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The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA ligases. DNA ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.

Objective The aim of this study was to examine DNA ligase activity and expression of DNA damage response pathway (DDR) genes in patients with stable angina (SA) and non-ST elevation myocardial infarction (NSTEMI) and determine whether they correlate with plaque morphology. Background Patients with coronary artery disease (CAD) have evidence of deoxyribonucleic acid (DNA) damage in peripheral blood mononuclear cells (PBMCs). It is unclear whether this represents excess damage or defective DNA repair activity. Methods DNA ligase activity and the expression of 22 DDR genes were measured in PBMCs of patients (both SA (n = 47) and NSTEMI (n = 42)) and in age and gender-matched controls (n = 35). Target lesion anatomical assessment was undertaken with frequency domain optical coherent tomography. Results DNA ligase activity was different across the three groups of patients (control = 119 ± 53, NSTEMI = 115.6 ± 85.1, SA = 81 ± 55.7 units/g of nuclear protein; ANOVA p = 0.023). Pair wise comparison demonstrated that this significance is due to differences between the control and SA patients (p = 0.046). Genes involved in double strand break repair and nucleotide excision repair pathways were differentially expressed in patients with SA and NSTEMI. In SA patients, fibrocalcific plaques were strongly associated with GTSE1, DDB1, MLH3 and ERCC1 expression. By contrast, in NSTEMI patients the strongest association was observed between fibrous plaques and ATM and XPA expression. Conclusion PBMCs from patients with CAD exhibit differences in DNA ligase activity and expression of DDR genes. Expression levels of certain DDR genes are strongly associated with plaque morphology and may play a role in plaque development and progression.

Deoxyribonucleic acid (DNA) damage and repair signaling cascades are related to the development of atherosclerosis. Pathological studies have demonstrated that healed coronary plaque rupture (HCPR) contributes to plaque progression and predisposes to sudden ischemic cardiac death. The objective of this study is to investigate the relationship between HCPR detected by optical coherence tomography (OCT) and DNA ligase. Forty-two patients with both OCT and DNA ligase were prospectively enrolled. The population included patients with stable angina pectoris (SA) and non-ST-elevation myocardial infarction (NSTEMI). It was found that the prevalence of HCPR was greater in subjects with higher DNA ligase activity (correlation coefficient 0.36, p = 0.019). The presence of HCPR in patients with NSTEMI was greater than in patients with SA per OCT analysis; however, there was no statistical difference in this limited population (22.53% versus 12.83%, respectively, p = 0.116). DNA repair activity by DNA ligase was associated with HCPR in advanced coronary artery plaque by OCT.

XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments. DOI: http://dx.doi.org/10.7554/eLife.22900.001, eLife digest DNA in human and other animal cells is organised into structures called chromosomes. One of the most dangerous types of DNA damage is a double-strand break, where both strands of the DNA helix are broken in the same place. If this damage is not repaired it can be serious enough to kill the cell. If the DNA is repaired badly, part of one chromosome can become attached to another – a defect known as a chromosome translocation. Fortunately, cells are equipped with machineries that can recognise and fix these breaks. One of these processes is known as “non-homologous end joining” and it involves a set of proteins including two known as XRCC4 and XLF. These proteins work like a bandage, holding together the broken DNA until it is repaired. Both proteins have long tails, but the role of these structures was not clear. During DNA repair, the cell chemically modifies the tails of these proteins by a process called phosphorylation. However, previous studies have found that it is possible to prevent the modification of the tail of one of the proteins, or even remove the tail entirely, without affecting the repair process. Here, Normanno et al. investigated the effect of blocking the modification of the tails of both proteins at the same time. For the experiments, the tails were both altered in various places to either block or mimic the phosphorylation that normally occurs during DNA repair. Mimicking the phosphorylation of both tails affected the ability of XRCC4 and XLF to stay attached to the DNA, suggesting that the phosphorylation helps these proteins to detach from the DNA once the repair is complete. Furthermore, in human embryonic kidney cells the altered proteins were less able to repair DNA damage in response to a drug that causes double-strand breaks. These findings improve our understanding of how cells repair their DNA to maintain a complete set of genetic information. Defects in DNA repair are linked to conditions where the brain does not develop properly, whilst some cancer therapies deliberately inflict double-strand breaks to kill cancer cells. In the future, these findings may lead to improvements in radiotherapy and other treatments for human diseases. DOI: http://dx.doi.org/10.7554/eLife.22900.002

Phaeocystis globosa virus 16T is a giant virus that belongs to the so-called nucleo-cytoplasmic large DNA virus (NCLDV) group. Its linear dsDNA genome contains an almost full complement of genes required to participate in viral base excision repair (BER). Among them is a gene coding for a bimodular protein consisting of an N-terminal Polβ-like core fused to a C-terminal domain (PgVPolX), which shows homology with NAD-dependent DNA ligases. Analysis of the biochemical features of the purified enzyme revealed that PgVPolX is a multifunctional protein that could act as a >Swiss army knife> enzyme during BER since it is endowed with: 1) a template-directed DNA polymerization activity, preferentially acting on DNA structures containing gaps; 2) 5′-deoxyribose-5-phosphate (dRP) and abasic (AP) site lyase activities; and 3) an NAD-dependent DNA ligase activity. We show how the three activities act in concert to efficiently repair BER intermediates, leading us to suggest that PgVPolX may constitute, together with the viral AP-endonuclease, a BER pathway. This is the first time that this type of protein fusion has been demonstrated to be functional., Ministry of Economy and Competitiveness grant BFU2014-53791-P to M.V., and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa”

The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase.

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation or V(D)J recombination of the immunoglobulin genes. The breaks often leave mismatched or nonligatable ends, and NHEJ must repair the breaks with high efficiency and minimal nucleotide loss. Here, the NHEJ proteins Ku, DNA-dependent protein kinase catalytic subunit, XRCC4/Ligase IV, and Cernunnos/XRCC4-like factor joined mismatched and noncohesive DNA ends in the absence of processing factors. Depending on the mismatch, Cernunnos stimulated joining 8- to 150-fold. For substrates with a blunt end and a 3′ overhanging end, Ku, XRCC4/Ligase IV, and Cernunnos ligated the 3′ overhanging hydroxyl group to the 5′ phosphate of the blunt end, leaving the other strand unjoined. This activity provides a mechanism for retaining 3′ overhang sequences, as observed during V(D)J recombination in vivo . Thus, Cernunnos/XRCC4-like factor promotes a mismatched end (MEnd) DNA ligase activity to facilitate joining and to preserve DNA sequence. Furthermore, MEnd ligase activity may have applications in recombinant DNA technology.

DNA ligase is an enzyme essential for DNA replication, repair, and recombination in all organisms. Bacterial DNA ligases catalyze a NAD(+)-dependent DNA ligation reaction, i.e., the formation of a phosphodiester bond between adjacent 3'-OH and 5'-phosphate termini of dsDNA. Due to their essential nature, unique cofactor requirement, and widespread existence in nature, bacterial DNA ligases appear to be valuable targets for identifying novel antibacterial agents. To explore bacterial DNA ligases as antibacterial targets and further characterize them, we developed a simple, robust, homogeneous time-resolved fluorescence resonance energy transfer assay (TR-FRET) for measuring Streptococcus pneumoniae DNA ligase activity. This assay involves the use of one dsDNA molecule labeled with biotin and another dsDNA molecule labeled with Cy5, an acceptor fluorophore. During ligation reactions, the donor fluorophore europium (Eu(3+)) labeled with streptavidin was added to the assay mixtures, which bound to the biotin label on the ligated products. This in turn resulted in the FRET from Eu(3+) to Cy5 due to their close proximity. The formation of ligation products was measured by monitoring the emission at 665nm. This assay was validated by the experiments showing that the DNA ligase activity required NAD(+) and MgCl(2), and was inhibited by NMN and AMP, products of the ligase reaction. Using this assay, we determined the K(m) values of the enzyme for dsDNA substrates and NAD(+), and the IC(50) values of NMN and AMP, examined the effects of MgCl(2) and PEG(8000) on the enzyme activity, optimized the concentrations of Eu(3+) in the assay, and validated its utilities for high-throughput screening and biochemical characterizations of this class of enzymes.

Drug resistance is an obstacle preventing success of cancer chemotherapy. Resistance of vaccinia virus towards the topoisomerase II (topo II) targeting anti-cancer drug etoposide has been mapped to the viral DNA ligase gene. The present study was performed to elucidate if the DNA ligase activity, besides topo II levels, was altered in human lymphatic leukaemia cell strains with different levels of etoposide resistance. At measurements of DNA ligase activity with specific substrates, to distinguish between different DNA ligases, a reduced DNA ligase activity was observed in the resistant substrains. In contrast, the initial step of the ligation process, formation of DNA ligase--AMP complex, did not decrease in the resistant cell strains, suggesting an alteration in a later reaction leading to a deteriorated DNA ligation. The results suggest that decreased DNA ligase activity, besides topo II alterations, may contribute to etoposide resistance of the investigated CEM cells. The relevance of this finding will be further investigated.

DNA damage and repair activity is increasingly recognized in atherogenesis. By facilitating DNA strand joining by catalyzing phosphodiestere bond formation, DNA ligase is crucial in single/double stranded DNA break repair. We aimed to identify differential DNA ligase activity as well as a

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5′-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus lig A gene. Orthologues of the putative S. aureus NAD + -dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli , and the enzyme was purified to near homogeneity. NAD + -dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32 P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD + -dependent DNA ligase from B. stearothermophilus , two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

A gene encoding DNA ligase ( lig Tk ) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, has been cloned and sequenced, and its protein product has been characterized. lig Tk consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig Tk was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig Tk was closely related to the ATP-dependent DNA ligase from Methanobacterium thermoautotrophicum ΔH, a moderate thermophilic archaeon, along with putative DNA ligases from Euryarchaeota and Crenarchaeota . We expressed lig Tk in Escherichia coli and purified the recombinant protein. Recombinant Lig Tk was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg 2+ . The optimum pH of Lig Tk was 8.0, the optimum concentration of Mg 2+ , which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K + was 10 to 30 mM. Lig Tk did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100°C. Unexpectedly, Lig Tk displayed a relatively small, but significant, DNA ligase activity when NAD + was added as the cofactor. Treatment of NAD + with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD + solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig Tk with NAD + . This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.

Summary A cDNA encoding the DNA ligase I homologue has been isolated from Arabidopsis thaliana using a degenerate PCR approach. The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protein with a theoretical molecular mass of 87.8 kDa and an isoelectric point (pI) of 8.20. Alignment of the A. thaliana DNA ligase protein sequence with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae showed good sequence homology (42–45% identity, 61–66% similarity), particularly around the active site. Sequence data indicate that the Arabidopsis DNA ligase is the homologue of the animal DNA ligase I species. Functional analysis of the cDNA clone demonstrated its ability to complement the conditional lethal phenotype of an S. cerevisiae cdc9 mutant defective in DNA ligase activity, confirming that the cloned sequence encodes an active DNA ligase. The level of the DNA ligase transcript was not increased in A. thaliana seedlings in response to DNA damage induced by a period of enhanced UV-B irradiation. However, the cellular level of the DNA ligase mRNA transcript does correlate with the replicative state of plant cells.

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris–HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

DNA ligases represent a fundamental class of enzymes required by all organisms to maintain the structural integrity of the genome. The cellular processes of DNA replication, recombination, and repair generate breaks in the phosphate backbone structure of DNA that can compromise the stability of the genome and threaten the loss of genetic information and the introduction of deleterious chromosomal mutations. DNA breaks are ultimately repaired through the catalytic activity of DNA ligases, which form a phosphodiester bond between adjacent nucleotides in duplex DNA. DNA ligase activity requires a nucleotide cofactor and follows a unique three-step reaction mechanism that involves covalent modification of the ligase enzyme and the DNA substrate. X-ray crystal structure analysis has captured a variety of DNA ligases at different stages of the ligation reaction that demonstrate the unique covalent reaction intermediates, provide insights into the mechanism of the ligase reaction, and illustrate the extensive conformational flexibility and dynamic nature of the modular domain architecture of DNA ligases.

Nitric oxide-induced modifications of DNA occur either by directly altering DNA chemically through reactive nitrogen oxide species (RNOS) or indirectly by inhibiting various repair processes. DNA ligases are enzymes which rejoin single-strand breaks and are critical for DNA integrity during processes such as gene transcription and repair. The eukaryotic and T4 DNA ligases are active in the presence of ATP and act in two steps: the formation of protein-AMP intermediates, then the ligation of DNA breaks. When T4 DNA ligase was exposed to the NO generator DEA/NO (Et2N[NO(NO)]Na), a concentration- and time-dependent inhibition of these two steps, adenylylation of the protein and ligation of the substrate, was observed. This inhibition was abated by the presence of cysteine, suggesting that RNOS, rather than NO, mediated the inhibition of the ligase activity. As mammalian and T4 DNA ligases act by the same mechanism, the inhibition of DNA ligase may explain the increase in single-strand breaks reported for cells exposed to NO and provides a mechanism to increase DNA lesions without direct chemical modification of DNA by NO or RNOS.

DNA ligase 1 (AtLIG1) is the only essential DNA ligase activity in Arabidopsis and is implicated in the important processes of DNA replication, repair and recombination and in transgene insertion during Agrobacterium-mediated plant transformations. The mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through the control of translation initiation from either the first (M1) or second (M2) in-frame AUG codons respectively. Translation from a third in-frame AUG codon (M3) occurs on transcripts in which M1 and M2 are mutagenized to stop codons. Wild-type AtLIG1–GFP constructs (where GFP stands for green fluorescent protein) can be targeted in planta to both the nucleus and mitochondria. AtLIG1–GFP translation from M1 specifically targets the fusion protein only to mitochondria in planta, whereas translation from M2 or M3 targets the fusion protein only to the nucleus. Interestingly, the AtLIG1–GFP fusion protein in which translation is initiated from M1 contains both an N-terminal mtPS (mitochondrial targeting presequence) and a nuclear localization signal; nonetheless, this protein is only targeted to the mitochondria. This result raises intriguing questions on the translational control mechanisms that regulate how the protein products of a single transcript are targeted to more than one cellular compartment.

Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.

Approximately 80% of the DNA ligase activity in HeLa cell extracts is associated with the 21 S enzyme complex that functions in simian virus 40 DNA replication in vitro (Malkas et al., Biochemistry 29, 6362-6374., 1990). The DNA ligase associated with the 21 S complex was purified extensively and its physical, enzymic and immunological properties characterized as DNA ligase I. The association of DNA ligase I with the 21 S complex of enzymes for DNA synthesis provides evidence for the physiological function of this DNA ligase in DNA replication in human cells.

The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).

A multiprotein DNA processing complex isolated from Deinococcus radiodurans contains the DNA repair protein PprA, an ATP-type DNA repair ligase (LigB) encoded by the drB0100 gene, and protein kinase activity. An ATP-dependent DNA end-joining activity was detected in the complex. To elucidate the function of the drB0100 gene, we generated the deletion mutant for the DR_B0100 ORF. The mutant exhibited a nearly 2-log cycle reduction in growth rate when exposed to a 10 000 Gray dose of γ-radiation, and a significant loss in mitomycin C and methylmethane sulphonate tolerance as compared with wild type. Functional complementation of these phenotypes required the wild-type copy of drB0100 along with other genes such as drb0099 and drb0098, organized downstream in the operon. The in vitro DNA ligase activity of LigB was stimulated severalfold by PprA in the presence of the recombinant DRB0098 protein. However, this activity did not improve when PprA was substituted with purified DRB0099 protein or when DRB0098 protein was substituted with the DRB0099 protein in the presence of PprA in solution. These results suggest that PprA and DRB0098 protein are required for LigB function. Furthermore, they also suggest that the LigB operon components contribute to radiation resistance and double-strand break (DSB) repair in D. radiodurans.

We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].

DNA lesions in mammalian cells may be induced by reactive oxygen species, alkylation, and ionizing radiation. This damage can then be repaired via the base excision repair (BER) pathway, which includes single strand break repair (SSBR). Thus, the BER (SSBR) pathway plays a critical role in maintaining genomic integrity, and may help us to better understand the mechanisms of aging, tumor formation, and degenerative diseases. AP site (apurinic/apyrimidinic site) or damaged base excision, nucleotide insertion and ligation are included in the BER (SSBR) pathway, which are facilitated by different enzymes at each step. Most previous in vitro BER studies have used modified radiolabeled (32)P oligonucleotide substrates. Which is a very conventional method for in vitro BER assay. However, the use of radioactive isotope material was limited in various laboratories which are unable to handle radioactive hazard. In this study, we developed a novel technique using real-time quantitative PCR (qPCR) to quantify BER activity in in vitro assays. Single strand breaks, DNA ligase activity, and glycosylase activity were detected to establish the feasibility and advantages of this qPCR technique for in vitro BER profiling.

Chromosome integrity and DNA polymerase activity are negatively affected by improper storage of maize seeds. We have extended our studies on the effect of improper storage on DNA metabolism by analysing the effect of hot–dry and hot–humid conditions on DNA ligase activity. An assay to measure ligase activity was established by using linearized plasmid DNA as substrate and protein extracts from maize axes. Optimal conditions were established for thein vitroassay. Enzyme activity was importantly reduced under both storage conditions. Activity recovered 6–9 h after imbibition. A model for loss of viability and vigour due to damage of DNA metabolism is proposed.

Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity. This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide. Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa. Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers. DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro. In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle. In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected. Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination. These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication.

Human cDNA clones encoding the major DNA ligase activity in proliferating cells, DNA ligase I, were isolated by two independent methods. In one approach, a human cDNA library was screened by hybridization with oligonucleotides deduced from partial amino acid sequence of purified bovine DNA ligase I. In an alternative approach, a human cDNA library was screened for functional expression of a polypeptide able to complement a cdc9 temperature-sensitive DNA ligase mutant of Saccharomyces cerevisiae. The sequence of an apparently full-length cDNA encodes a 102-kDa protein, indistinguishable in size from authentic human DNA ligase I. The deduced amino acid sequence of the human DNA ligase I cDNA is 40% homologous to the smaller DNA ligases of S. cerevisiae and Schizosaccharomyces pombe, homology being confined to the carboxyl-terminal regions of the respective proteins. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is transcribed from a single-copy gene on chromosome 19.

DNA ligase I is the major DNA ligase activity in proliferating mammalian cells. The protein has been purified to apparent homogeneity from calf thymus. It has a monomeric structure and a blocked N-terminal residue. DNA ligase I is a 125-kDa polypeptide as estimated by sodium dodecyl sulfate-gel electrophoresis and by gel chromatography under denaturing conditions, whereas hydrodynamic measurements indicate that the enzyme is an asymmetric 98-kDa protein. Immunoblotting with rabbit polyclonal antibodies to the enzyme revealed a single polypeptide of 125 kDa in freshly prepared crude cell extracts of calf thymus. Limited digestion of the purified DNA ligase I with several reagent proteolytic enzymes generated a relatively protease-resistant 85-kDa fragment. This domain retained full catalytic activity. Similar results were obtained with partially purified human DNA ligase I. The active large fragment represents the C-terminal part of the intact protein, and contains an epitope conserved between mammalian DNA ligase I and yeast and vaccinia virus DNA ligases. The function of the N-terminal region of DNA ligase I is unknown.

International audience; Partial purification of the DNA ligase from three human tissues (liver, thymus and lymphoblasts) revealed that each cell type contains several different polypeptides bearing a DNA ligase I activity. Their apparent molecular weights estimated after SDS PAGE, 130 kDa, 100 kDa and 80 kDa, are in agreement with previous reports. These polypeptides are related by proteolysis to a single higher molecular weight protein of 200 kDa which does not show DNA ligase activity but that could be a preprotein.

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 degrees C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 degrees C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.

Publisher Summary Arsenic (As) is not a mutagen but a human carcinogen though it inhibits DNA repair and is a comutagen. Human AG06 keratinocytes treated with micromolar As exhibit dose and time-dependent loss of DNA ligase function. However, purified human DNA ligase I, ligase III, and other repair enzymes, such as DNA polymerase β, are not inhibited by less than millimolar arsenite, As(III), the most toxic form of As found in the environment. DNA ligase activity in extracts from untreated keratinocytes is also insensitive to less than millimolar As. Pyruvate dehydrogenase, on the other hand, is inhibited by micromolar As and probably determines As-induced cytotoxicity. Simultaneous treatment of AG06 cells with an alkylating agent, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), plus As produces a synergistic increase in viability (dye uptake) at low doses and a synergistic increase in toxicity at high doses. Micromolar As also modulates cellular redox levels and induces a variety of cellular stress response genes. Keratinocytes treated with As exhibit both a time- and dose-dependent increase in cellular GSH levels and alterations in the relative activity of several GSH-dependent enzymes. These As-induced changes in cellular redox capacity and DNA repair activity are not directly related to toxicity. Maximal induction of GSH and DNA repair occurs after a treatment with subtoxic concentrations of As. At submicromolar concentrations, As also induces hyperproliferation of keratinocytes, both in vivo and in vitro. Results in this chapter suggest that As modulates DNA repair and redox levels primarily through post-translation or transcriptional mechanisms.

Here we demonstrate that the Saccharomyces cerevisiae DNA ligase activity, which we previously designated DNA ligase II, is encoded by the genomic DNA sequence YOR005c. Based on its homology with mammalian LIG4, this yeast gene has been named DNL4 and the enzyme activity renamed Dnl4. In agreement with others, we find that DNL4 is not required for vegetative growth but is involved in the repair of DNA double-strand breaks by non-homologous end joining. In contrast to a previous report, we find that a dnl4 null mutation has no effect on sporulation efficiency, indicating that Dnl4 is not required for proper meiotic chromosome behavior or subsequent ascosporogenesis in yeast. Disruption of the DNL4 gene in one strain, M1-2B, results in temperature-sensitive vegetative growth. At the restrictive temperature, mutant cells progressively lose viability and accumulate small, nucleated and non-dividing daughter cells which remain attached to the mother cell. This novel temperature-sensitive phenotype is complemented by retransformation with a plasmid-borne DNL4 gene. Thus, we conclude that the abnormal growth of the dnl4 mutant strain is a synthetic phenotype resulting from Dnl4 deficiency in combination with undetermined genetic factors in the M1-2B strain background.

The molecular mechanism of arsenic toxicity is believed to be due to the ability of arsenite [As(III)] to bind protein thiols. Numerous studies have shown that arsenic is cytotoxic at micromolar concentrations. Micromolar As can also induce chromosomal damage and inhibit DNA repair. The mechanism of arsenic-induced genotoxicity is very important because arsenic is a human carcinogen, but not a mutagen, and there is a need to establish recommendations for safe levels of As in the environment. We have measured the dose-response for arsenic inhibition of several purified human DNA repair enzymes, including DNA polymerase beta, DNA ligase I and DNA ligase III and have found that most enzymes, even those with critical SH groups, are very insensitive to As. Many repair enzymes are activated by millimolar concentrations of As(III) and/or As(V). Only pyruvate dehydrogenase, one of eight purified enzymes examined so far, is inhibited by micromolar arsenic. In contrast to the purified enzymes, treatment of human cells in culture with micromolar arsenic produces a significant dose-dependent decrease in DNA ligase activity in nuclear extracts from the treated cells. However, the ligase activity in extracts from untreated cells is no more sensitive to arsenic than the purified enzymes. Our results show that direct enzyme inhibition is not a common toxic effect of As and that only a few sensitive enzymes are responsible for arsenic-induced cellular toxicity. Thus, arsenic-induced co-mutagenesis and inhibition of DNA repair is probably not the result of direct enzyme inhibition, but may be an indirect effect caused by As-induced changes in cellular redox levels or alterations in signal transduction pathways and consequent changes in gene expression.

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit in vitro the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.

Supramolecular structures composed of padlock probes and primers were used to perform rolling circle amplification (RCA) which was achieved by metal ion (Hg2+ or Ag+) induced DNA ligase activity. In the presence of Hg2+ (or Ag+), the specific and strong interaction between thymidine–thymidine and Hg2+ (or cytosine–cytosine and Ag+) at the terminal of the padlock probe enabled the circularization of the padlock probe with primer in the aid of DNA ligase. An RCA process was then accomplished by DNA polymerase/dNTPs. The RCA product containing multiple tandem repeats could hybridize with a large number of molecular beacons (reporter), resulting in an enhanced fluorescence signal. This proposed single-input YES gate enabled the sensitive and selective detection of Hg2+ (or Ag+). Additionally, based on the principle of DNA hybridization and displacement, a NOT logic gate was constructed by designing a double-stranded fluorescence probe as reporter. Significantly, this assay was further applied to the construction of a complete set of two-input molecular-scale logic gates and three advanced logic devices.

DNA replication in mammalian cells occurs in discrete nuclear foci called 'replication factories'. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non-S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)-DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N-terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub-nuclear compartmentalization.

Chronic exposure to dimethylnitrosamine produces hepatic tumors through recurrent DNA alkylation, whereas acute exposure can cause liver necrosis through mechanisms that remain largely unknown. Our laboratory recently demonstrated that DNA fragmentation occurs early on and may be a causal event in dimethylnitrosamine-induced necrosis in liver. A challenge to interpreting these results is that up to 30% of liver cells are non-parenchymal and could account for the observed DNA fragmentation. In the present study, we have examined whether dimethylnitrosamine induces early genomic DNA fragmentation in cultured mouse hepatocytes. Hepatic parenchymal cells isolated from male ICR mice were cultured in Williams E medium. DNA damage was assessed quantitatively as a fragmented fraction that was not sedimented at 27,000 x g, and qualitatively from agarose gel electrophoresis. Cellular response to DNA damage was assessed by measuring induction of the DNA repair enzyme DNA ligase. Toxic cell death was estimated from release of lactate dehydrogenase (LDH) or adenine nucleotides from cells prelabeled with [3H]adenine. Dimethylnitrosamine produced a twofold increase in [3H]adenine release by 6 h and LDH release at 36 h. DNA fragmentation and DNA ligase activity increased by as early as 1 h. The Ca(2+)-endonuclease inhibitor aurintricarboxylic acid and the Ca2+ chelator ethylenediamine tetraacetic acid (EDTA) prevented DNA fragmentation through 6 h and virtually abolished cytotoxicity through 30 h. DNA ligase induction was strongly associated with DNA fragmentation. Early increases in DNA fragmentation and DNA ligase were highly correlated with later toxic cell death. Such results strongly suggest that dimethylnitrosamine-induced fragmentation of DNA in target parenchymal cells is a causal factor in the toxic death of these liver cells.

Single-stranded DNA can fold into well-defined sequence-dependent tertiary structures that specifically bind a variety of target molecules, raising the possibility that some folded single-stranded DNAs might exhibit catalytic activities similar to those of ribozymes and protein enzymes. Derivatives of the hammerhead ribozyme that contain a majority of deoxyribonucleotides retain the ability to cleave RNA, and a 'deoxyribozyme' was generated by leaving all essential ribonucleotides of the hammerhead on the RNA 'substrate'. Recently in vitro selection has been used to isolate a DNA sequence that shows Pb(2+)-dependent RNA-cleaving activity. Here we report the isolation by in vitro selection of a small single-stranded DNA that is a Zn2+/Cu(2+)-dependent metalloenzyme. The enzyme catalyses the formation of a new phosphodiester bond by the condensation of the 5'-hydroxyl of one oligodeoxynucleotide and a 3'-phosphorimidazolide on another oligodeoxynucleotide, and shows multiple turnover ligation.

International audience; We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.

The activity of DNA ligase, the enzyme involved in ligation of DNA fragments and also in DNA repair is inhibited by Ara-C. The exposure of two human leukemic cell lines, K562 and HL-60 to 10−5 M Ara-C for 3 h, induces a decrease of DNA ligase activity by 40% in K562 and 92% in HL-60. This decreased activity is due to an inhibition by Ara-CTP of the ligase-adenylate complex generation, the crucial step in the action of this enzyme. The activity of the semi-purified ligase as well as the formation of ligase-adenylate complex are decreased in the presence of Ara-CTP. These results demonstrate that Ara-C via its active form Ara-CTP inhibits DNA ligase activity through the inhibition of the ligase-adenylate complex. Other inhibitors of DNA synthesis, such as hydroxyurea, do not exert the same inhibitory effect. The inhibition of DNA ligase activity may be partly responsible for the cytotoxicity of Ara-C.

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.

DNA ligase has been purified to near-homogeneity from the extract of bovine thymus with a yield of 5%. The purified enzyme catalyzed the joining of single-stranded breaks in duplex DNA at a rate of 33 nmol of phosphodiester bonds/min/mg of protein. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double diffusion analysis. The enzyme is composed of a single polypeptide with a molecular weight of about 130,000. The enzyme has a Stokes radius of 52 A, a sedimentation coefficient of about 5 S, and a frictional ratio of 1.6. Apparent Km values for ATP and Mg2+ are 2 microM and 0.9 mM, respectively. Antibody against bovine thymus DNA ligase was prepared by injecting a rabbit with the purified enzyme. Immunochemical titrations revealed that the increased activity of DNA ligase observed after partial hepatectomy of rat and 16-fold higher activity level of mouse Ehrlich tumor cells compared with the host liver are due to a change in the enzyme quantity but not to a change in the catalytic efficiency of the enzyme molecule. Wide variations in the level of DNA ligase activity in extracts from various tissues of rat and mouse were accompanied by proportionate changes in the quantity of immunochemically reactive protein. The antibody inhibited DNA ligase activity from bovine tissues with 20-fold higher efficiency, compared with the enzyme from the rodent tissues. The enzyme activity from chick embryo was unaffected by the antibody.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.