Non-homologous End Joining Repair Proteins Research Articles

Nonhomologous end joining repair pathway molecules as predictive biomarkers for patients with oral squamous cell carcinoma

Nonhomologous end-joining (NHEJ) is critical for the repair of either pathologic double-strand breaks (DSBs) and/or for the repair of physiologic DSBs created during radiotherapy to kill the tumor cell. Therefore, patients with higher expression of NHEJ repair proteins might develop resistance to ionizing radiation, allowing the disease to recur. As cancer of the oral cavity is a serious health problem globally, the present study aimed to examine the expression of Ku70/80, X-ray repair cross-complementing protein 4 (XRCC4) and DNA ligase IV-core molecules of the NHEJ pathway in patients with oral cancer. Protein expression of Ku70/80, XRCC4, and DNA ligase IV were studied by Immunohistochemistry and mRNA expression of Ku70 and Ku80 were studied using reverse transcription polymerase chain reaction. Data were analyzed statistically using SPSS. A univariate survival analysis revealed an association of Ku70 mRNA with shorter overall survival (OS). While protein expression of XRCC4 showed an association with reduced relapse-free survival and shorter OS. Multivariate survival analysis demonstrated that XRCC4 and DNA ligase IV are independent prognosticators for predicting adverse disease outcomes. Strong expression of repair proteins - XRCC4 and DNA ligase IV is associated with unfavorable disease outcome in patients with oral squamous cell carcinoma.

EIF3a Regulation of NHEJ Repair Protein Synthesis and Cellular Response to Ionizing Radiation.

Translation initiation in protein synthesis regulated by eukaryotic initiation factors (eIFs) is a crucial step in controlling gene expression. eIF3a has been shown to regulate protein synthesis and cellular response to treatments by anticancer agents including cisplatin by regulating nucleotide excision repair. In this study, we tested the hypothesis that eIF3a regulates the synthesis of proteins important for the repair of double-strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a upregulation sensitized cellular response to IR while its downregulation caused resistance to IR. eIF3a increases IR-induced DNA damages and decreases non-homologous end joining (NHEJ) activity by suppressing the synthesis of NHEJ repair proteins. Furthermore, analysis of existing patient database shows that eIF3a expression associates with better overall survival of breast, gastric, lung, and ovarian cancer patients. These findings together suggest that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating the synthesis of DNA repair proteins and, thus, eIIF3a likely contributes to the outcome of cancer patients treated with DNA-damaging strategies including IR.

The NAE inhibitor pevonedistat interacts with the HDAC inhibitor belinostat to target AML cells by disrupting the DDR

AbstractTwo classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3–internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor–κB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencing–defined poor-prognostic cancer hotspot mutations, and CD34+/CD38−/CD123+ populations, but not normal CD34+ progenitors. Finally, combined treatment markedly reduced tumor burden and significantly prolonged animal survival (P < .0001) in AML xenograft models with negligible toxicity, accompanied by pharmacodynamic effects observed in vitro. Collectively, these findings argue that MLN4924 and belinostat interact synergistically by reciprocally disabling the DDR in AML/MDS cells. This strategy warrants further consideration in AML/MDS, particularly in disease with unfavorable genetic aberrations.

Abstract 3029: Role of eIF3a expression in cellular sensitivity to radiation treatment

Abstract Translation initiation involving eukaryotic translation initiation factors (eIFs) is a crucial mechanism in controlling gene expression. eIF3a has been shown previously to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of DNA repair proteins, which in turn regulates cellular response to ionizing radiation (IR) treatments. We found that eIF3a overexpression sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) repair activity by suppressing expression level of NHEJ repair proteins such as DNAPKcs. Consistently, eIF3a knockdown decreases IR-induced DNA damage and increases Non-Homologous End Joining (NHEJ) repair activity by enhancing expression level of NHEJ repair proteins. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies. Note: This abstract was not presented at the meeting. Citation Format: Rima A. Tumia. Role of eIF3a expression in cellular sensitivity to radiation treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2015-3029

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

Abstract LB-133: Orchestrated recruitment of histone methyltransferases to DNA double strand breaks facilitates 53BP1 binding and proficient repair

Abstract While selective binding of the tandem tudor domains of p53 binding protein 1 (53BP1) to dimethylated histone H4 lysine 20 (H4K20me2) at DNA double strand breaks (DSBs) is well-documented to be an essential event for proficient DSB repair, the enzyme(s) responsible for H4K20 methylation at DSBs remained unknown. Contrary to previous reports implicating the MMSET/NSD2 methyltransferase in this pathway, we demonstrate that MMSET is dispensable for H4K20 methylation, 53BP1 accumulation and DSB repair. Alternatively, we discovered that the PR-Set7 H4K20 monomethyltransferase (H4K20me1) is an essential component of nonhomologous end joining (NHEJ) which functions early and directly in DSB repair. PR-Set7 was found to mobilize immediately to DSBs and PR-Set7 enzymatic activity was necessary for H4K20 methylation, 53BP1 accumulation and proficient DSB repair. Identification of PR-Set7-associated proteins revealed interactions between PR-Set7 and several NHEJ repair proteins. Consistent with these findings, the rapid recruitment of PR-Set7 to induced DSBs was dependent on direct binding to the NHEJ Ku70/Ku86 heterodimer. PR-Set7-mediated H4K20me1 was found to be necessary, but not sufficient, for H4K20me2 and 53BP1 recruitment at induced DSBs and an undamaged ectopic locus. In addition, we also discovered that the Suv4-20 H4K20 dimethyltransferases mobilize to DSBs and were required for H4K20me2 and 53BP1 accumulation. Importantly, Suv4-20 was not sufficient for H4K20me2 and 53BP1 localization at induced DSBs or an undamaged ectopic locus in cells lacking a catalytically competent PR-Set7. Therefore, these results illuminate an orchestrated molecular pathway whereby PR-Set7-mediated H4K20me1 “primes” the DSB to expedite subsequent catalysis by Suv4-20 for the generation of H4K20me2 at the lesion necessary for 53BP1 binding. The collective findings identify PR-Set7 as an essential early component of NHEJ and reveal a novel temporal PR-Set7-dependent pathway that facilitates H4K20me2 and 53BP1 accumulation at DSBs necessary for proficient DSB repair. Furthermore, these results and preliminary studies indicate that small molecule inhibitors targeting PR-Set7 and/or Suv4-20 will likely be clinically effective radio- and chemo-sensitizing agents in cancer treatment. Citation Format: Creighton T. Tuzon, Tanya M. Spektor, Lauren M. Congdon, Xiaodong Kong, Peggie Cheung, Alex J. Kuo, Kyoko Yokomori, Or Gozani, Judd C. Rice. Orchestrated recruitment of histone methyltransferases to DNA double strand breaks facilitates 53BP1 binding and proficient repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-133. doi:10.1158/1538-7445.AM2014-LB-133

Oxidative DNA Damage Response in Helicobacter pylori-Infected Mongolian Gerbils.

Helicobacter pylori (H. pylori) induced DNA damage which may be related to gastric cancer development. The DNA damage response coordinates DNA repair, cell-cycle transition, and apoptosis through activation of DNA damage response molecules. The damaged DNA is repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). In the present study, we investigated the changes of HR DNA repair proteins (ataxia-telangiectasia-mutated; ATM, ATM and Rad3-related; ATR), NHEJ repair proteins (Ku70/80), cell cycle regulators (Chk1, Chk2), and apoptosis marker (p53/p-p53) were determined in H. pylori-infected Mongolian gerbils. In addition, the effect of an antioxidant N-acetylcysteine (NAC) on H. pylori-induced DNA damage response was determined to assess the involvement of oxidative stress on DNA damage of the animals infected with H. pylori. One week after intragastric inoculation with H. pylori, Mongolian gerbils were fed with basal diet with or without 3% NAC for 6 weeks. After 6 week, the expression levels of DNA repair proteins (Ku70/80, ATM, ATR), cell cycle regulators (Chk1, Chk2) and apoptosis marker (p-p53/p53) were increased in gastric mucosa of Mongolian gerbils, which was suppressed by NAC treatment. In conclusion, oxidative stress mediates H. pylori-induced DNA damage response including NHEJ and HR repairing processes, cell cycle arrest and apoptosis in gastric mucosa of Mongolian gerbils.

Breadth by depth: Expanding our understanding of the repair of transposon-induced DNA double strand breaks via deep-sequencing

The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed.

Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate

Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4

DNA double-strand break (DSB) repair by nonhomologous end joining (NHEJ) requires the assembly of several proteins on DNA ends. Although biochemical studies have elucidated several aspects of the NHEJ reaction mechanism, much less is known about NHEJ in living cells, mainly because of the inability to visualize NHEJ repair proteins at DNA damage. Here we provide evidence that a pulsed near IR laser can produce DSBs without any visible alterations in the nucleus, and we show that NHEJ proteins accumulate in the irradiated areas. The levels of DSBs and Ku accumulation diminished in time, showing that this approach allows us to study DNA repair kinetics in vivo. Remarkably, the Ku heterodimers on DNA ends were in dynamic equilibrium with Ku70/80 in solution, showing that NHEJ complex assembly is reversible. Accumulation of XRCC4/ligase IV on DSBs depended on the presence of Ku70/80, but not DNA-PK(CS). We detected a direct interaction between Ku70 and XRCC4 that could explain these requirements. Our results suggest that this assembly constitutes the core of the NHEJ reaction and that XRCC4 may serve as a flexible tether between Ku70/80 and ligase IV.