ATP-dependent DNA ligases

SUMMARY: By catalyzing the joining of breaks in the phosphodiester backbone of duplex DNA, DNA ligases play a vital role in the diverse processes of DNA replication, recombination and repair. Three related classes of ATP-dependent DNA ligase are readily apparent in eukaryotic cells. Enzymes of each class comprise catalytic and non-catalytic domains together with additional domains of varying function. DNA ligase I is required for the ligation of Okazaki fragments during lagging-strand DNA synthesis, as well as for several DNA-repair pathways; these functions are mediated, at least in part, by interactions between DNA ligase I and the sliding-clamp protein PCNA. DNA ligase III, which is unique to vertebrates, functions both in the nucleus and in mitochondria. Two distinct isoforms of this enzyme, differing in their carboxy-terminal sequences, are produced by alternative splicing: DNA ligase IIIalpha has a carboxy-terminal BRCT domain that interacts with the mammalian DNA-repair factor XrccI, but both alpha and beta isoforms have an amino-terminal zinc-finger motif that appears to play a role in the recognition of DNA secondary structures that resemble intermediates in DNA metabolism. DNA ligase IV is required for DNA non-homologous end joining pathways, including recombination of the V(D)J immunoglobulin gene segments in cells of the mammalian immune system. DNA ligase IV forms a tight complex with Xrcc4 through an interaction motif located between a pair of carboxy-terminal BRCT domains in the ligase. Recent structural studies have shed light on the catalytic function of DNA ligases, as well as illuminating protein-protein interactions involving DNA ligases IIIalpha and IV.     

Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks

DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.     

Mammalian DNA ligases

DNA joining enzymes play an essential role in the maintenance of genomic integrity and stability. Three mammalian genes encoding DNA ligases, LIG1, LIG3 and LIG4, have been identified. Since DNA ligase II appears to be derived from DNA ligase III by a proteolytic mechanism, the three LIG genes can account for the four biochemically distinct DNA ligase activities, DNA ligases I, II, III and IV, that have been purified from mammalian cell extracts. It is probable that the specific cellular roles of these enzymes are determined by the proteins with which they interact. The specific involvement of DNA ligase I in DNA replication is mediated by the non-catalytic amino-terminal domain of this enzyme. Furthermore, DNA ligase I participates in DNA base excision repair as a component of a multiprotein complex. Two forms of DNA ligase III are produced by an alternative splicing mechanism. The ubiqitously expressed DNA ligase III-α forms a complex with the DNA single-strand break repair protein XRCC1. In contrast, DNA ligase III-β, which does not interact with XRCC1, is only expressed in male meiotic germ cells, suggesting a role for this isoform in meiotic recombination. At present, there is very little information about the cellular functions of DNA ligase IV.     

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.     

DNA ligase III

DNA ligases are crucial for most DNA transactions, including DNA replication, repair, and recombination. Recently, DNA ligase III (Lig3) has been demonstrated to be crucial for cell survival due to its catalytic function in mitochondria. This review summarizes these recent results and reports on a hitherto unappreciated widespread phylogenetic presence of Lig3 in eukaryotes, including in some organisms before the divergence of metazoa. Analysis of these putative Lig3 homologs suggests that many of them are likely to be found in mitochondria and to be critical for mitochondrial function.     

Human DNA ligase IV and the ligase IV/XRCC4 complex: analysis of nick ligation fidelity

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.     

Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair.

DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage. Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9. Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.     

Structural and mechanistic conservation in DNA ligases

DNA ligases are enzymes required for the repair, replication and recombination of DNA. DNA ligases catalyse the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA. Despite their occurrence in all organisms, DNA ligases show a wide diversity of amino acid sequences, molecular sizes and properties. The enzymes fall into two groups based on their cofactor specificity, those requiring NAD⁺ for activity and those requiring ATP. The eukaryotic, viral and archael bacteria encoded enzymes all require ATP. NAD⁺-requiring DNA ligases have only been found in prokaryotic organisms. Recently, the crystal structures of a number of DNA ligases have been reported. It is the purpose of this review to summarise the current knowledge of the structure and catalytic mechanism of DNA ligases.     

DNA ligase 1 deficient plants display severe growth defects and delayed repair of both DNA single and double strand breaks

Background  

DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability.     

Two DNA-binding and nick recognition modules in human DNA ligase III

Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bind specifically to nicked DNA over intact duplex DNA. One of these modules comprises the zinc finger domain and DNA-binding domain, which function together as a single DNA binding unit. The catalytic core of ligase III is the second DNA nick-binding module. Both binding modules are required for ligation of blunt ended DNA substrates. Although the zinc finger increases the catalytic efficiency of nick ligation, it appears to occupy the same binding site as the DNA ligase III catalytic core. We present a jackknife model for ligase III that posits conformational changes during nick sensing and ligation to extend the versatility of the enzyme.     

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

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.     

Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.     

Interplay between DNA Polymerases and DNA Ligases: Influence on Substrate Channeling and the Fidelity of DNA Ligation

DNA ligases are a highly conserved group of nucleic acid enzymes that play an essential role in DNA repair, replication, and recombination. This review focuses on functional interaction between DNA polymerases and DNA ligases in the repair of single- and double-strand DNA breaks, and discusses the notion that the substrate channeling during DNA polymerase-mediated nucleotide insertion coupled to DNA ligation could be a mechanism to minimize the release of potentially mutagenic repair intermediates. Evidence suggesting that DNA ligases are essential for cell viability includes the fact that defects or insufficiency in DNA ligase are casually linked to genome instability. In the future, it may be possible to develop small molecule inhibitors of mammalian DNA ligases and/or their functional protein partners that potentiate the effects of chemotherapeutic compounds and improve cancer treatment outcomes.     

Biochemical and genetic analysis of the four DNA ligases of mycobacteria

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.     

Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase,an enzyme essential for DNA replication,repair, and recombination

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.     

Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4

Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.     

DNA ligase III: A spotty presence in eukaryotes,but an essential function where tested

DNA ligases are crucial for most DNA transactions, including DNA replication, repair and recombination. Recently, DNA ligase III (Lig3) has been demonstrated to be crucial for cell survival due to its catalytic function in mitochondria. This review summarizes these recent results and reports on a hitherto unappreciated widespread phylogenetic presence of Lig3 in eukaryotes, including in some organisms before the divergence of metazoa. Analysis of these putative Lig3 homologs suggests that many of them are likely to be found in mitochondria and to be critical for mitochondrial function.Key words: genetics, molecular biology, DNA ligases, genome integrity, DNA repair     

Biochemical characterization of two DNA ligases from Deinococcus radiodurans

Two genes encoding a NAD(+)-dependent DNA ligase (LigA) and an ATP-dependent DNA ligase (LigB) were identified in the genome of the extremely radioresistant bacterium, Deinococcus radiodurans (DR). The recombinant enzymes expressed in Escherichia coli, were purified to homogeneity and characterized. The optimal temperature and pH value of the two DNA ligases were 60 ( degrees )C and 7.0, respectively. Their optimal concentration of MgCl(2) was 5mM. Their half-lifes of heat inactivation at 100 ( degrees )C were about 3 min and 5 min, respectively. In addition, the results showed that DRLigB displayed higher activity than DRLigA at stick and blunt ended joining of DNA, indicating that DRLigB is a key DNA ligase of D. radiodurans in DNA recombination and double-strand break repair.     

The Arabidopsis AtLIG4 gene is required for the repair of DNA damage,but not for the integration of Agrobacterium T-DNA

The joining of breaks in the chromosomal DNA backbone by ligases in processes of replication, recombination and repair plays a crucial role in the maintenance of genomic stability. Four ATP-dependent ligases, designated DNA ligases I–IV, have been identified in higher eukaryotes, and each one has distinct functions. In mammals and yeast, DNA ligase IV is exclusively involved in the repair of DNA double-strand breaks by non-homologous end joining. Recently, an Arabidopsis thaliana orthologue of the yeast and mammalian DNA ligase IV gene was found and termed AtLIG4. Here we describe the isolation and functional characterisation of a plant line with a T-DNA insertion in the AtLIG4 gene. Plants homozygous for the T-DNA insertion did not display any growth or developmental defects and were fertile. However, mutant seedlings were hypersensitive to the DNA-damaging agents methyl methanesulfonate and X-rays, demonstrating that AtLIG4 is required for the repair of DNA damage. Recently, we showed that a yeast lig4 mutant is deficient in Agrobacterium T-DNA integration. However, using tumorigenesis and germline transformation assays, we found that the plant AtLIG4 mutant is not impaired in T-DNA integration. Thus, in contrast to yeast, DNA ligase IV is not required for T-DNA integration in plants.     

Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions.

Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick.