Polyoma virus minichromosomes: associated enzyme activities.

Polyoma minichromosomes were isolated and fractionated on glycerol gradients as described by Gourlie et al. (J. Virol. 38:805-814, 1981). Specific assays for DNa polymerases alpha, beta, and gamma, DNA topoisomerase I, and RNase H were carried out on each fraction. The number of units of activity in each fraction was compared with the number of total polyoma and replicative intermediate DNA molecules in each fraction determined by quantitative electron microscopy (M. R. Krauss and R. M. Benbow, J. Virol. 38:815-825, 1981). DNA polymerase alpha cosedimented with polyoma replicative intermediate DNA molecules. DNA polymerase beta and DNA topoisomerase I activities sedimented with mature polyoma minichromosomes. Although the bulk of RNase H activity sedimented in the minichromosome region, the peak of activity was found one fraction behind the peak of mature minichromosomes. Virtually no DNA polymerase gamma activity cosedimented with polyoma minichromosomes.     

Polyoma virus minichromosomes: associated DNA molecules

Electron microscopy was used to identify and quantitate DNA molecules associated with 3H-labeled polyoma minichromosomes which had been fractionated on a sucrose gradient. The percentage of replicating DNA molecules observed in the fractions of the gradient normally designated the replicative intermediate region was up to ninefold higher than in fractions from the mature region. Nevertheless, because of the higher overall concentration of polyoma DNA molecules in the mature region, nearly as many replicating DNA molecules were computed to be in the mature region as in the replicative intermediate region. The replicating molecules in the mature region was predominantly early replicative intermediates. Almost all late replicative intermediates were found in the replicative intermediate region. Under aqueous spreading conditions, a substantial fraction of the replicating DNA structures appeared to be asymmetrical or otherwise unusual, suggesting that extensive single-stranded regions may exist in replicating polyoma minichromosomes.     

Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.     

Effects of VM26, a specific inhibitor of type II DNA topoisomerase, on SV40 chromatin replication in vitro.

We have examined the influence of VM26 (teniposide), a specific inhibitor of mammalian type II DNA topoisomerase, on the replication of SV40 minichromosomes in vitro. The replication system we used consists of replicative intermediate SV40 chromatin as substrate which is converted to mature SV40 chromatin in the presence of ATP, deoxynucleotides and a protein extract from uninfected cells. The addition of 100 microM VM26 to this system reduces DNA synthesis to 70 to 80 percent of the control and leads to an accumulation of 'late replicative intermediates'. The VM26 induced block of replication was not released by the addition of large quantities of type I DNA topoisomerase. We conclude, that type II DNA topoisomerase is essential for the final replication steps leading from late Cairns structures of replicative intermediates to monomeric minichromosomes. It appears that type I DNA topoisomerase can function as a swivelase during most of the replicative elongation phase, but must later be replaced by type II DNA topoisomerase.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

DNAaseI-hypersensitive minichromosomes of SV40 possess an elastic torsional strain in DNA.

Previously, we have shown that DNA in a small fraction (2-5%) of SV40 minichromosomes was torsionally strained and could be relaxed by treating minichromosomes with topoisomerase I. This fraction was enriched with endogeneous RNA polymerase II (Luchnik et al., 1982, EMBO J., 1, 1353). Here we show that one and the same fraction of SV40 minichromosomes is hypersensitive to DNAase I and is relaxable by topoisomerase I. Moreover, this fraction completely loses its hypersensitivity to DNAase I upon relaxation. The possibility that this fraction of minichromosomes can be represented by naked DNA is ruled out by the results of studying the kinetics of minichromosome digestion by DNAase I in comparison to digestion of pure SV40 DNA and by measuring the buoyant density of SV40 chromatin in equilibrium CsCl gradient. Our data obtained with SV40 minichromosomes may be relevant to the mechanism responsible for DNAase I hypersensitivity in the loops or domains of cellular chromatin.     

DNA ligases from rat liver. Purification and partial characterization of two molecular forms

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.     

Evidence for participation of a multiprotein complex in yeast DNA replication in vitro

Fractions containing a high molecular weight form (Mr approximately equal to 2 X 10(6] of the activity that replicates in vitro both the 2-micron yeast DNA plasmid and the chromosomal autonomously replicating sequence ars 1 can be prepared from cells of the budding yeast Saccharomyces. Protein complexes from the fractions associate in vitro with the replication origins of these DNA elements, as determined by electron microscopy. In the present study, the high molecular weight replicative fraction has been characterized in further detail. The DNA synthetic activity in the high molecular weight fraction was bound to the DNA and could be isolated with it. This binding of the replicating activity to the DNA was greatly reduced in the absence of the 2-micron origins of replication. Association of the protein complexes with DNA depended on the amount of replicating activity added, was sensitive to 0.2 M KCl, and exhibited a requirement for rATP and deoxyribonucleoside triphosphates. It was not blocked, however, by the DNA polymerase inhibitor aphidicolin or by the RNA polymerase inhibitor alpha-amanitin. The lack of inhibition by aphidicolin suggests that the deoxyribonucleoside triphosphates may function as cofactors in the binding of protein complexes to DNA or as substrates for a polymerizing activity such as a primase. Binding of the protein complexes as well as actual DNA replication were heat sensitive in the high molecular weight fraction prepared from the temperature-sensitive mutant of the cell division cycle cdc 8. This suggests that the cdc 8 gene product is present in a replicative protein complex and strengthens the conclusion that the presence of the protein complexes on the DNA is associated with replication. Using independent enzyme assays, several other possible replication proteins (including DNA polymerase I, DNA ligase, DNA primase, and DNA topoisomerase II) have been identified directly in the high molecular weight replicative fraction. All of these results provide support for the idea that a protein complex (or replisome ) is involved in the replication of both the extrachromosomal 2-micron DNA and chromosomal DNA in yeast.     

Polyoma virus minichromosomes: characterization of the products of in vitro DNA synthesis.

A two-dimensional (neutral-alkali) agarose gel electrophoretic system was used to separate three families of replicative intermediate (RI) polyoma virus DNA molecules (form I, form II, and form III RIs). Two of these families, form II and III RIs, are the result of artifactual nicking of one of the parental strands of supercoiled RIs (form I RIs) during in vitro replication of soluble minichromosomes. Kinetic studies in vitro showed that the nicked RIs serve as templates for limited DNA synthesis. The nicked species are not converted into normal products, however. The nicking reaction, which appears to be specific for the parental strands, is dependent on magnesium ions and occurs concurrently with the in vitro synthesis of DNA.     

Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity.

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.     

DNA-dependent adenosinetriphosphatase C1 from mouse FM3A cells has DNA helicase activity.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).     

Purification and properties of two DNA ligases from human placenta

Two DNA ligase activities have been separated, purified, and characterized. The resolution of the two enzymes from crude extracts was initially achieved through Polymin P precipitation. The ligation activity precipitating with the nucleic acids on treatment with Polymin P is designated as DNA ligase I, and an activity remaining in the supernatant fraction, as DNA ligase II. DNA ligase I and II are ATP and Mg2+-dependent enzymes with pH optima of 7.8 and 8.0 and isoelectric points of 6.9 and 7.6, respectively. The purified I and II DNA ligase activities have molecular weights of 83,000 and 89,000, respectively. Both activities are inhibited by dATP and inorganic pyrophosphate. However, in the presence of optimum rATP levels, dATP stimulates DNA ligase II activity, whereas DNA ligase I is inhibited under the same conditions. Both activities are DNA specific and ligation follows reaction steps similar to those described for the Escherichia coli DNA ligase.     

Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA.     

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.     

Mammalian DNA ligases. Serological evidence for two separate enzymes.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.     

Efficient resolution of replicated poxvirus telomeres to native hairpin structures requires two inverted symmetrical copies of a core target DNA sequence.

The terminal hairpin sequences of the linear double-stranded DNA genome of the leporipoxvirus Shope fibroma virus (SFV) has been cloned in Saccharomyces cerevisiae and in recombination-deficient Escherichia coli as a palindromic insert within circular plasmid vectors. This sequence configuration is equivalent to the inverted repeat structure detected as a telomeric replicative intermediate during poxvirus replication in vivo. Previously, it has been shown that when circular plasmids containing this palindromic insert were transfected into SFV-infected cells, efficient replication and resolution generated linear minichromosomes with bona fide viral hairpin termini (A. M. DeLange, M. Reddy, D. Scraba, C. Upton, and G. McFadden, J. Virol. 59:249-259, 1986). To localize the minimal target DNA sequence required for efficient resolution, a series of staggered unidirectional deletions were constructed at both ends of the inverted repeat. Analyses of the resolution efficiencies of the various clones indicate that up to 240 base pairs (bp) centered at the symmetry axis were required for maximal resolution to minichromosomes. To investigate the role of the AT-rich central axis sequences, which in SFV include 8 nonpalindromic bp, a unique AflII site at the symmetry axis was exploited. Bidirectional deletions extending from this AflII site and insertions of synthetic oligonucleotides into one of the deletion derivatives were constructed and tested in vivo. The efficiency with which these plasmids resolved to linear minichromosomes with hairpin termini has enabled us to define the minimal target DNA sequence as two inverted copies of an identical DNA sequence between 58 and 76 bp in length. The nonpalindromic nucleotides, which, after resolution, constitute the extrahelical residues characteristic of native poxviral telomeres, were not required for resolution. The close resemblance of the SFV core target sequence to the analogous region from the orthopoxvirus vaccinia virus is consistent with a conserved mechanism for poxviral telomere resolution.     

In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.

Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate.     

DNA synthesis in nucleotide-permeable Escherichia coli cells. VII. Conversion of phi chi-174 DNA to its replicative form

On incubation with deoxynucleoside triphosphates and rATP, ether-treated (nucleotide-permeable) cells convert the single-stranded DNA of adsorbed bacteriophage φX174 particles to the double-stranded replicative forms. The main final product is the doubly-closed replicative form, RFI; a minor product is the relaxed form II. Interruptions in the nascent complementary strand of the viral DNA result in pieces corresponding to 5 to 10% of the unit length of the viral DNA. Pieces of similar size were previously seen in studies of the replication synthesis of Escherichia, coli DNA in ether-treated cells. Since the conversion of the single-stranded φX174 DNA to replicative form is known to be mediated entirely by host factors, it is argued that the viral single strands are replicated by macromolecular factors involed in the replication of E. coli DNA and that this is the reason why new φX174 DNA appears in short pieces. Possible consequences of this interpretation for an understanding of duplex replication are discussed. The joining of the short pieces of complementary φX174 DNA is inhibited at low deoxynucleoside triphosphate concentration (1 μM) but not by nicotinamide mononucleotide, which inhibits the NAD-dependent DNA ligase and blocks the conversion of RFII to RFI in ether-treated cells. The results are discussed with respect to previous studies on cell-DNA synthesis (Geider, 1972). It is argued that there are two polynucleotide joining mechanisms, of which only one requires NAD-dependent ligase action.