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.     

The DNA ligase III zinc finger stimulates binding to DNA secondary structure and promotes end joining

The ability to rejoin broken chromosomes is fundamental to the maintenance of genetic integrity. Mammalian cells possess at least five DNA ligases, including three isoforms of DNA ligase III (Lig-3). Lig-3 proteins differ from other DNA ligases in the presence of an N-terminal zinc finger (Zn-f) motif that exhibits extensive homology with two zinc fingers in poly(ADP-ribose) polymerase (PARP). Here we report that the Zn-f confers upon Lig-3 the ability to bind DNA duplexes harbouring a variety of DNA secondary structures, including single-strand gaps and single-strand flaps. Moreover, the Zn-f stimulates intermolecular end joining of duplexes that harbour these structures up to 16-fold. The Zn-f also stimulates end joining between duplexes lacking secondary structure, but to a lesser extent (up to 4-fold). We conclude that the Zn-f may enable Lig-3 to rejoin chromosomal DNA strand breaks located at sites of clustered damage induced by ionising radiation or near to secondary structure intermediates of DNA metabolism.     

Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.     

Role of the DNA ligase III zinc finger in polynucleotide binding and ligation.

Mammalian DNA ligase III exists as two distinct isoforms denoted alpha and beta. Both forms possess a motif that is homologous to the putative zinc finger present in poly(ADP-ribose) polymerase. Here, the role of this motif in the binding and ligation of nicked DNA and RNA substrates in vitro has been examined in both isoforms. Disruption of the putative zinc finger did not affect DNA ligase III activity on nicked DNA duplex, nor did it abolish DNA ligase III-alpha activity during DNA base excision repair in a cell-free assay. In contrast, disruption of this motif reduced 3-fold the activity of both DNA ligase III isoforms on nicked RNA present in RNA/DNA homopolymers. Furthermore, whereas disruption of the motif did not prevent binding of DNA ligase III to nicked DNA duplex, binding to nicked RNA homopolymers was reduced approximately 10-fold. These results suggest that the putative zinc finger does not stimulate DNA ligase III activity on simple nicked DNA substrates, but indicate that this motif can target the binding and activity of DNA ligase III to nicked RNA homopolymer. The implications of these results to the cellular role of the putative zinc finger are discussed.     

A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability

The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.     

XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly (ADP-ribose) polymerase, and DNA ligase III is a novel molecular 'nick-sensor' in vitro.

The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-beta in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multiprotein complex.     

The closed structure of an archaeal DNA ligase from Pyrococcus furiosus

DNA ligases join single-strand breaks in double-stranded DNA, and are essential to maintain genome integrity in DNA metabolism. Here, we report the 1.8 A resolution structure of Pyrococcus furiosus DNA ligase (PfuLig), which represents the first full-length atomic view of an ATP-dependent eukaryotic-type DNA ligase. The enzyme comprises the N-terminal DNA-binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes. The closed conformation of the two "catalytic core" domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a non-covalently bound AMP molecule. This domain rearrangement results from the "domain-connecting" role of the helical extension conserved at the C termini in archaeal and eukaryotic DNA ligases. The DNA substrate in the human open-ligase is replaced by motif VI in the Pfu closed-ligase. Both the shapes and electrostatic distributions are similar between motif VI and the DNA substrate, suggesting that motif VI in the closed state mimics the incoming substrate DNA. Two basic residues (R531 and K534) in motif VI reside within the active site pocket and interact with the phosphate group of the bound AMP. The crystallographic and functional analyses of mutant enzymes revealed that these two residues within the RxDK sequence play essential and complementary roles in ATP processing. This sequence is also conserved exclusively among the covalent nucleotidyltransferases, even including mRNA-capping enzymes with similar helical extensions at the C termini.     

Conversion of a DNA ligase into an RNA capping enzyme.

In eukaryotes, newly synthesised mRNA is 'capped' by the addition of GMP to the 5" end by RNA capping enzymes. Recent structural studies have shown that RNA capping enzymes and DNA ligases have similar protein folds, suggesting a conserved catalytic mechanism. To explore these similarities we have produced a chimeric enzyme comprising the N-terminal domain 1 of a DNA ligase fused to the C-terminal domain 2 of a mRNA capping enzyme. This report shows that this hybrid enzyme retains adenylation activity, characteristic of DNA ligases but, remarkably, the chimera has ATP-dependent mRNA capping activity. This is the first observation of ATP-dependent RNA capping. These results suggest that nucleotidyltransferases may have evolved from a common ancestral gene.     

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.     

Structure and mechanism of RNA ligase

T4 RNA ligase 2 (Rnl2) exemplifies an RNA ligase family that includes the RNA editing ligases (RELs) of Trypanosoma and Leishmania. The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3'-OH and 5'-PO4 RNA ends by Rnl2, but not for ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA ligase domain. We report the 1.9 A crystal structure of the ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA ligases, DNA ligases, and mRNA capping enzymes.     

Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins

Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21(CIP1/WAF1), xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.     

Structure of the adenylation domain of an NAD+-dependent DNA ligase.

BACKGROUND: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD+4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target. RESULTS: The crystal structure of the adenylation domain of the NAD+-dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 A resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD+-binding site. CONCLUSIONS: Comparison of the structure of the NAD+4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.     

Mammalian DNA ligases. Catalytic domain and size of DNA ligase I.

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.     

Structure-function analysis of yeast tRNA ligase

Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.     

DNA ligase I mediates essential functions in mammalian cells.

DNA replication, repair, and recombination are essential processes in mammalian cells. Hence, the application of gene targeting to the study of these DNA metabolic pathways requires the creation of nonnull mutations. We have developed a method for introducing partially defective mutants in murine embryonic stem cells that circumvents the problem of cellular lethality of targeted mutations at essential loci. Using this approach, we have determined that mammalian DNA ligase I is essential for cell viability. Thus, DNA ligases II and III are not redundant with DNA ligase I for the function(s) associated with cell proliferation. Partial complementation of the lethal DNA ligase I null mutation allowed the creation of deficient embryonic stem cell lines. We found that a wild-type DNA ligase I cDNA, as well as a variant DNA ligase I cDNA, was able to rescue the lethality of the homozygous null mutation, whereas an N-terminal deletion mutant consisting of the minimal DNA ligase I catalytic domain was not. This observation demonstrates that sequences outside the DNA ligase I catalytic domain are essential for DNA ligase I function in vivo.     

A DNA ligase from the psychrophile Pseudoalteromonas haloplanktis gives insights into the adaptation of proteins to low temperatures.

The cloning, overexpression and characterization of a cold-adapted DNA ligase from the Antarctic sea water bacterium Pseudoalteromonas haloplanktis are described. Protein sequence analysis revealed that the cold-adapted Ph DNA ligase shows a significant level of sequence similarity to other NAD+-dependent DNA ligases and contains several previously described sequence motifs. Also, a decreased level of arginine and proline residues in Ph DNA ligase could be involved in the cold-adaptation strategy. Moreover, 3D modelling of the N-terminal domain of Ph DNA ligase clearly indicates that this domain is destabilized compared with its thermophilic homologue. The recombinant Ph DNA ligase was overexpressed in Escherichia coli and purified to homogeneity. Mass spectroscopy experiments indicated that the purified enzyme is mainly in an adenylated form with a molecular mass of 74 593 Da. Ph DNA ligase shows similar overall catalytic properties to other NAD+-dependent DNA ligases but is a cold-adapted enzyme as its catalytic efficiency (kcat/Km) at low and moderate temperatures is higher than that of its mesophilic counterpart E. coli DNA ligase. A kinetic comparison of three enzymes adapted to different temperatures (P. haloplanktis, E. coli and Thermus scotoductus DNA ligases) indicated that an increased kcat is the most important adaptive parameter for enzymatic activity at low temperatures, whereas a decreased Km for the nicked DNA substrate seems to allow T. scotoductus DNA ligase to work efficiently at high temperatures. Besides being useful for investigation of the adaptation of enzymes to extreme temperatures, P. haloplanktis DNA ligase, which is very efficient at low temperatures, offers a novel tool for biotechnology.     

Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI.

A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.     

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.     

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.