Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Thermodynamics of human DNA ligase I trimerization and association with DNA polymerase beta

The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites for self-association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol beta interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol beta did not. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain.     

Specificity and fidelity of strand joining by Chlorella virus DNA ligase

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.     

Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.     

Characterization of a baculovirus-encoded ATP-dependent DNA ligase

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [alpha-32P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) . poly(dT)12-18, or poly(rA) . poly(dT)12-18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.     

Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.     

Mammalian abasic site base excision repair. Identification of the reaction sequence and rate-determining steps

Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5'-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase beta (beta-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP endonuclease, beta-pol, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5' "nicking" of the AP site-containing DNA strand by AP endonuclease, beta-pol performs DNA synthesis prior to removal of the 5'-dRP moiety in the gap. Removal of the dRP flap is strictly required for DNA ligase I to seal the resulting nick. Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system. Instead, the rate-limiting step in the reconstituted system was found to be removal of dRP (i.e. dRP lyase), catalyzed by the amino-terminal domain of beta-pol. This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed.     

Role of a BRCT domain in the interaction of DNA ligase III-alpha with the DNA repair protein XRCC1

The BRCT domain (for BRCA1 carboxyl terminus) is a protein motif of unknown function, comprising approximately 100 amino acids in five conserved blocks denoted A-E. BRCT domains are present in the tumour suppressor protein BRCA1 [1-3], and the domain is found in over 40 other proteins, defining a superfamily that includes DNA ligase III-alpha and the essential human DNA repair protein XRCC1. DNA ligase III-alpha and XRCC1 interact via their carboxyl termini, close to or within regions that contain a BRCT domain [4]. To examine whether the primary role of the carboxy-terminal BRCT domain of XRCC1 (denoted BRCT II) is to mediate the interaction with DNA ligase III-alpha, we identified the regions of the domain that are required and sufficient for the interaction. An XRCC1 protein in which the conserved D-block tryptophan was disrupted by point mutation retained the ability to interact with DNA ligase III-alpha, so this tryptophan must mediate a different, although conserved, role. XRCC1 in which the weakly conserved C-block was mutated lost the ability to interact with DNA ligase III-alpha. Moreover, 20 amino acids spanning the C-block of BRCT II conferred full DNA ligase III-alpha binding activity upon an unrelated polypeptide. An XRCC1 protein in which this 20mer was deleted could not maintain normal levels of DNA ligase III-alpha in transfected rodent cells, a phenotype associated with defective repair [5]. In summary, these data demonstrate that a BRCT domain can mediate a biologically important protein-protein interaction, and support the existence of additional roles.     

A catalytic domain of eukaryotic DNA topoisomerase I

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.     

Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.     

In vitro activity of a new oral triazole, BMS-207147 (ER-30346)

One viral strand of phi Lf, a filamentous phage of Xanthomonas campestris pv.campestris, the open reading frame (ORF440) behind gene VI was identified as gene I. This gene codes for pI protein (440 aa, 48 kDa) which was shown to be membrane-bound in the phi Lf-infected host cell by Western blot analysis using the antibody raised against the protein expressed in Escherichia coli. Its predicted amino acid sequence has a nucleotide-binding motif in the N-terminal 97 aa and a membrane-spanning domain (aa 221 to 236). These structural features are characteristic of pIs of several filamentous phages which are transmembrane proteins required for phage assembly. Thus far, nine phi Lf genes have been identified which are organized in the order GII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI, similar to the genome organization of E. coli filamentous phages.     

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.     

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.     

Biochemical characterization of a protein inhibitor for DNA ligase I from human cells. Regulation/replication/repair/recombination

An inhibitor for DNA ligase I has recently been purified from human cells. This inhibitor of 55-75 kDa forms a reversible complex with DNA ligase I, but has no effect on DNA ligase II and T4 DNA ligase, suggesting that it may play a regulatory role for DNA replication and repair. This report shows that the inhibitor was sensitive to heating at 52 degrees C and to trypsin treatment, indicating that it is a heat-labile protein. The inhibitor affected the ligation of double- and single-strand breaks in natural and synthetic DNA, but had no effect on the formation of the ligase-AMP complex and on the subsequent reaction following the formation of the AMP-DNA complex. These data indicate that the major mechanism of action for the inhibitor is the blocking of the second step of the reaction, in which the AMP moiety is transferred from the ligase-AMP to DNA. The site of interaction for the enzyme is therefore localized in a domain associated with the DNA binding or the AMP-transferring function.     

Thioredoxin from Streptomyces aureofaciens controls coiling of plasmid DNA

A number of potential functions of thioredoxin have been proposed in literature, including a role for DNA replication. The aim of our study was to investigate the effects of thioredoxin from Streptomyces aureofaciens (Trx S.a.) on plasmid DNA. Trx S.a. was incubated with plasmid forms and the incubation product(s) characterized on agarose gels. To compare Trx activity with enzymes with known DNA modifying activities, topoisomerase I, II (gyrase) and T4 DNA ligase were incubated with plasmid DNA in parallel. For the demonstration of nick removal a PCR technique was used. Trx S.a. bound non-specifically to plasmid DNA relaxing supercoiled circle closed form (CCC form) with subsequent formation of the circle closed form (CC form) as a major product. The amplification of a specific DNA template, possible only after nick removal, took place following incubation with Trx. The effect of topoisomerase I on plasmid DNA resembled Trx S.a. activity. We propose the following mechanism for CCC relaxation: Binding of Trx leads to a break of one strand and CC is formed by stepwise relaxation, ending with nick removal. The concomitant finding of open circle form (OC form) generation after incubation with Trx may indicate the generation of an intermediate due to the postulated strand break at initiation. This control of coiling may play a role in the DNA replication machinery, providing CC as a readily available substrate for DNA polymerases. In addition, Trx may serve in DNA repair mechanisms by its nonspecific binding to DNA and nick removing activity.     

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.     

Activation of nuclear factor kappa B inflammatory bowel disease

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.     

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.