorly understood. Recent studies have implicated a role for DNA polymerase theta (Pol θ), an A-family polymerase is essential for non-canonical NHEJ pathway. Emerging evidence indicates X-family polymerases, mammalian DNA polymerases lambda (λ) and mu (μ) and yeast Pol 4 promote DNA end-joining. Our laboratory has recently provided evidence for a role for DNA polymerase beta (Pol β), another X-family polymerase, in V(D)J recombination, a process that requires end-joining. Here, using a recently developed fluorescence based assay that monitors non-canonical NHEJ and HR, we provide evidence that Pol β plays a role in the non-canonical NHEJ process. DNA sequencing at the break point junctions revealed Pol β-depleted cells have fewer small deletions than control cells, but significantly greater numbers of insertions and large deletions. We further demonstrate that Pol β-depleted cells have increased sensitivity to DNA damaging agents that induce double-strand breaks and that there is persistent accumulation of DSBs in these cells. In combination, our results suggest that Pol β is critical for double-strand break repair. Citation Format: Sreerupa Ray, Michelle DeVeaux, Gregory Breuer, Ranjit Bindra, Daniel Zelterman, Joann Sweasy. DNA polymerase beta participates in DNA end-joining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2017-2492

Sensitizing thermochemotherapy with a PARP1-inhibitor.

Cis-diamminedichloroplatinum(II) (cisplatin, cDDP) is an effective chemotherapeutic agent that induces DNA double strand breaks (DSBs), primarily in replicating cells. Generally, such DSBs can be repaired by the classical or backup non-homologous end joining (c-NHEJ/b-NHEJ) or homologous recombination (HR). Therefore, inhibiting these pathways in cancer cells should enhance the efficiency of cDDP treatments. Indeed, inhibition of HR by hyperthermia (HT) sensitizes cancer cells to cDDP and in the Netherlands this combination is a standard treatment option for recurrent cervical cancer after previous radiotherapy. Additionally, cDDP has been demonstrated to disrupt c-NHEJ, which likely further increases the treatment efficacy. However, if one of these pathways is blocked, DSB repair functions can be sustained by the Poly-(ADP-ribose)-polymerase1 (PARP1)-dependent b-NHEJ. Therefore, disabling b-NHEJ should, in principle, further inhibit the repair of cDDP-induced DNA lesions and enhance the toxicity of thermochemotherapy. To explore this hypothesis, we treated a panel of cancer cell lines with HT, cDDP and a PARP1-i and measured various end-point relevant in cancer treatment. Our results demonstrate that PARP1-i does not considerably increase the efficacy of HT combined with standard, commonly used cDDP concentrations. However, in the presence of a PARP1-i, ten-fold lower concentration of cDDP can be used to induce similar cytotoxic effects. PARP1 inhibition may thus permit a substantial lowering of cDDP concentrations without diminishing treatment efficacy, potentially reducing systemic side effects.

Cell-ECM interactions control DDR.

Fundamental to an improvement of cancer cure rates is a better understanding of the underlying molecular mechanisms driving resistance to standard treatments such as radio- and chemotherapy. A powerful and promising approach to sensitize cancer cells to therapy is represented by Paul Ehrlich's old and still up-to-date concept of the “magic bullet”. Reviewing the complexity of the “hallmarks of cancer” provides clarity about the needs to employ pathophysiological models for our examinations and consider multi-targeting concepts [1]. For identifying novel potential cancer targets, we need to unravel the molecular biology of how cancer cells develop, manifest and interact with their microenvironment. These actions are not only promoted by intrinsic genetic and epigenetic alterations but also through a tightly regulated interplay between cancer cells and stromal components documented as key determinants of various hallmarks, including resistance to cell death, sustainability of proliferative signals, and activation of invasion and metastasis. Among this large plethora of autocrine and paracrine interactions, integrin-mediated adhesion to the extracellular matrix (ECM) is one of the most influencing characteristics eliciting cancer cell therapy resistance. Integrins are transmembrane receptors consisting of one alpha and one beta subunit [2]. Overall, 18 α and 8 β subunits form 24 different integrin cell adhesion receptors. Their dual functionality, ECM binding for structure and for signaling, and their location at subcellular sites pinpoint them as highly interesting molecules. Structurally, a linkage of ECM/integrin/actin cytoskeleton/nuclear membrane/chromatin connects the outside with the inside of the cell [3]. Signaling-wise, outside-in and inside-out communication by integrins significantly contributes to the regulation of cell survival, proliferation, migration or apoptosis. However what turns integrins into potent cancer targets? First, integrins are overexpressed in different tumor entities relative to the corresponding healthy tissue. Secondly, integrins are critically involved in the prosurvival, promigratory and therapy resistance mechanisms, and third, integrins are surface receptors that are easily druggable and utilizable for imaging. Different preclinical studies convincingly show that inhibition of integrin receptors sensitizes tumor cells to chemo- and radiotherapy [4,5]. As is often the case, clinical trials were unable to recapitulate these findings due to insufficient integrin inhibition. Thus, a new strategy proposed by us and others is to inhibit all β1 integrin associated integrin receptors. As β1 integrins form by far the biggest group of integrin receptors, β1 integrin targeting enables the deactivation of a wide variety of integrins at once. In our case, we used the monoclonal inhibitory anti-β1 integrin antibody AIIB2 in head and neck squamous cell carcinomas (HNSCC). We previously showed that β1 integrins signal via the focal adhesions kinase (FAK)/Cortactin/c-Jun N-terminal kinase 1 (JNK1) signaling axis for increased survival after radiation [4]. Hence, we asked whether there is a connection between β1 integrin signaling and DNA repair. Despite one report on suppression of Bleomycin-induced DNA damage by integrin signaling [6], there was a lack of knowledge about integrin signaling and repair of radiogenic DNA double strand breaks (DSBs). Cells exhibit two pathways for the repair of DSBs: non-homologous end-joining (NHEJ) and homologous recombination. NHEJ is distinguished by classical NHEJ (C-NHEJ), which depends on DNA-PKcs, and backup NHEJ (B-NHEJ), which depends on PARP-1 [7]. In our study using 3D matrix-based cell culture and tumor xenograft models, we focused on the impact of β1 integrins on C- and B-NHEJ [8]. We found increased DSBs after β1 integrin inhibition and radiation in foci and comet assay analysis. Mechanistically, β1 integrins signal via the FAK/JNK1 axis to the repair of radiogenic DSBs. Interestingly, β1 integrin inhibition impaired C-NHEJ whereas B-NHEJ was not influenced. We found enhanced γH2AX- and pDNA-PKcs Thr2609-positive DSBs and decreased Ku70, Rad50 and Nbs1 expression but no PARP-1 modifications after AIIB2-mediated β1 integrin inhibition. Further experiments clearly indicated FAK and JNK1 to be part of a signaling cascade connecting β1 integrins to the C-NHEJ machinery. In terms of the concept of synthetic lethality, we exploited the independence of PARP from β1 integrins by inhibiting PARP with Olaparib. Intriguingly, this combination therapy with AIIB2 and Olaparib resulted in an additive decrease of clonogenic radiation survival, thereby suggesting a new approach to overcome radioresistance in HNSCC. Figure 1 β1 integrin-mediated signaling modulates DNA repair To our knowledge, this is the first study showing a mechanistic connection between β1 integrin targeting and impaired DNA repair, thus connecting the ECM/integrin interplay with radiation-induced DNA repair processes. In conclusion, we show that the β1 integrin-mediated adhesion to the ECM co-determines both the repair of radiogenic DSBs and the clonogenic radiation survival. Further characterization of β1 integrin-mediated prosurvival signaling including the interrelation between β1 integrin and homologous recombination is necessary for a safe, effective and reasonable translation into the clinic.

DNA Ligases I and III Cooperate in Alternative Non-Homologous End-Joining in Vertebrates

Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3.

Systems Modelling of NHEJ Reveals the Importance of Redox Regulation of Ku70/80 in the Dynamics of DNA Damage Foci

The presence of DNA double-stranded breaks in a mammalian cell typically activates the Non-Homologous End Joining (NHEJ) pathway to repair the damage and signal to downstream systems that govern cellular decisions such as apoptosis or senescence. The signalling system also stimulates effects such as the generation of reactive oxygen species (ROS) which in turn feed back into the damage response. Although the overall process of NHEJ is well documented, we know little of the dynamics and how the system operates as a whole. We have developed a computational model which includes DNA Protein Kinase (DNA-PK) dependent NHEJ (D-NHEJ) and back-up NHEJ mechanisms (B-NHEJ) and use it to explain the dynamic response to damage induced by different levels of gamma irradiation in human fibroblasts. Our work suggests that the observed shift from fast to slow repair of DNA damage foci at higher levels of damage cannot be explained solely by inherent stochasticity in the NHEJ system. Instead, our model highlights the importance of Ku oxidation which leads to increased Ku dissociation rates from DNA damage foci and shifts repair in favour of the less efficient B-NHEJ system.

Factors determining DNA double-strand break repair pathway choice in G2 phase.

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.

ATM-independent, high-fidelity nonhomologous end joining predominates in human embryonic stem cells

We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.

BCR/ABL downregulates DNA-PKCS-dependent and upregulates backup non-homologous end joining in leukemic cells

Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PK(CS); D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PK(CS) (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PK(CS), did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.

The type and yield of ionising radiation induced chromosomal aberrations depend on the efficiency of different DSB repair pathways in mammalian cells

In order to evaluate the relative role of two major DNA double strand break repair pathways, i.e., non-homologous end joining (NHEJ) and homologous recombination repair (HRR), CHO mutants deficient in these two pathways and the parental cells (AA8) were X-irradiated with various doses. The cells were harvested at different times after irradiation, representing G 2, S and G 1 phase at the time of irradiation, The mutant cell lines used were V33 (NHEJ deficient), Irs1SF, 51-D1 (HRR deficient). In addition to parental cell line (AA8), a revertant of V33, namely V33-155 was employed. Both types of mutant cells responded with increased frequencies of chromosomal aberrations at all recovery times in comparison to the parental and revertant cells. Mutant cells deficient in NHEJ were more sensitive in all cell stages in comparison to HRR deficient mutant cells, indicating NHEJ is the major repair pathway for DSB repair through out the cell cycle. Both chromosome and chromatid types of exchange aberrations were observed following G 1 irradiation (16 and 24 h recovery). Interestingly, configurations involving both chromosome (dicentrics) and chromatid exchanges were encountered in G 1 irradiated V33 cells. This may indicate that unrepaired DSBs accumulate in G 1 in these mutant cells and carried over to S phase, where they are repaired by HRR or other pathways such as B-NHEJ (back up NHEJ), which appear to be highly error prone. Both NHEJ and HRR, which share some of the same proteins in their pathways, are involved in the repair of DSBs leading to chromosomal aberrations, but with a major role of NHEJ in all stages of cell cycle.

Repair of radiation induced DNA double strand breaks by backup NHEJ is enhanced in G2

In higher eukaryotes DNA double strand breaks (DSBs) are repaired by homologous recombination (HRR) or non-homologous end joining (NHEJ). In addition to the DNA-PK dependent pathway of NHEJ (D-NHEJ), cells employ a backup pathway (B-NHEJ) utilizing Ligase III and PARP-1. The cell cycle dependence and coordination of these pathways is being actively investigated. We examine DSB repair in unperturbed G1 and G2 phase cells using mouse embryo fibroblast (MEF) mutants defective in D-NHEJ and/or HRR. WT and Rad54 −/− MEFs repair DSBs with similar efficiency in G1 and G2 phase. LIG4 −/−, DNA- PKcs −/−, and Ku70 −/− MEFs show more pronounced repair defects in G1 than in G2. LIG4 −/−/ Rad54 −/− MEFs repair DSBs as efficiently as LIG4 −/− MEFs suggesting that the increased repair efficiency in G2 relies on enhanced function of B-NHEJ rather than HRR. In vivo and in vitro plasmid end joining assays confirm an enhanced function of B-NHEJ in G2. The results show a new and potentially important cell cycle regulation of B-NHEJ and generate a framework to investigate the mechanistic basis of HRR contribution to DSB repair.