Eukaryotic DNA ligases

Recent studies on eukaryotic DNA ligases are briefly reviewed. The two distinguishable enzymes from mammalian cells, DNA ligase I and DNA ligase II, have been purified to homogeneity and characterized biochemically. Two distinct DNA ligases have also been identified in Drosophila melanogaster embryos. The genes encoding DNA ligases from Schizosaccharomyces pombe, Saccharomyces cerevisiae and vaccinia virus have been cloned and sequenced. These 3 proteins exhibit about 30% amino acid sequence identity; the 2 yeast enzymes share 53% amino acid sequence identity or conserved changes. Altered DNA ligase I activity has been found in cell lines from patients with Bloom's syndrome, although a causal link between the enzyme deficiency and the disease has not yet been proven.     

Vaccinia virus encodes a polypeptide with DNA ligase activity.

Vaccinia virus gene SalF 15R potentially encodes a polypeptide of 63 kD which shares 30% amino acid identity with S. pombe and S. cerevisiae DNA ligases. DNA ligase proteins can be identified by incubation with alpha-(32P)ATP, resulting in the formation of a covalent DNA ligase-AMP adduct, an intermediate in the enzyme reaction. A novel radio-labelled polypeptide of approximately 61 kD appears in extracts from vaccinia virus infected cells after incubation with alpha-(32P)ATP. This protein is present throughout infection and is a DNA ligase as the radioactivity is discharged in the presence of either DNA substrate or pyrophosphate. DNA ligase assays show an increase in enzyme activity in cell extracts after vaccinia virus infection. A rabbit antiserum, raised against a bacterial fusion protein of beta-galactosidase and a portion of SalF 15R, immune-precipitates polypeptides of 61 and 54 kD from extracts of vaccinia virus-infected cells. This antiserum also immune-precipitates the novel DNA ligase-AMP adduct, thus proving that the observed DNA ligase is encoded by SalF 15R.     

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.     

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.     

An engineered mutant of vaccinia virus DNA topoisomerase I is sensitive to the anti-cancer drug camptothecin.

Although highly homologous to the other eukaryotic type I DNA topoisomerases, vaccinia virus DNA topoisomerase I is distinct in its resistance to the anti-cancer drug camptothecin. After comparison of available sequences of sensitive and resistant type I topoisomerases, the aspartic acid at position 221 of vaccinia virus topoisomerase I is mutated to a valine. The resulting mutant protein is partially active. In contrast to the wild type enzyme, the relaxation of supercoiled DNA is inhibited by camptothecin. Its cleavage reaction with DNA is enhanced by camptothecin due to inhibition of religation of DNA. This demonstrates that even though the size of vaccinia virus is only about one-third that of the other camptothecin-sensitive topoisomerases, it has a potential interaction site for camptothecin.     

Properties of DNA ligase from uninfected and virus-infected HeLa cells.

HeLa cells contain a high M.W. form of DNA ligase which can be completely converted to a low M.W. form. Stokes radius, frictional ratio, sedimentation coefficient, molecular weight, pH dependence, and heat inactivation rate of the two forms have been studied. The major properties of the two forms of DNA ligase in HeLa cells (in particular molecular weights and pH dependence) resemble those of the "dimer" and "monomer" structures described in cultured human cells (Pedrali, G., Spadari, S., Ciarrocchi, G., Pedrini, M., Falaschi, A. (1973) Eur. J. Biochem., 39 343) .In synchronized HeLa cells, the DNA ligase shows a two fold increase during S phase and parallels the increase in the DNA synthesis rate. DNA ligase increases in parallel with viral DNA synthesis after infection of HeLa cells with vaccinia and Herpes virus but its cofactor requirements and physical properties (including the dimer leads to monomer conversion) are unchanged, suggesting that the newly formed ligase is not virus-coded.     

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.     

Purification and characterization of DNA ligase I from the trypanosomatid Crithidia fasciculata.

A DNA ligase has been purified approximately 5000-fold, to near homogeneity, from the trypanosomatid Crithidia fasciculata. The purified enzyme contains polypeptides with molecular masses of 84 and 80 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both polypeptides formed enzyme-adenylate complexes in the absence of DNA, contained an epitope that is highly conserved between human and bovine DNA ligase I and yeast and vaccinia virus DNA ligases, and were identified in fresh lysates of C. fasciculata by antibodies raised against the purified protein. Hydrodynamic measurements indicate that the enzyme is an asymmetric protein of approximately 80 kDa. The purified DNA ligase can join oligo(dT) annealed to poly(dA), but not oligo(dT) annealed to poly(rA), and can ligate blunt-ended DNA fragments. The enzyme has a low Km for ATP of 0.3 microM. The DNA ligase absolutely requires ATP and Mg2+, and is inhibited by N-ethylmaleimide and by KCI. Substrate specificity, Km for ATP, and the conserved epitope all suggest that the purified enzyme is the trypanosome homologue of DNA ligase I.     

Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line.

The vaccinia virus D4R open reading frame, encoding the essential DNA repair enzyme uracil DNA glycosylase, was expressed in two permanent cell lines, the rabbit kidney cell line RK13 and the human fibroblast cell line 293. The temperature-sensitive vaccinia virus mutant ts4149, which maps within D4R, was able to grow under restrictive conditions in both of these transformed cell lines. Cell clones complemented D4R function to various degrees, demonstrating complementation of an essential vaccinia virus gene by a cell line constitutively expressing the essential function. Thus, the complementing host cells allowed the rescue of a virus defective in the D4R gene, demonstrating that this system may be used for the propagation of defective cytoplasmic DNA viruses. The defective virus grew to high yields only in the engineered cell lines. The data support the hypothesis that early gene products, such as uracil DNA glycosylase, supplied in trans can fully complement essential viral functions.     

Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I.

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.     

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.     

A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.

Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.     

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.     

Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli

The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter. Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme. A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria. DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc. Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA. The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.     

Polynucleotide Ligase Activity in Cells Infected with Simian Virus 40, Polyoma Virus, or Vaccinia Virus

The conversion of simian virus 40 (SV40) component II deoxyribonucleic acid to component I has been used to assay polynucleotide ligase in extracts of tissue culture cells. All cell types examined, including chicken, hamster, mouse, monkey, and human cells, contained adenosine triphosphate-dependent ligase. After infection of mouse embryo, monkey kidney, and HeLa cells with polyoma virus, SV40, and vaccinia virus, respectively, the enzyme activity increased, but its cofactor requirement was unchanged. In vaccinia virus-infected cells, the increased activity was localized in the cytoplasm. Ligase induction occurred in the presence of cytosine arabinoside but was prevented by puromycin. Rifampicin blocked the production of infectious vaccinia particles but had little effect on the induction of ligase.     

DNA strand exchange catalyzed by proteins from vaccinia virus-infected cells.

Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex. The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product. Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps. The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa. The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+. One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing. As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors. Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail. The recovery of these structures shows that the enzyme catalyzes true strand exchange. There is also a unique polarity to the strand exchange reaction. The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand. Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product. Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.     

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.     

Domain structure of vaccinia DNA ligase.

The 552 amino acid vaccinia virus DNA ligase consists of three structural domains defined by partial proteolysis: (i) an amino-terminal 175 amino acid segment that is susceptible to digestion with chymotrypsin and trypsin; (ii) a protease-resistant central domain that contains the active site of nucleotidyl transfer (Lys-231); (iii) a protease-resistant carboxyl domain. The two protease-resistant domains are separated by a protease-sensitive interdomain bridge from positions 296 to 307. Adenylyltransferase and DNA ligation activities are preserved when the N-terminal 200 amino acids are deleted. However, the truncated form of vaccinia ligase has a reduced catalytic rate in strand joining and a lower affinity for DNA than does the full-sized enzyme. The 350 amino acid catalytic core of the vaccinia ligase is similar in size and protease-sensitivity to the full-length bacteriophage T7 DNA ligase.     

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.