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At least 19 recordsLinked to original sources
PubMed2026

BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Humans↗
PubMed2026

Non-syndromic premature ovarian insufficiency associated with monoallelic LIG4 mutation via haploinsufficiency.

BACKGROUND: Premature ovarian insufficiency (POI) is a heterogeneous reproductive disorder, with genetic factors, particularly defects in DNA damage response pathways, increasingly implicated in its pathogenesis. DNA ligase IV (LIG4) is a key enzyme in the non-homologous end joining (NHEJ) pathway responsible for repairing DNA double-strand breaks (DSBs). However, its role in non-syndromic POI remains unclear. This study aimed to investigate the potential contribution of LIG4 variants to non-syndromic POI. RESULTS: Whole-exome sequencing identified a heterozygous frameshift variant in LIG4 (c.1271_1275del) in a three-generation Han Chinese family with non-syndromic POI, which co-segregated with affected individuals. AlphaFold-based structural modeling predicted truncation of the C-terminal XRCC4 interaction region. Functional experiments demonstrated that the mutant LIG4 protein showed reduced stability and was predominantly mislocalized to the cytoplasm of cells. In ovarian KGN cells, LIG4 depletion reduced cell viability, induced stress-associated cellular senescence, and impaired DNA damage repair capacity. In LIG4 knockout 293T cells, co-transfection of wild-type and mutant constructs revealed dose-dependent functional impairment, resulting in increased apoptosis under basal conditions and after phleomycin induced DNA damage, together with delayed repair of DSBs. Reanalysis of public single-cell RNA sequencing data further showed stage specific upregulation of LIG4 during oocyte maturation. Co-expression network analysis revealed enrichment in the Fanconi anemia pathway, phosphatidylinositol 3-kinase signaling pathway, and glycan metabolism. CONCLUSIONS: Our findings suggest that monoallelic LIG4 mutations may represent a potential genetic etiology for non-syndromic POI with sex-limited penetrance. While further validation in more physiologically relevant models is warranted, our data indicate that LIG4 haploinsufficiency may impair DSB repair and disrupt molecular pathways crucial for oocyte maturation and survival, highlighting a potential role of the NHEJ pathway in maintaining human ovarian function.

Humans↗
PubMed2026

Comprehensive In Silico Analysis Identifies MSTO1 and LIG1 as Candidate Biomarkers With Diagnostic and Prognostic Relevance in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and remains a major cause of cancer-related mortality worldwide. Its poor clinical outcomes are largely attributed to late-stage diagnosis and the limited accuracy of currently available diagnostic and prognostic biomarkers. Therefore, identifying novel molecular markers with improved sensitivity, specificity, and therapeutic relevance is essential for enhancing early detection and guiding personalized treatment strategies. AIMS: To identify and prioritize novel candidate HCC biomarkers with diagnostic and prognostic value and potential therapeutic vulnerability using integrated multi-omics, survival, functional dependency, and tumor microenvironment analyses. METHODS AND RESULTS: We examined the mRNA and protein expression levels of 8 DEGs in HCC tissues in the TCGA and CPTAC datasets using UALCAN, which showed that MSTO1 and LIG1 were overexpressed consistently in HCC relative to normal liver tissues. Moreover, elevated expression levels of these genes were significantly associated with higher tumor grade and advanced stage. Kaplan-Meier plotter survival data confirmed that increased expression of MSTO1 and LIG1 was associated with poorer overall survival. The DepMap CRISPR knockout data confirmed a functional dependency of both genes in HCC cell lines. CBioPortal analyses provided characterization of genomic alterations and enabled enrichment analysis of co-expressed genes, and the TCGA-UALCAN pan-cancer analyses supported the assessment of tissue specificity across tumor types. TIMER3 analyses linked candidate gene expression with immune cell infiltration patterns. Diagnostic performance by ROC analysis showed excellent discrimination for MSTO1 (AUC = 0.987) and good discrimination for LIG1 (AUC = 0.897). Multivariate Cox regression with Benjamini-Hochberg FDR correction across the eight genes supported MSTO1 as a candidate independent prognostic factor after adjustment for tumor stage, grade, etiology, age, and sex (HR = 1.29, p = 0.035), whilst LIG1 showed no independent prognostic value. Promoter methylation of MSTO1 and ADH4, assessed via UALCAN, showed that both genes were significantly differentially methylated in the promoter region of primary HCC tissues compared with normal liver tissues. Our study also confirmed the biological and clinical relevance of established HCC biomarkers: TERT, IRAK1, and ADH4. CONCLUSION: MSTO1 and LIG1 emerged as candidate diagnostic biomarkers in HCC. Additionally, MSTO1 showed a candidate prognostic association with overall survival that remained significant after adjusting for tumor stage, grade, and etiology, as well as patients' age, but not after further adjustment for AFP status. Functional data also highlighted MSTO1 as a candidate therapeutic dependency. On the other hand, LIG1 showed no independent prognostic association in either multivariate model. Their differential expression and functional essentiality in HCC cell lines highlighted their value for further experimental and independent-cohort validation before potential integration into biomarker development pipelines aimed at improving early detection and targeted therapy in HCC.

Humans↗
PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗
PubMed2026

Revealing the Association of LIG1 Genetic Variants With Pterygium Susceptibility and Demographic Characteristics in a Taiwanese Population.

BACKGROUND/AIM: Pterygium is a common ocular surface disorder associated with long-term ultraviolet exposure and/or oxidative DNA damage. Accumulated evidence suggests that defects in DNA repair pathways may contribute to pterygium risk. DNA ligase I (LIG1), a key enzyme involved in DNA replication and base excision repair, has been involved in the etiology of several human diseases, including cancers. However, its role in pterygium has never been examined before. This study aimed at exploring the association between the LIG1 genotypes and pterygium risk in a Taiwanese population. PATIENTS AND METHODS: The hospital-based case-control study was conducted including 165 patients with pterygium and 320 age- and sex-matched non-pterygium controls. LIG1 rs20579 genotypes were accessed utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Stratified analysis and the calculation of odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were used for evaluating the associations between genotypes and pterygium risk. RESULTS: Individuals carrying the homozygous variant AA genotype exhibited a significantly elevated risk of pterygium compared with those carrying the GG genotype (OR=2.74, 95% CI=1.17-6.43, p=0.0305). Under the recessive model, the AA genotype conferred a 2.65-fold elevated risk (95% CI=1.14-6.18, p=0.0350). The variant A allele was also associated with increased susceptibility (OR=1.46, 95% CI=1.03-2.07, p=0.0413). Stratified analyses revealed significant associations specifically among individuals aged ≥60 years (OR=5.64, 95% CI=1.67-19.04, p=0.0039) and males (OR=5.23, 95% CI=1.74-15.73, p=0.0034), but not those of younger ages or females. CONCLUSION: The LIG1 rs20579 genotype is significantly associated with pterygium susceptibility in Taiwanese individuals, particularly among elderly and male subjects. These findings support the involvement of impaired DNA repair machinery in pterygium pathogenesis and suggest that LIG1 rs20579 may serve as a novel genetic biomarker for risk assessment and early detection of pterygium.

Humans↗
PubMed1977

DNA synthesis and degradation in UV-irradiated toluene treated cells of E. coli K12: the role of polynucleotide ligase.

Toluene treated cells have been used to study the processes of DNA synthesis and DNA degradation in ultra-violet irradiated Escherichia coli K12. Synthesis and degradation are both shown to occur extensively if polynucleotide ligase is inhibited, and to occur to a much lesser extent if ligase activity is optimal. Extensive UV-induced DNA synthesis in toluene-treated cells requires ATP for the initial incision step, and DNA polymerase I. Extensive degradation also depends on the early ATP-dependent incision step, and the subsequent degradation shows a partial requirement for ATP. Curtailment of degradation by ligase requires DNA polymerase activity, but is not dependent upon DNA polymerase I. Apparently this process can be carried out with equal facility by either DNA polymerase II or polymerase III. These observations suggest that extensive DNA polymerase I-dependent repair synthesis and extensive DNA degradation are facets of two divergent pathways of excision repair, both of which depend upon the early uvrABC determined ATP-dependent incision step.

DNA Repair↗
PubMed1976

A deoxyribonucleic acid kinase from nuclei of rat liver. Purification and properties.

A DNA kinase has been partially purified from rat liver nuclei by a procedure which also yields DNA ligase. The kinase uses ATP to phosphorylate specifically the 5'-hydroxyl termini of oligodeoxynucleotides and of single- or double-stranded DNA, yielding 5'-phosphate termini and ADP. The kinase is inactive on RNA, or on oligodeoxynucleotides of chain length less than approximately 10 to 12 residues. The kinase requires a divalent cation (Mg2+, Mn2+, Co2+, Zn2+, Ni2+, or Ca2+) for activity and has an acidic pH optimum. It is inhibited by a variety of nucleotides as well as by very low levels of inorganic and organic sulfate compounds and sulfate analogues. The molecular weight of the kinase is estimated to be 8 times 10(4) from gel filtration.

Animals↗
PubMed1978

Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.

Two active components alpha and beta of micrococcus luteus DNA gyrase, of peptide weights of 115,000 and 97,000, respectively, have been purified. Each individual component exhibits little DNA gyrase activity; the ATP-dependent negative supercoiling of a covalently closed circular DNA duplex is catalyzed by a combination of the two. Covalent closure by Escherichia coli ligase of a circular DNA containing single-chain scissions, when carried out in the presence of a combination of the DNA gyrase components alpha and beta, gives a positively supercoiled DNA upon removal of the bound protein molecules. ATP was not present during the ligase treatment; therefore the positive supercoiling of DNA observed is a result of the binding of gyrase molecules, presumably as multi-subunit oligomers, during the ligation step. This is in contrast to the negative supercoiling of DNA catalyzed by gyrase in the presence of ATP. A model in which negative supercoiling of DNA is achieved by ATP-modulated repetitive wrapping of the DNA around gyrase is described. The model also suggests a plausible mode of action by which translocation of a DNA along its helix axis can be actively driven by an ATPase.

Adenosine Triphosphate↗
PubMed1975

Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [Söderhäll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.

Adenosine Monophosphate↗
PubMed1975

Kinetics and effect of salts and polyamines on T4 polynucleotide ligase.

The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.

Adenosine Monophosphate↗
PubMed1977

Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates.

Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor. We have identified adenylylated DNA and RNA reaction intermediates in which the AMP moiety is attached by a pyrophosphate bond to the 5'-phosphoryl group of the donor. A large amount of DNA-adenylate accumulates during the reaction and the dependence of joining and adenylylation on chain length are similar. The adenylylated donor is joined by ligase to an acceptor in the absence of ATP, and AMP is released stoichiometrically in this reaction. The acceptor is not only a substrate in the reaction but also a cofactor for adenylylation of the donor; in the absence of a 3'-hydroxyl group the activated intermediate does not form. The activated DNA need not join to the acceptor that initially stimulated activation but can also join to another acceptor. This process of acceptor exchanges has proven useful for promoting the cyclization of small DNA substrates and the synthesis of DNA co-polymers.

Coliphages↗
PubMed1975

In vitro repair of UV-or x-irradiated bacteriophage T4 DNA by extract from blue-green alga Anacystis nidulans.

The cell-free extract from blue-green alga Anacystis nidulans contains enzymatic activities which repair in vitro transforming DNA of bacteriophage T4 damaged by UV light or X-rays. The repair effect of the extract was observed with double-stranded irradiated DNA but not with denatured irradiated DNA. The level of restoration of the transforming activity depends on the protein concentration in the reaction mixture and on the dose of irradiation. A fraction of DNA lesions induced by X-rays is repaired by a NAD-dependent polynucleotide ligase present in the extract. The repair of UV-induced lesions is the most efficient in the presence of magnesium ions, NAD, ATP and the four deoxynucleoside triphosphates. The results indicate that the repair of UV-irradiated DNA is performed with the participation of DNA polymerase and polynucleotide ligase which function in the cell-free extract of the algae on the background of a low deoxyribonuclease activity.

Adenosine Triphosphate↗
PubMed1976

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA.

Four site-specific endodeoxyribonucleases have been partially purified from extracts of bacteriophage T5-infected Escherichia coli by gel filtration and affinity chromatography on single- and double-stranded DNA. The enzymes were detected and characterized by agarose gel electrophoresis of alkali-denatured digestion products. None of the four is found in uninfected cells. In the presence of a divalent cation, all four endonucleases make ligase-repairable, single-chain interruptions at specific sites in the duplex DNA of several bacteriophages (lambda, T7, and T5) and a mammalian virus (adenovirus 2). These activities are not stimulated by ATP. None of the four is active on single-stranded DNA. The fragments produced by each enzyme from ligase-repaired T5 DNA do not correspond to those derived from mature T5 DNA. Each of the enzymes is able to cleave the intact strand of T5 DNA.

Coliphages↗
PubMed1979

The synthesis of oligodeoxyribonucleotides using RNA ligase.

T4 RNA ligase catalyzes the addition of a single deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of oligodeoxyribonucleotides (Hinton et al. (1978) Biochemistry 17, 5091). We have determined improved conditions for this reaction which give yields equal to or greater than 85% when any of five common deoxyribonucleoside bisphosphate (pdAp, pdCp, pdGp, pdTp, or pdUp) are added to dA(PDA)4. A low ATP concentration, which is constantly maintained by a regeneration system composed of phosphocreatine, creatine kinase, and myokinase, contributes to the attainment of high yields. The addition of RNase A and spermine also enhances the rates and yields of the reactions. These conditions facilitate the use of RNA ligase as a reagent for the stepwise synthesis of DNA of defined sequence.

Base Sequence↗
PubMed1975

A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.

Adenosine Triphosphate↗
PubMed1979

Split genes and RNA splicing.

A number of genes in higher organisms and in their viruses appear to be split. That is, they have "nonsense" stretches of DNA interspersed within the sense DNA. The cell produces a full RNA transcript of this DNA, nonsense and all, and then appears to splice out the nonsense sequences before sending the RNA to the cytoplasm. In this article what is known about these intervening sequences and about the processing of the RNA is outlined. Also discussed is their possible use and how they might have arisen in evolution.

Animals↗
PubMed1979

Addition of oligonucleotides to the 5'-terminus of DNA by T4 RNA ligase.

Bacteriophage T4-induced RNA ligase catalyzes the controlled template-independent addition of RNA to the 5'-phosphoryl end of large DNA molecules. Restriction enzyme-generated fragments of Co1E1 DNA with completely basepaired or cohesive ends and linear single-stranded øX174 viral DNA were all good substrates. DNA molecules from 10 to 6000 nucleotides long were quantitatively joined in an hour to a number of different RNA homopolymers. The most efficient of these was A(pA)5; I(pI)5 and C(pC)5 were also utilized while U(pU)5 was not. The optimum ribohomopolymer length was six nucleotides. Joining of ribohomopolymers between 10 and 20 nucleotides long occurred at approximately 1/2 the maximal rate and a trimer was the shortest substrate. Thus RNA ligase provides a method for generating extensions of predetermined length and base composition at the 5'-end of large DNA molecules that complements the available procedures for extending the 3'-hydroxyl terminus of DNA.

Coliphages↗