Non-homologous End Joining DNA Repair Research Articles

Negative feedback loop in the activation of non-homologous end joining DNA repair pathway in Helicobacter pylori infected patients with gastritis

This study aimed to investigate the activation of error-prone DNA repair pathway in response to Helicobacter pylori infection. Relative changes in the expression levels of genes involved in the non-homologous end-joining pathway (NHEJ) in H. pylori-infected (Cases) and non-infected patients (Controls) with chronic gastritis were measured. A significant increase in the relative expression level of TP53, and significant decrease in the relative transcription of lncRNA LINP1 and XRCC5 were detected in the case group. The transcription of Lig4 and XRCC6 was increased in the case group, which was not statistically significant. The Spearman’s Correlation Coefficient analysis showed a significant positive-correlation between the transcriptional levels of LINP1 and XRCC4/XRCC5/Lig4, and between XRCC5 and TP53/Lig4 both in the case and control groups. Moreover, a significant positive correlation between LinP1 and XRCC6 in the case, and a significant positive correlation between XRCC4 and Lig4, and a negative correlation between TP53 and LinP1/XRCC4/XRCC5 in the control group was detected. Although a relative difference was detected in transcriptional levels of the NHEJ gene mediators, downregulation of LinP1 in H. pylori-infected patients proposed the activation of a negative feedback loop, which may interfere with the NHEJ activity at the early stages of gastritis.

Investigation of the association between polymorphisms in DNA repair enzymes and STEMI

STEMI (ST-elevation myocardial infarction), a condition in which DNA damage likely contributes to its pathogenesis, is among the most severe manifestations of coronary artery disease. There are very few publications in this field in the literature. In our study, we examined the association between four polymorphisms in repair enzymes (LIG4 Thr9Ile, XRCC6 promoter C-57G, XPA -4A/G, OGG1 Ser326Cys) involved in three distinct DNA repair mechanisms [NHEJ (non-homologous end joining)], NER (nucleotide excision repair), and BER (base excision repair), and their impact on the risk of STEMI. This study involved 185 patients diagnosed with STEMI and included 155 healthy controls. The genotyping of SNPs was conducted through the PCR-RFLP (Polymerase chain reaction-restriction fragment length polymorphism) method for the following variants: XRCC6 (rs2267437), XPA (rs1800975), LIG4 (rs1805388), and OGG1 (rs1052133). No significant differences were observed in the genotype distributions of the XRCC6 and OGG1 variations between the control and patient groups. On the other hand, our findings indicate that individuals carrying the mutant G allele for XPA polymorphism and the mutant T allele for LIG4 polymorphism are susceptible to STEMI. Our findings demonstrate the significance of NHEJ and NER DNA repair processes in the pathogenesis of STEMI, as evidenced by the observed relationship between LIG4 and XPA polymorphisms.

Inhibition of Aberrantly Overexpressed Polo-like Kinase 4 Is a Potential Effective Treatment for DNA Damage Repair-Deficient Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (LMS) is an aggressive sarcoma and a subset of which exhibits DNA repair defects. Polo-like kinase 4 (PLK4) precisely modulates mitosis, and its inhibition causes chromosome missegregation and increased DNA damage. We hypothesize that PLK4 inhibition is an effective LMS treatment. Genomic profiling of clinical uterine LMS samples was performed, and homologous recombination (HR) deficiency scores were calculated. A PLK4 inhibitor (CFI-400945) with and without an ataxia telangiectasia mutated (ATM) inhibitor (AZD0156) was tested in vitro on gynecologic sarcoma cell lines SK-UT-1, SKN, and SK-LMS-1. Findings were validated in vivo using the SK-UT-1 xenograft model in the Balb/c nude mouse model. The effects of CFI-400945 were also evaluated in a BRCA2-knockout SK-UT-1 cell line. The mechanisms of DNA repair were analyzed using a DNA damage reporter assay. Uterine LMS had a high HR deficiency score, overexpressed PLK4 mRNA, and displayed mutations in genes responsible for DNA repair. CFI-400945 demonstrated effective antitumor activity in vitro and in vivo. The addition of AZD0156 resulted in drug synergism, largely due to a preference for nonhomologous end-joining DNA repair. Compared with wild-type cells, BRCA2 knockouts were more sensitive to PLK4 inhibition when both HR and nonhomologous end-joining repairs were impaired. Uterine LMS with DNA repair defects is sensitive to PLK4 inhibition because of the effects of chromosome missegregation and increased DNA damage. Loss-of-function BRCA2 alterations or pharmacologic inhibition of ATM enhanced the efficacy of the PLK4 inhibitor. Genomic profiling of an advanced-stage or recurrent uterine LMS may guide therapy.

Abstract 7128: Novel Ku-DNA binding inhibitors impact on the cellular and in vivo DNA damage response to radio- and radiomimetic-therapy

Abstract DNA-PK, the DNA-dependent protein kinase, is a validated target for cancer therapeutics that plays a central role in the non-homologous end joining (NHEJ) DNA repair pathway and the DNA damage response (DDR). NHEJ is a key mechanism of DNA double-strand breaks (DSB) repair, especially those induced by ionizing radiation (IR). Ku 70/80 heterodimer is required as initiator in this process, acting as an essential DNA-binding component of DNA-PK that senses DNA damage. Blocking the DNA-PK kinase activity in combination with DSB inducing agents has been widely used as a therapeutic strategy to drive clinical efficacy of radiation therapy for cancer treatment. We have reported a unique approach of DNA-PK inhibition by developing Ku-DNA binding inhibitors (Ku-DBi’s), small molecule inhibitors that target the interaction between Ku70/80 and DNA. Ku-DBi’s demonstrated a direct interaction with Ku70/80, a potent inhibition of Ku-DNA binding and DNA-PK catalytic activity, and in vitro and cellular NHEJ inhibitory activity. This results in the potentiation of non-small cell lung cancer (NSCLC) cellular sensitivity to DSB generating therapies, as a function of inhibiting DNA-PKcs autophosphorylation and dysregulation of signaling to the DDR. In this study, we have expanded our structure activity relationship analyses to focus on optimizing selectivity, cellular uptake and Ku inhibitory activity. The new series of Ku-DBi’s include oxindole derivatives of the X80 core structure that achieve these goals. These novel compounds display increased cellular uptake while retaining potent Ku inhibitory activity and enhanced cellular inhibition on DNA-DSB repair. Ku-DBi’s optimization has enabled in vivo analyses of Ku-DBi - ionizing radiation combination therapy in human xenograft models of non-small cell lung cancer. The impact of Ku-DBi treatment on therapeutic efficacy, toxicity, Ku/DNA-PK inhibition, and DDR signaling will be presented. These data represent a significant advance in the development of Ku-DNA binding inhibitors and their therapeutic intervention for cancer treatment. Citation Format: Pamela L. Mendoza-Munoz, Dineshsinha Chauhan, Navnath S. Gavande, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Novel Ku-DNA binding inhibitors impact on the cellular and in vivo DNA damage response to radio- and radiomimetic-therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7128.

Abstract 5825: Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer

Abstract Rare cell populations manage to evade the effects of cancer therapies, ultimately leading to drug resistance and subsequent cancer relapse. These resilient populations, often referred to as "persisters," exhibit distinct molecular characteristics when compared to the broader tumor population. In the current standard of clinical care for non-small cell lung cancer (NSCLC), patients typically undergo a series of standard treatments, such as surgery followed by chemotherapy/immunotherapy, and then targeted therapy, based on the disease stages. This continues until drug resistance emerges, with little attention given to the state of these persister cells in response to these treatments. In the case of drug resistance in KRAS-mutated NSCLC, ongoing clinical investigations are exploring the combined efficacy of KRAS G12Ci inhibitors with drugs that target mitogen-activated protein kinase (MAPK) feedback reactivation, immunotherapy, and chemotherapy. Despite the myriad of combination therapies, persister populations invariably emerge. The manner in which these persisters evolve to withstand therapies remains elusive. We employed a DNA barcode lineage tracing analysis coupled with mathematical modeling to decode persister evolution to therapies targeting different pathways, e.g., KRAS+ SHP2, KRAS+CDK4/6, and KRAS+MCL-1. Our model reveals that rapidly emerging persister populations are predetermined and do not exhibit clear genetic alterations. In some cells, common persisters exist in response to different therapies. Single-cell ATAC-RNA sequencing showed that these persisters are enriched in chromatin-binding of transcription factors that regulate classic non-homologous end-joining (cNHEJ) DNA repair (ZNF384), interferon response (IRF1), and stem-like characteristics (FOXP1), along with the activation of an IFN-γ transcriptional signature. These findings underscore the connection between specific DNA repair processes, chromosomal instability, and resistance to KRAS G12Ci. In other cells, however, distinct persister populations exist to different therapies, leading to the hypothesis that these diverse persister populations can be targeted orthogonally or sequentially to achieve tumor regression. In summary, our study unveils the heterogeneity in persister evolution trajectories in KRAS NSCLC. Our platform identifies targetable persister populations in NSCLC models and offers a proof-of-concept for non-standard strategies to combat drug resistance based on persister cell states. Citation Format: Chendi Li, Christopher J. Graser, Qian Qin, Anahita Nimbalkar, Usman Syed, Barbara Karakyriakou, Sarah Clark, Anne Y. Saiki, Paul E. Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron Hata. Non-genetic determinants driving the evolutional trajectories of drug-tolerant-persisters in KRAS Non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5825.

Concomitant inhibition of the thioredoxin system and nonhomologous DNA repair potently sensitizes Philadelphia-positive lymphoid leukemia to tyrosine kinase inhibitors.

Breakpoint cluster region-Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients, 50% of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B-ALL cells, particularly in combination with other drugs. Here, we present that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib-treated, PRDX1-deficient cells revealed that the nonhomologous end-joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B-ALL cells. Accordingly, PRDX1-deficient Ph+ B-ALL cells were susceptible to NHEJ inhibitors. Finally, we demonstrated the potent efficacy of a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors against Ph+ B-ALL cell lines and primary cells, which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B-ALL.

Non-homologous End-joining Genotype, mRNA Expression, and DNA Repair Capacity in Childhood Acute Lymphocytic Leukemia.

The capacity for non-homologous end-joining (NHEJ) repair plays a pivotal role in maintaining genome stability and in carcinogenesis. However, there is little literature on the involvement of NHEJ-related genes in childhood acute lymphocytic leukemia (ALL). Our study aimed to elucidate the impact of polymorphisms of X-ray repair cross-complementing group 4 (XRCC4) (rs6869366, rs2075685, rs2075686, rs28360071, rs3734091, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and DNA ligase IV (LIG4) rs1805388, on the odds of childhood ALL. Genotypes NHEJ-related genes of 266 cases and 266 controls were determined, and the genotype-phenotype correlation was investigated by examining mRNA transcript expression and the capacity for overall and precise NHEJ repair. The variant genotypes of XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 were significantly associated with increased odds of childhood ALL. Further analysis based on susceptibility genotypes showed no significant differences in mRNA transcript expression levels among childhood ALL cases with various putative high-risk genotypes, except XRCC6 rs5751129. Moreover, the overall NHEJ repair capacity was similar among carriers of different XRCC4, XRCC5, and XRCC6 genotypes. However, it is worth noting that individuals carrying the variant C allele at XRCC6 rs5751129 exhibited lower precise NHEJ repair capacity compared to those with the wild-type T allele. Our study identified significant associations between XRCC4 rs3734091, rs28360071, XRCC5 rs828907, and XRCC6 rs5751129 genotypes and childhood ALL. Notably, lower transcriptional expression and reduced precise NHEJ repair capacity were observed in patients carrying the C allele of XRCC6 rs5751129. Further investigations are required to gain deeper insights into childhood ALL development.

The BAP1 nuclear deubiquitinase is involved in the nonhomologous end-joining pathway of double-strand DNA repair through interaction with DNA-PK.

BRCA1-associated protein 1 (BAP1) has emerged as a major tumor suppressor gene in diverse cancer types, notably in malignant pleural mesothelioma (DPM), and has also been identified as a germline cancer predisposition gene for DPM and other select cancers. However, its role in the response to DNA damage has remained unclear. Here, we show that BAP1 inactivation is associated with increased DNA damage both in Met-5A human mesothelial cells and human DPM cell lines. Through proteomic analyses, we identified PRKDC as an interaction partner of BAP1 protein complexes in DPM cells and 293 T human embryonic kidney cells. PRKDC encodes the catalytic subunit of DNA protein kinase (DNA-PKcs) which functions in the nonhomologous end-joining (NHEJ) pathway of DNA repair. Double-stranded DNA damage resulted in prominent nuclear expression of BAP1 in DPM cells and phosphorylation of BAP1 at serine 395. A plasmid-based NHEJ assay confirmed a significant effect of BAP1 knockdown on cellular NHEJ activity. Combination treatment with X-ray irradiation and gemcitabine (as a radiosensitizer) strongly suppressed the growth of BAP1-deficient cells. Our results suggest reciprocal positive interactions between BAP1 and DNA-PKcs, based on phosphorylation of BAP1 by the latter and deubiquitination of DNA-PKcs by BAP1. Thus, functional interaction of BAP1 with DNA-PKcs supports a role for BAP1 in NHEJ DNA repair and may provide the basis for new therapeutic strategies and new insights into its role as a tumor suppressor.

CRISPR/Cas9-mediated homology donor repair base editing system to confer herbicide resistance in maize (Zea mays L.)

Weed infestation is a significant concern to crop yield loss, globally. The potent broad-spectrum glyphosate (N-phosphomethyl-glycine) has a widely utilized herbicide, acting on the shikimic acid pathway within chloroplast by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This crucial enzyme plays a vital role in aromatic amino acid synthesis. Repurposing of CRISPR/Cas9-mediated gene-editing was the inflection point for generating novel crop germplasm with diverse genetic variations in essential agronomic traits, achieved through the introduction of nucleotide substitutions at target sites within the native genes, and subsequent induction of indels through error-prone non-homologous end-joining DNA repair mechanisms. Here, we describe the development of efficient herbicide-resistant maize lines by using CRISPR/Cas9 mediated site-specific native ZmEPSPS gene fragment replacement via knock-out of conserved region followed by knock-in of desired homologous donor repair (HDR-GATIPS-mZmEPSPS) with triple amino acid substitution. The novel triple substitution conferred high herbicide tolerance in edited maize plants. Transgene-free progeny harbouring the triple amino acid substitutions revealed agronomic performances similar to that of wild-type plants, suggesting that the GATIPS-mZmEPSPS allele substitutions are crucial for developing elite maize varieties with significantly enhanced glyphosate resistance. Furthermore, the aromatic amino acid contents in edited maize lines were significantly higher than in wild-type plants. The present study describing the introduction of site-specific CRISPR/Cas9- GATIPS mutations in the ZmEPSPS gene via genome editing has immense potential for higher tolerance to glyphosate with no yield penalty in maize.

Abstract A027: Development of small molecule inhibitors of the Ku-DNA interaction: Impacts on NHEJ, DDR signaling, optimizing genome editing technologies, and therapeutic intervention for the treatment of cancer

Abstract DNA-PK, the DNA-dependent protein kinase is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role in the non-homologous end joining (NHEJ) DNA repair pathway. NHEJ is responsible for the repair of DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR). The generation of DNA DSBs drives clinical efficacy of radiation therapy and numerous DNA damaging chemotherapeutic drugs used to treat cancer. Modulating the DSB repair pathways is an effective mechanism to increase clinical efficacy of IR and certain chemotherapeutic regimens. We have taken the novel approach to inhibit target the DDR and NHEJ by developing small molecule inhibitors of the DSB sensor Ku, and its interaction with DNA. The Ku heterodimer serves as initiator of the NHEJ pathway following DSB induction and is the key DNA-binding component of the DNA-PK. We have created an extensive library of Ku DNA binding inhibitors (Ku-DBis) and demonstrate a direct interaction with the Ku heterodimer and potent inhibition of Ku-DNA binding and DNA-PK catalytic activity. In vitro analysis of a series of Ku truncation mutants defines the necessary domains for Ku-DBi activity. Ku-DBi’s show potent inhibition of in vitro and cellular NHEJ and, as a result, potentiate cellular sensitivity to DSB-inducing agents. Enhancement of sensitivity is a function of inhibiting DNA-PKcs autophosphorylation and dysregulation of signaling to the DDR. Recent chemical optimization has identified independent pharmacophores that drive off-target cytotoxicity, direct Ku inhibitory potency, and enhance cellular uptake activity. These data served as the basis for creation of a novel series of Ku-DBi’s towards interrogating NHEJ and DDR signaling in vivo, optimizing genome editing technologies, and therapeutic intervention for the treatment of cancer. Citation Format: John J. Turchi, Pamela L. Mendoza-Munoz, Pamela S. VanderVere-Carozza, Navnath S. Gavande, Joseph R. Dynlacht, Joy E. Garrett, Dineshsinha Chauhan, Katherine S. Pawelczak. Development of small molecule inhibitors of the Ku-DNA interaction: Impacts on NHEJ, DDR signaling, optimizing genome editing technologies, and therapeutic intervention for the treatment of cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A027.

The SAP domain of Ku facilitates its efficient loading onto DNA ends.

The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku-DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and sliding of Ku along DNA. Our findings revealed that the C-terminal SAP domain of Ku70 facilitates the initial phases of the Ku-DNA interaction but does not affect the sliding process. This suggests that the SAP domain may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of the SAP domain, we generated Arabidopsis plants expressing Ku lacking the SAP domain. Intriguingly, despite the decreased efficiency of the ΔSAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed with which Ku loads onto telomeres or DNA double-strand breaks is not the decisive factor in stabilizing these DNA structures.

260 Insertion of the CFTR cDNA in the CFTR locus in human bronchial epithelial cells is improved by inhibiting the nonhomologous end joining DNA repair pathway

260 Insertion of the CFTR cDNA in the CFTR locus in human bronchial epithelial cells is improved by inhibiting the nonhomologous end joining DNA repair pathway

Uncovering the Molecular Drivers of NHEJ DNA Repair-Implicated Missense Variants and Their Functional Consequences.

Variants in non-homologous end joining (NHEJ) DNA repair genes are associated with various human syndromes, including microcephaly, growth delay, Fanconi anemia, and different hereditary cancers. However, very little has been done previously to systematically record the underlying molecular consequences of NHEJ variants and their link to phenotypic outcomes. In this study, a list of over 2983 missense variants of the principal components of the NHEJ system, including DNA Ligase IV, DNA-PKcs, Ku70/80 and XRCC4, reported in the clinical literature, was initially collected. The molecular consequences of variants were evaluated using in silico biophysical tools to quantitatively assess their impact on protein folding, dynamics, stability, and interactions. Cancer-causing and population variants within these NHEJ factors were statistically analyzed to identify molecular drivers. A comprehensive catalog of NHEJ variants from genes known to be mutated in cancer was curated, providing a resource for better understanding their role and molecular mechanisms in diseases. The variant analysis highlighted different molecular drivers among the distinct proteins, where cancer-driving variants in anchor proteins, such as Ku70/80, were more likely to affect key protein-protein interactions, whilst those in the enzymatic components, such as DNA-PKcs, were likely to be found in intolerant regions undergoing purifying selection. We believe that the information acquired in our database will be a powerful resource to better understand the role of non-homologous end-joining DNA repair in genetic disorders, and will serve as a source to inspire other investigations to understand the disease further, vital for the development of improved therapeutic strategies.

CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species.

Sporothrix brasiliensis is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of Sporothrix species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in Sporothrix has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in S. brasiliensis, S. schenckii, and S. chilensis. Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene pks1, which is a proposed virulence factor of Sporothrix species. To improve in locus integration of deletion constructs, we deleted the KU80 homolog that is critical for non-homologous end-joining DNA repair. The use of Δku80 strains from S. brasiliensis enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in Sporothrix species. IMPORTANCE Sporotrichosis caused by Sporothrix brasiliensis is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in Sporothrix, this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of Sporothrix species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis.

Activation of the DNA damage response pathway in the infected gastric tissue with Helicobacter pylori: a case-control study.

Gastritis is among the most common human diseases worldwide. Although the involvement of Helicobacter pylori infection as a class I human carcinogen for gastric cancer progression is accepted, it is not well known how gastritis progression to atrophy and stomach cancer occurs. In this case-control study, the potential link of H. pylori infection with alteration in the transcription of genes involved in DNA Damage Response pathways was investigated among the patients with gastritis. To measure the difference in the relative mRNA expression level of ATM, CHEK2, TP53, DCLRE1C, POLM, and XRCC4 genes between H. pylori-infected and non-infected patients, gastric biopsies of 30 H. pylori infected patients with moderate chronic gastritis and 30 non-infected patients with mild chronic gastritis were analyzed. Up-regulation of genes linked to non-homologous end joining (NHEJ) pathway (DCLRE1C, POLM, and XRCC) was shown in 40% (8.44 fold ± 13.91), 63.33% (15.72 fold ± 33.08) and 50% (9.99 fold ± 21.55), respectively, and also to DDR pathway (ATM, CHEK2, and TP53) in 33% (2.42 fold ± 3.17), 40% (2.86 fold ± 3.61) and 50% (5.00 fold ± 6.52), respectively. No correlation was detected between alteration in the transcription level of the studied genes and age or gender. Our results provide new data that may support the potential involvement of H. pylori infection in the activation of genes involved in DNA damage response, mainly through a non-homologous end-joining DNA repair system that might be linked to mutagenesis in the pre-cancerous gastric tissue.

Noncanonical functions of Ku may underlie essentiality in human cells

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

The Contribution of DNA Ligase 4 Genetic Variations to Taiwanese Lung Cancer.

Impaired non-homologous end-joining DNA repair capacity may have a significant role in maintaining genome integrity and triggering carcinogenesis. However, the specific impact of DNA ligase 4 (Lig4) genotypes remains unclear. This study aimed to assess the contribution of Lig4 genotypes to the risk of developing lung cancer. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to examine the genotypes of Lig4 rs1805388, and their association with lung cancer risk was evaluated in a case-control study consisting of 358 lung cancer cases and 716 age- and sex-matched cancer-free control subjects. The distribution of CC, CT, and TT genotypes for Lig4 rs1805388 among the cases was 45.0%, 41.6%, and 13.4%, respectively, compared to 58.0%, 36.3%, and 5.7% among the controls (p for trend=1.98×10-6). Allelic analysis indicated that individuals carrying the T-allele for Lig4 rs1805388 had a 1.66-fold higher risk of developing lung cancer compared to those carrying the wild-type C-allele [95% confidence interval (CI)=1.36-2.02, p=4.04×10-7]. Moreover, a significant interaction was observed between the Lig4 rs1805388 genotype and smoking status (p=1.32×10-7). These findings suggest that the CT and TT variant genotypes of Lig4 rs1805388, combined with cigarette smoking, may contribute to a higher risk of developing lung cancer.

MiR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

Trichoderma harzianum marker-free strain construction based on efficient CRISPR/Cas9 recyclable system: A helpful tool for the study of biological control agents

This article reports the first successful marker-free genetic engineering of Trichoderma harzianum, a widely studied biocontrol agent with great potential for use in sustainable agriculture. T. harzianum has been shown to control plant diseases, promote plant growth, and improve plant tolerance to abiotic stress. A specific CRISPR/Cas9 gene-editing system, based in self-replicative AMA1-plasmid, was used to generate T. harzianum mutant strains, targeting the albA (pks4) and ku70 genes, involved in melanin synthesis and non-homologous end joining DNA repair pathway, respectively. The resulting albA-deficient strain displayed a loss of pigmentation in their conidia, confirming phenotypically and genotypically the efficacy of the CRISPR/Cas9 system in generating targeted gene knockouts in T. harzianum. A marker-free Δku70 strain was also constructed, with the maintenance of its original fitness, providing a helpful tool for precise genetic engineering in T. harzianum, due to NHEJ-deficiency, able to be used in in vitro and in vivo (in planta) studies. The study highlights the potential of using CRISPR/Cas9 technology to facilitate the study of the molecular processes involved in the biocontrol and plant growth-promoting properties of T. harzianum.

Non-Homologous End-Joining Pathway Genotypes Significantly Associated with Nasopharyngeal Carcinoma Susceptibility.

Defects in the non-homologous end-joining (NHEJ) DNA repair pathway lead to genomic instability and carcinogenesis. However, the roles of individual NHEJ genes in nasopharyngeal carcinoma (NPC) etiology are not well-understood. The aim of this study was to assess the contribution of NHEJ genotypes, including XRCC4 (rs6869366, rs3734091, rs28360071, rs28360317, rs1805377), XRCC5 (rs828907, rs11685387, rs9288518), XRCC6 (rs5751129, rs2267437, rs132770, rs132774), XRCC7 rs7003908, and Ligase4 rs1805388, to NPC risk, with 208 NPC patients and 416 controls. Genotype-phenotype correlations were also investigated by measuring mRNA and protein expression in adjacent normal tissues and assessing the NHEJ repair capacity in blood lymphocytes from 43 NPC patients. The results showed significant differences in the distributions of variant genotypes at XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 between the cases and controls. The variant genotypes of these three polymorphisms were associated with significantly increased NPC risks. NPC patients with the risk genotypes at XRCC6 rs2267437 had significantly reduced expression levels of both mRNA and protein, as well as a lower NHEJ repair capacity, than those with the wild-type genotype. In conclusion, XRCC4 rs3734091, rs28360071, and XRCC6 rs2267437 in the NHEJ pathway were associated with NPC susceptibility. XRCC6 rs2267437 can modulate mRNA and protein expression and the NHEJ repair capacity.