Properties of herpes simplex virus type 1 and type 2 DNA polymerase

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.     

Phylogenomics of type II DNA topoisomerases

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.     

Stimulation of cellular DNA synthesis by wild type and mutant bovine papillomavirus DNA

Microinjection of recombinant plasmids containing bovine papillomavirus type 1 DNA into the nuclei of mouse C127 cells results in the stimulation of cellular DNA synthesis. Mutations in the viral E2 gene have no apparent effect on this activity even though the same mutations prevent efficient C127 cell focus formation and inhibit transactivation by this gene.     

DNA damage and repair in type 2 diabetes mellitus

DNA damage may be associated with type 2 diabetes mellitus (T2DM) and its complications mainly through oxidative stress. Little is known about DNA repair disturbances potentially contributing to the overall extent of DNA damage in T2DM, which, in turn, may be linked with genomic instability resulting in cancer. To assess whether DNA repair may be perturbed in 2DM we determined: (1) the level of endogenous basal DNA damage, this means damage recognized in the alkaline comet assay (DNA strand breaks and alkali labile sites) as well as endogenous oxidative and alkylative DNA damage (2) the sensitivity to DNA-damaging agents hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of T2DM patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair was evaluated by the alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. The levels of basal endogenous and oxidative DNA damage in diabetes patients were higher than in control subjects. There was no difference between the level of alkylative DNA in the patients and the controls. Diabetes patients displayed higher susceptibility to hydrogen peroxide and doxorubicin and decreased efficacy of repairing DNA damage induced by these agents than healthy controls. Our results suggest that type 2 diabetes mellitus may be associated not only with the elevated level of oxidative DNA damage but also with the increased susceptibility to mutagens and the decreased efficacy of DNA repair. These features may contribute to a link between diabetes and cancer and metrics of DNA damage and repair, measured by the comet assay, may be markers of risk of cancer in diabetes.     

Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases

Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase–quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions −2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme–drug complex.     

Theoretical models of DNA topology simplification by type IIA DNA topoisomerases

It was discovered 12 years ago that type IIA topoisomerases can simplify DNA topology—the steady-state fractions of knots and links created by the enzymes are many times lower than the corresponding equilibrium fractions. Though this property of the enzymes made clear biological sense, it was not clear how small enzymes could selectively change the topology of very large DNA molecules, since topology is a global property and cannot be determined by a local DNA–protein interaction. A few models, suggested to explain the phenomenon, are analyzed in this review. We also consider experimental data that both support and contravene these models.     

Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA

Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.     

Nature of the single-stranded DNA in replicating adenovirus type 5 DNA.

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.     

DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies.     

Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.     

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions.

Both bacteriophage T4 and human type I DNA ligases in the presence of a mixture of ATP, AMP and PPi altered the topological properties of a supercoiled substrate by a step-wise reaction eventually leading to a population of fully relaxed, covalently closed products. In the presence of only AMP and PPi DNA products containing nicks with 3'OH/5'P termini accumulated in the presence of bacteriophage T4 DNA ligase, suggesting reversal of the entire joining reaction, but not in the presence of human DNA ligase I. Both DNA ligases became deoxyadenylylated in the presence of dATP, but the joining reaction did not proceed to completion. However, with both enzymes the full relaxing reaction took place in the presence of dAMP alone and in the presence of a mixture of dATP, dAMP and PPi. In no case could the joining reaction be reversed by dAMP and PPi. Related experiments with modified derivatives of deoxyribonucleoside 5'-triphosphates and PPi gave results in accord with these observations. The AMP dependent DNA relaxation catalysed by DNA ligases was insensitive to the presence of exonuclease III. These results indicate that controlled relaxation of the substrate by both DNA ligases occurs as a separate reaction rather than by simple reversal of the joining reaction. These findings support the hypothesis that in vivo the DNA topoisomerising ligases relax their substrate at the replication fork both during and separately from ligation of a pre-existing nick.     

Synthesis and properties of a new type DNA dendrimer.

We have designed a new type of a DNA dendrimer which has rigid branched structure. The branching molecule was prepared from 1,3,5-tribromobenzene. The dendrimer unit, in which three oligonucleotide-chains, two molecules of T15 and one molecule of A15, linked to the branching molecule, was synthesized by an automated DNA synthesizer. The properties of the dendrimer unit and dendrimer formation by inter-molecular association of T15 and A15 chains of the dendrimer unit will be presented.