Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1.

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

Plasmid containing a DNA ligase gene from Haemophilus influenzae

A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2 . Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant.     

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid Mth ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K_m 1.1 µM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD⁺ cannot. Mth ligase activity is thermophilic in vitro, with optimal nick-joining at 60°C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase–adenylate intermediate (step 1) and hence cannot seal a 3′-OH/5′-PO₄ nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase–adenylate formation, but defective in activating the nick via formation of the DNA–adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA–adenylate, could be elicited for the wild-type Mth ligase by inclusion of calcium as the divalent cation cofactor. Mth ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that Mth ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.     

Uptake of heterologous DNA by Haemophilus influenzae.

With the use of highly competent Haemophilus influenzae cells, it was possible to demonstrate the uptake of heterologous DNAs. However, these DNAs, as expected, were only 1% or less as effective when competing for uptake with Haemophilus DNA. Escherichia coli DNA was removed from solution by competent cells to the extent expected if all the E. coli DNA particles contained at least one uptake recognition signal. The data were consistent with a model in which there was one uptake signal per 20 X 10(6) to 30 X 10(6) daltons of E. coli DNA. Since H. influenzae DNA has many more recognition signals, approximately one per 2 X 10(6) daltons (Danner et al., Gene 77:311-318, 1980; K. Vogt and S. H. Goodgal, submitted for publication), it has been suggested that the slower rate of E. coli DNA binding and the so-called specificity of Haemophilus DNA binding are due to the number of recognition signals per molecule of DNA as well as the nature of the DNA receptor (Vogt and Goodgal, submitted for publication). The specificity of native H. influenzae DNA binding does not apply to the uptake of denatured DNA in the transforming system (low pH) for denatured DNA.     

Bleomycin-induced DNA repair by Saccharomyces cerevisiae ATP-dependent polydeoxyribonucleotide ligase.

In contrast to ligase-deficient (cdc9) Saccharomyces cerevisiae, which did not rejoin bleomycin-induced DNA breaks, ligase-proficient (CDC9) yeast cells eliminated approximately 90% of DNA breaks within 90 to 120 min after treatment. Experimental conditions restricted enzymatic removal of the unusual 3'-phosphoglycolate termini in DNA cleaved by bleomycin and involved doses producing equivalent numbers of DNA breaks or doses producing equivalent killing.     

Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.     

Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii

Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes auto-adenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90 degrees C and nick-joining activity at 70 to 90 degrees C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.     

Bacteriophage N3 of Haemophilus influenzae. IV. Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae cells by N3 phage or transfection by DNA from phage were examined. After infection by whole phage three forms of intracellular phage DNA were observed by sedimentation velocity analysis. These forms are probably twisted circles, open circles and linear duplexes. In transfection only about 15% of the phage DNA is efficiently taken up by the competent cells. After entry of phage DNA into wild-type cells in transfection the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. These presumably concatamer forms are generated by recombination. In strain rec-1 the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in the wild-type cells. Since rec-1 is transfected with much lower efficiency than the wild-type our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for efficient transfection. These results also show that although phage N3 codes for its own recombination system it cannot operate in the early stages of transfection and succesful transfection is entirely dependent upon the host recombination system.     

NAD+-dependent DNA ligase encoded by a eukaryotic virus.

We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+). Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+). Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate. We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).     

Mechanism of DNA degradation by the ATP-dependent DNase from Hemophilus influenzae Rd.

The rate of production of acid-soluble material during degradation of duplex DNA by Hemophilus influenzae ATP-dependent DNAse (Hind exonuclease V) has been shown to be directly dependent upon the Mg2+ concentration in the reaction mixture. At high concentrations of Mg2+ (5 to 20 mM), DNA degradation to acid-soluble products is rapid and the rate of ATP hydrolysis is slightly depressed. At low concentrations of Mg2+ (0.1 to 0.5 mM), the enzyme rapidly hydrolyzes ATP and converts up to 35% of linear duplex DNA to single-stranded material while degrading less than 0.2% of the DNA to acid-soluble products. We refer to this enzymatic production of single-stranded DNA as the "melting" activity. Under the conditions of our assay, the initial melting reaction is processive, lasting about 70s on phage T7 DNA. Using DNAs with several different lengths, we have established that the duration of the initial reaction is dependent upon DNA length, requiring approximately 1 s per 0.18 mum. The products of the initial reaction on phage T7 DNA are somewhat heterogeneous, consisting of short duplex fragments approximately 0.5 mum long, purely single-stranded products up to 7 mum long, and longer duplex fragments 3 to 11 mum in length, some of which have single-stranded tails. Nearly half of the single-stranded material remains linked to a duplex segment of DNA after the inital processive reaction. We propose that Hind exo V initiates attack at the DNA termini and then acts in a processive manner, migrating along the DNA molecule, converting some regions to single-stranded material by the combined action of the melting activity and limited phosphodiester cleavage, while leaving other regions double-stranded. At the completion of its processive movement through a single DNA molecule, it is released and then recycles onto either intact molecules or the partially degraded products, continuing in this manner until the DNA is finally reduced to oligonucleotides.     

Characterization of an ATP-dependent DNA strand transferase from human cells.

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.     

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.     

Cleavage of bacteriophage M13 DNA by Haemophilus influenzae endonuclease-R

The restriction enzyme from Haemophilus influenzae, endonuclease-R, has only one cleavage site on the double-stranded replicative form DNA of bacteriophage M13. Circular replicative forms are broken to yield full-length liniar M13-DNA molecules (RF-III). The cleavage site appears to be specific as the RF-III molecules, produced by endonuclease-R, cannot be circularized by denaturation and renaturation.     

Host specificity of DNA in Haemophilus influenzae: DNA restriction enzyme from H. influenzae Rf232.

A restriction endonuclease has been partially purified from Haemophilus influenzae Rf232 containing the genetically determined system of restriction and modification of DNA. The enzyme requires ATP for the degradation of transfecting phage DNA.     

Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules.

The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.