Human DNA ligases I and III have stand-alone end-joining capability,but differ in ligation efficiency and specificity

Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3′-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5′-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg²⁺, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg²⁺ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.     

Early embryonic lethality due to targeted inactivation of DNA ligase III

DNA ligases catalyze the joining of strand breaks in the phosphodiester backbone of duplex DNA and play essential roles in DNA replication, recombination, repair, and maintenance of genomic integrity. Three mammalian DNA ligase genes have been identified, and their corresponding ligases play distinct roles in DNA metabolism. DNA ligase III is proposed to be involved in the repairing of DNA single-strand breaks, but its precise role has not yet been demonstrated directly. To determine its role in DNA repair, cellular growth, and embryonic development, we introduced targeted interruption of the DNA ligase III (LIG3) gene into the mouse. Mice homozygous for LIG3 disruption showed early embryonic lethality. We found that the mutant embryonic developmental process stops at 8.5 days postcoitum (dpc), and excessive cell death occurs at 9.5 dpc. LIG3 mutant cells have relatively normal XRCC1 levels but elevated sister chromatid exchange. These findings indicate that DNA ligase III is involved in essential DNA repair activities required for early embryonic development and therefore cannot be replaced by other DNA ligases.     

TRIM37 promotes gallbladder cancer proliferation by activating the Wnt/β-catenin pathway via ubiquitination of Axin1

BackgroundGallbladder cancer (GBC) is among the most lethal malignancies in the world, with a prognosis that is extremely poor. The results of previous studies suggest that tripartite motif containing 37 (TRIM37) contributes to the progression of numerous types of cancer. Nevertheless, there is little knowledge about the molecular mechanisms and functions of TRIM37 in GBC.MethodsA clinical significance assessment was conducted on TRIM37 following its detection by immunohistochemistry. In vitro and in vivo functional assays were performed to investigate the role of TRIM37 in GBC.ResultsIn this study, TRIM37 is upregulated in GBC tissues, which is associated with decreased histological differentiation, advanced TNM stage, and shorter overall survival rates. In vitro, TRIM37 knockdown inhibited cell proliferation and promoted apoptosis, and in vivo, TRIM37 knockdown suppressed GBC growth. Contrary to this, cell proliferation is increased in GBC cells when overexpression of TRIM37 is expressed. Mechanistic investigations revealed that TRIM37 promotes GBC progression through activation of the Wnt/β‑catenin signaling pathway via degradation of Axin1.ConclusionThe present study suggests that TRIM37 contributes to the development of GBC and thus provides an important biomarker for predicting GBC prognosis and an effective target for therapeutic intervention.     

Disconnecting XRCC1 and DNA ligase III

DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease.     

LIG1 syndrome mutations remodel a cooperative network of ligand binding interactions to compromise ligation efficiency

Human DNA ligase I (LIG1) is the main replicative ligase and it also seals DNA breaks to complete DNA repair and recombination pathways. Immune compromised patients harbor hypomorphic LIG1 alleles encoding substitutions of conserved arginine residues, R771W and R641L, that compromise LIG1 activity through poorly defined mechanisms. To understand the molecular basis of LIG1 syndrome mutations, we determined high resolution X-ray structures and performed systematic biochemical characterization of LIG1 mutants using steady-state and pre-steady state kinetic approaches. Our results unveil a cooperative network of plastic DNA-LIG1 interactions that connect DNA substrate engagement with productive binding of Mg²⁺ cofactors for catalysis. LIG1 syndrome mutations destabilize this network, compromising Mg²⁺ binding affinity, decreasing ligation efficiency, and leading to elevated abortive ligation that may underlie the disease pathology. These findings provide novel insights into the fundamental mechanism by which DNA ligases engage with a nicked DNA substrate, and they suggest that disease pathology of LIG1 syndrome could be modulated by Mg²⁺ levels.     

Disconnecting XRCC1 and DNA ligase III

DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease.Key words: DNA repair, nervous system, neurodegeneration, DNA ligase III, DNA damage, XRCC1, mitochondria, mtDNA     

Serine/Threonine Kinase CHEK1-Dependent Transcriptional Regulation of RAD54L Promotes Proliferation and Radio Resistance in Glioblastoma

Accumulating evidence indicates that Checkpoint kinase 1 (CHEK1) plays an essential role in tumor cells and that it could induce cell proliferation and could be related to prognosis in multiple types of cancer. However, the biological role and molecular mechanism of CHEK1 in GBM still remain unclear. In this study, we identified that CHEK1 expression was enriched in glioblastoma (GBM) tumors and was functionally required for tumor proliferation and that its expression was associated to poor prognosis in GBM patients. Mechanically, CHEK1 induced radio resistance in GBM cells, and CHEK1 knockdown increased cell apoptosis when combined with radiotherapy via regulation of the DNA repair/recombination protein 54L (RAD54L) expression. Therapeutically, we found that CHEK1 inhibitor attenuated tumor growth both in vitro and in vivo. Collectively, CHEK1 promotes proliferation, induces radio resistance in GBM, and could become a potential therapeutic target for GBM.     

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.     

Downregulated circRNA_CDKN1A promotes gallbladder cancer progression through activation of the NF-κB pathway

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial–mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A–NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.     

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

DNA ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1, which was recruited onto chromatin. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signaling promotes the recruitment of LIG3 on chromatin and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.     

MUC3A promotes non-small cell lung cancer progression via activating the NFκB pathway and attenuates radiosensitivity

Mucin 3A (MUC3A) is highly expressed in non-small cell lung cancer (NSCLC), but its functions and effects on clinical outcomes are not well understood. Tissue microarray of 92 NSCLC samples indicated that high levels of MUC3A were associated with poor prognosis, advanced staging, and low differentiation. MUC3A knockdown significantly suppressed NSCLC cell proliferation and induced G1/S accumulation via downregulating cell cycle checkpoints. MUC3A knockdown also inhibited tumor growth in vivo and had synergistic effects with radiation. MUC3A knockdown increased radiation-induced DNA double strain breaks and γ-H2AX phosphorylation in NSCLC cells. MUC3A downregulation inhibited the BRCA-1/RAD51 pathway and nucleus translocation of P53 and XCRR6, suggesting that MUC3A promoted DNA damage repair and attenuated radiation sensitivity. MUC3A knockdown also resulted in less nucleus translocation of RELA and P53 in vivo. Immunoprecipitation revealed that MUC3A interacted with RELA and activated the NFκB pathway via promoting RELA phosphorylation and interfering the binding of RELA to IκB. Our studies indicated that MUC3A was a potential oncogene and associated with unfavorable clinical outcomes. NSCLC patients with a high MUC3A level, who should be more frequent follow-up and might benefit less from radiotherapy.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target.     

DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme''s SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3.     

A noncatalytic function of the ligation complex during nonhomologous end joining

Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)–DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)–X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4–Cer-XLF complexes on DNA ends.