Analysis of herpes simplex virus DNA synthesized in infected nuclei by chromatography on benzoylated naphthoylated DEAE cellulose columns.

The nature of the DNA molecules synthesized in nuclei of herpes simplex virus (HSV)-infected cells in vivo and in vitro was studied by chromatography on BND-cellulose columns after shearing to DNA fragments of 10 to 20 X 10(6) daltons. The incorporation of labelled precursors occurs in the DNA fragments containing single-stranded regions, presumably the replication forks. Prolongation of DNA synthesis leads to the accumulation of labelled DNA fragments that lack single-stranded sequences. Analysis of the isolated DNA fragments by density centrifugation in CSCl gradients revealed that most of the labelled DNA molecules are of virus specificity and the minority are cellular DNA fragments. Double-stranded virus DNA fragments and virus DNA fragments containing single-stranded sequences band in CSCl gradients at a density of 1-718 g/ml, the density of virion DNA. This suggests that the replicating HSV DNA molecules have the same density as the virion DNA and contain relatively little single-stranded DNA. The synthesis of HSV DNA molecules under in vitro conditions in isolated nuclei occurs by incorporation of the precursors into DNA fragments with single-stranded regions. The synthesis of cellular DNA in nuclei from hydroxyurea and cytosine arabinoside treated cells also occurs by elongation of nascent DNA chains.     

Low infectivity of HSV-1 DNA caused by defective-interfering genomes

The infectivity of herpes simplex virus, type 1, strain ANG progeny DNA from standard virus infections and of progeny DNA from infections involving defective-interfering virus particles (DI DNA) was compared in transfection assays. No difference in infectivity of virus DNA isolated either from infected cells or from progeny virus was found for a given type of infection. However, the values for two types of infection differed markedly, with DI progeny DNA being less infectious by more than 2 log10. The low infectivity was mainly due to the presence of interfering DNA molecules in DI progeny DNA, regardless of whether intracellular DNA or DNA extracted from mature virions was analysed. The interfering capacity of DI progeny DNA did not depend on the integrity of the genomes. The physical proximity provided by simultaneous precipitation of infectious and of interfering DNA is an important factor influencing the degree to which DI DNA interferes. Interference by DI DNA in the transfection assay can be partly reversed by the addition of XbaI fragments of standard DNA; in control experiments this fragmented DNA was shown to lead to a reduction rather than to an enhancement of the infectivity of standard virus DNA.     

Integration of viral into chromosomal deoxyribonucleic acid in an inducible line of polyoma-transformed cells

RNA-DNA hybridization has shown that in poiyoma-transformed rat cells which can be induced to synthesize infectious virus (LPT cells), polyoma DNA sequences are associated with chromosomal DNARNA complementary to the viral DNA (cRNA) was synthesized in vitro, using purified viral DNA as a template and Escherichia coli RNA polymerase. High-molecular-weight chromosomal DNA was fractionated from linear and supercoiled viral DNA molecules by centrifugation of whole cells through alkaline glycerol gradients. Hybridization carried out between the cRNA and fractionated chromosomal DNA showed that the amount of RNA hybridized to the LPT DNA was two to three times larger than the amount hybridized to DNA from normal rat cells. cRNA was also hybridized, under the same conditions, with mixtures containing a constant amount of normal cell DNA and varying quantities of purified viral DNA. These assays have established that a linear relationship exists between the amount of cRNA specifically hybridized with a given sample of DNA and the quantity of viral DNA in the sample. Using this relationship, it is estimated that LPT chromosomal DNA contains 6–9 genome-equivalents of polyoma DNA per cell. This quantity represents 18–29% of the amount of polyoma DNA found in the cells, as determined by hybridization of cRNA with unfractionated LPT DNA.To exclude the possibility that the chromosomally associated viral DNA is an artifact due to incomplete removal of the extrachromosomal viral DNA, control experiments were performed in which the cells were superinfected with polyoma virus (m.o.i.-500; 3 hr infection). In these experiments, less than 1% of the viral DNA introduced into the cells by the superinfecting virus were found by the same techniques to be associated with chromosomal DNA. Other experiments show that LPT cells do not contain significant amounts of complex viral DNA molecules which sediment in the vicinity of chromosomal DNA. It is therefore suggested that viral and chromosomal DNA are bound to each other by bonds which cannot be disrupted by alkali treatment.     

Alteration of DNA tertiary structure by physical and chemical carcinogens: involvement in DNA repair processes

Parameters defining the topological state of DNA seem extremely important for describing the reactive state of the same DNA molecules. We have shown that physical and chemical DNA modifying agents alter the tertiary structure of DNA molecules. Variations in the tertiary structure of DNA were studied by one dimensional electrophoresis on an agarose gel of partially relaxed plasmid DNA topoisomers, a technique allowing the measurement of alterations in the degree of supercoiling equivalent to fractions of superhelical turns. Unwinding angles of -10.1 degrees or -8.7 degrees per pyrimidine or thymine dimer respectively, of -12 degrees per apurinic site, and of -3.4 degrees per methylated purine were obtained by titrating the number of damaged sites necessary to reduce the number of superhelical turns by one in each topoisomer. On the contrary, enzymatic methylation of the C-5 position of cytosine (a modified base present in prokaryotic and eukaryotic DNAs) did not alter the DNA tertiary structure. We have also shown that local alterations in DNA structure caused by UV-irradiation inhibit bacterial DNA topoisomerase I and DNA methylase, and that the topological state of DNA substrate influences the mode of methylation of Hpa II DNA methylase. These findings suggest that the natural topological state of DNA substrate (linear, relaxed, or covalently closed duplex DNA with varying degrees of supercoiling) influences the mode of action of enzymes possibly involved in DNA repair processes, while DNA structural alterations caused by DNA modifying agents might influence DNA repair processes in two ways: either by driving the interaction between repair enzymes and the modified sites of DNA, or by inhibiting or changing the mode of action of enzymes normally acting on unmodified DNA.     

Studies on DNA methyltransferase and alteration of the enzyme activity by chemical carcinogens

DNA in mammalian cells is enzymatically methylated at the 5-position of cytosine via S-adenosylmethionine and DNA methyltransferase. Several chemical carcinogens have been shown to inhibit this reaction, altering DNA methylation. We have been studying the mechanism by which carcinogens alter the methylation of DNA in order to better understand the cellular regulation of DNA methylase activity and to understand the role, if any, of DNA methylation in the carcinogenic process. We have utilized an in vitro assay for DNA methylase isolated from purified rat-liver nuclei. Ethionine, a liver carcinogen, given to rats 17 hr after partial hepatectomy inhibited the incorporation of [methyl-3H]-methionine into 5-methylcytosine residues of DNA. DNA isolated from these ethionine-treated rats was able to accept methyl groups from S-adenosylmethionine 8 times more than control DNA. It was further demonstrated that S-adenosylethionine competitively inhibited the DNA methylase resulting in hypomethylated DNA. N-Methyl-N-nitro-N-nitrosoguanidine reacted with the DNA methylase at the sulfhydryl sites inactivating the enzyme. Methylnitrosourea did not react directly with the methylase enzyme, but when reacted with DNA, the DNA methylase activity was inhibited by the carcinogen alkylated DNA. Sodium selenite also inhibited the enzyme non-competitively with a Ki of 6.7 microM. 5-Azacytidine prevented the 2 to 3 fold increase in DNA methylase seen 2 days following partial hepatectomy. All of these data with various carcinogens, altering DNA methylation by different mechanisms, support the hypothesis that DNA methylation plays a role in the initiation of carcinogenesis.     

一种可同时检测抗双链和单链DNA抗体的快速斑点免疫金渗滤试验

目的 建立一种可同时检测抗双链ＤＮＡ（ｄｓ－ＤＮＡ）和单链（ｓｓ－ＤＮＡ）抗体的快速斑点免疫渗滤试验（ＤＩＣ－ＦＡ）。方法 将ｄｓ－ＤＮＡ和ｓｓ－ＤＮＡ抗原结合在同一检测盒内,采用胶体金标记和快速膜斑点渗滤技术,同时检测抗ｄｓ－ＤＮＡ和ｓｓ－ＤＮＡ抗体。在结果：ＤＩＧＦＡ既可同步又可区别检测两种ＤＮＡ抗体,且检测ｄｓ－ＤＮＡ抗体的敏感性优于酶联免疫吸附法（ＥＬＩＳＡ）,结论：ＤＩＧＦＡ简便,快速,     

A cell-free system for studying a priming factor involved in repair of bleomycin-damaged DNA

A simple cell-free system for studying a priming factor involved in the repair of bleomycin-damaged DNA was established. The template-primer used for the repair DNA synthesis was prepared by treating the closed circular, superhelical form of pUC19 plasmid DNA with 2.2 microM bleomycin and 20 microM ferrous ions. Single-strand breaks were introduced into pUC19 DNA by the bleomycin treatment, and the DNA was consequently converted largely into the open circular form. A system for repair of this bleomycin-damaged DNA was constructed with a priming factor, DNA polymerase (DNA polymerase beta or Klenow fragment of DNA polymerase I), ATP, T4 DNA ligase and four deoxynucleoside triphosphates. After incubation, the conformation of the DNA was analyzed by agarose gel electrophoresis and electron microscopy. The open circular DNA was largely converted to the closed circular DNA, indicating that the single-strand breaks of DNA were repaired. When the priming factor was omitted, DNA repair did not occur. The present system seemed to be applicable to the study of priming factors involved in the repair of DNA with single-strand breaks caused not only by bleomycin but also by ionizing radiation or active oxygen.     

The evolution of new species of viral DNA during serial passage of simian virus 40 at high multiplicity

Viral DNA isolated during serial undiluted passage of SV40 was analysed by electrophoresis of fragments produced by H. influenzae restriction endonuclease and by DNA-DNA hybridization. Compared to parental SV40 DNA, early passage DNA showed alterations at many sites in the population of DNA molecules, although certain sites were more frequently altered than others as evidenced by the reduced yield of some original DNA fragments and the appearance of new fragments. With continued passage, new dominant species of viral DNA evolved which yielded simple restriction endonuclease digest patterns. DNA molecules from all passages retained SV40 DNA sequences; however, the percentage of total DNA sequences which corresponded to SV40 sequences diminished to about 30% or less in late passage DNA. Repetitive host cell DNA sequences were detected in a high percentage of late passage DNA molecules, but accounted for 20% or less of total sequences present. Therefore the new species of viral DNA which have evolved probably contain predominantly nonrepetitive host DNA sequences.     

Recognition and repair of chemically heterogeneous structures at DNA ends

Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean.” Rather, DNA breaks typically lack DNA 5′‐phosphate and 3′‐hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single‐strand and double‐strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical “cleansing” of DNA breaks to prevent genomic instability and disease, and promote progression of DNA‐ and RNA‐DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. Environ. Mol. Mutagen. 56:1–21, 2015. © 2014 Wiley Periodicals, Inc.     

Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.     

Molecular relationship of the DNA and RNA of intracytoplasmic A particles to mouse and mammary tumour virus genomes.

We have directly tested the hypothesis that single-stranded cytoplasmic A particle-associated DNA (ss CAP DNA) is a murine mammary tumour virus (MMTV) proviral intermediate by hybridizing 125I-labelled ss CAP DNA to MMTV RNA or to MMTV complementary DNA (cDNA). 125I-labelled CAP DNA did not form duplexes with either MMTV RNA or MMTV cDNA. In contrast, CAP RNA hybridized readily with MMTV cDNA. CAP RNA contained all the MMTV virus sequences, but at lower concentrations than in MMTV virus particles. Single-stranded CAP DNA hybridized readily with mouse DNA from several sources. A study of the rate of hybridization of CAP DNA to cell DNA at various driver to probe ratios showed that its rate of hybridization is similar to that of tumour cell DNA reassociation. Further, in reassociation studies accelerated by using the phenol emulsion reassociation technique (PERT), CAP DNA originally isolated as single-stranded DNA was shown to reanneal (70%), to protect 125I-cell DNA to the same extent (67%) and to do so with kinetics of reassociation equivalent to that of mouse DNA. Although CAP DNA isolates were slightly enriched for MMTV specific sequences when compared to total cellular DNA, we conclude that the majority of ss CAP-associated DNA is equivalent to a random sample of total tumour cell DNA.     

Herpes simplex virus necleic acid synthesis following infection of non-permissive XC cells.

DNA hybridization kinetic analysis of cellular DNA following high multiplicity infection of non-permissive XC cells by herpes simplex virus type I showed that HSV DNA penetrates to the nucleus of the cell but that the number of virus DNA copies present in each cell quickly begins to decline. There did not appear to be any net virus DNA synthesis and the loss of virus DNA copies continued until there was approximately one per haploid genome equivalent. HSV-2 likewise did not show any detectable virus DNA replication. The residual virus information was stable for more than 48 h. CsCl density gradient analysis of the infected cell DNA suggested an association between the HSV DNA and that of the cells. Network analysis also supported the suggestion that a stable association between the virus DNA and host DNA begins shortly after infection. Cell division resulted in the segregation of the virus DNA but not its loss from the cell population. Virus-specific RNA synthesis was easily detectable and 40 to 50% of a labelled DNA probe was converted to an RNA:DNA hybrid.     

Deoxyribonuclease I sensitivity of DNA replicated in permeable mouse sarcoma cells

To study chromatin structure at the sites of DNA replicated in permeable cells, deoxyribonuclease I (DNase I) sensitivity of newly replicated DNA in permeable mouse sarcoma cells was compared with that of newly replicated DNA in intact cells. About 35% of the DNA replicated in permeable cells was hypersensitive to DNase I, and the remaining DNA showed the same DNase I sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in intact cells, and was close to that of DNA replicated in the presence of cycloheximide. The sensitivity of DNA pulse-labeled with [3H]deoxythymidine triphosphate by replication in permeable cells was reduced significantly by chasing with cold deoxythymidine triphosphate. The present results suggest that chromatin structure at the sites of DNA replicated in permeable cells is similar to that at the sites of DNA replicated in living cells in the absence of protein synthesis, and that some structural change (possibly toward the maturation) of newly replicated chromatin occurs after the DNA replication in permeable cells.     

Bleomycin-induced DNA synthesis in a cell-free system using a permeable mouse sarcoma cell extract

To investigate factors involved in excision repair DNA synthesis, a soluble extract was prepared from permeable mouse sarcoma (SR-C3H/He) cells by homogenization and ultracentrifugation. DNA synthesis measured by using native calf thymus DNA as the template-primer and the extract as the polymerase source showed low activity. The DNA synthesis was enhanced more than ten-fold by the addition of an appropriate concentration of bleomycin, a radiomimetic DNA-damaging drug. Using selective inhibitors of DNA polymerases, it was shown that the DNA polymerase involved in the bleomycin-induced DNA synthesis was DNA polymerase beta. In addition to DNA polymerase beta, an exonuclease which converts bleomycin-damaged DNA into suitable template-primers for repair DNA synthesis appeared to be present in the permeable cell extract.     

Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells

To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s).     

Characterization of bacterial DNA binding to human neutrophil surface

Bacterial DNA activates neutrophils through a CpG- and TLR9-independent mechanism. Neutrophil activation does not require DNA internalization, suggesting that it results from the interaction of bacterial DNA with a neutrophil surface receptor. The aim of this study was to characterize the interaction of bacterial DNA with the neutrophil surface. Bacterial DNA binding showed saturation and was inhibited by unlabeled DNA but not by other polyanions like yeast tRNA and poly-A. Resembling the conditions under which bacterial DNA triggers neutrophil activation, binding was not modified in the presence or absence of calcium, magnesium or serum. Treatment of neutrophils with proteases not only dramatically reduced bacterial DNA binding but also inhibited neutrophil activation induced by bacterial DNA. Experiments performed with DNA samples of different lengths obtained after digestion of bacterial DNA with DNase showed that only DNA fragments greater than approximately 170-180 nucleotides competed bacterial DNA binding and retained the ability to trigger cell activation. Treatment of neutrophils with chemoattractants or conventional agonists significantly increased bacterial DNA binding. Moreover, neutrophils that underwent transmigration through human endothelial cell monolayers even in the absence of chemoattractants, exhibited higher binding levels of bacterial DNA. Together, our findings provide evidence that binding of bacterial DNA to neutrophils is a receptor-mediated process that conditions the ability of DNA to trigger cell activation. We speculate that neutrophil recognition of bacterial DNA might be modulated by the balance of agonists present at inflammatory foci. This effect might be relevant in bacterial infections with a biofilm etiology, in which extracellular DNA could function as a potent neutrophil agonist.     

Comparison of seven commercial DNA extraction kits for the recovery of <Emphasis Type="Italic">Brucella</Emphasis> DNA from spiked human serum samples using real-time PCR

We compared the relative recovery of extraction of bacterial DNA from serum using seven commercial kits (UltraClean DNA BloodSpin Kit, Puregene DNA Purification System, Wizard Genomic DNA Purification Kit, High Pure PCR Template Preparation Kit, GFX™ Genomic Blood DNA Purification Kit, NucleoSpin Tissue Kit, and QIAamp DNA Blood Mini Kit). Human serum samples were spiked with known concentrations of Brucella melitensis Rev 1; the DNA was extracted and tested in genus-specific LightCycler polymerase chain reaction (PCR). The UltraClean DNA BloodSpin Kit proved to be as sensitive as the QIAamp DNA Blood Mini Kit isolation method and could detect down to 100 fg of DNA, though only the former had no contamination. All the other procedures yielded DNA isolation results that were less sensitive and were always contaminated. Our results show that the UltraClean DNA Blood Spin Kit was the commercially available assay tested that yielded the best sensitivity, purity, and lack of contamination for Brucella DNA isolation from serum.     

Purification and characterization of DNA polymerases from Plasmodium berghei

DNA polymerases from the malaria parasite Plasmodium berghei were purified more than 50-fold. Several distinct enzymatic activities were isolated that could be distinguished by the use of various specific DNA polymerase inhibitors. In particular, subdivision into an aphidicolin-sensitive and an aphidicolin-resistant group was possible. Further analysis allowed a better comparison with host DNA polymerases and indicated that one aphidicolin-sensitive DNA polymerase resembled DNA polymerase alpha displaying processive DNA synthesis and using RNA primers, whereas another aphidicolin-sensitive DNA polymerase was distributive and only used DNA primers. Marked differences from the host enzymes do exist, however, such as insensitivity to BuPdGTP. Another P. berghei DNA polymerase was isolated that showed characteristics of a DNA polymerase beta-like enzyme, but which differed from host DNA polymerase beta in its insensitivity to dideoxynucleotides.     

Electron microscopy of herpes simplex virus DNA molecules isolated from infected cells by centrifugation in CsCl density gradients.

Herpes simplex virus (HSV) DNA molecules were isolated from infected BSC 1 cells and centrifuged in CsCl-ethidium bromide density gradients. Both newly labelled and mature virus DNA molecules were found to have a linear conformation. The morphology of virus DNA molecules at different stages of the virus growth cycle in BSC 1 cells, was studied by electron microscopy after separation of virus DNA from cellular DNA by centrifugation in CsCl gradients. In each sample, about 200 virus DNA molecules were photographed and the different morphological forms were studied. Four classes of virus DNA molecules were observed: (a) mature linear DNA molecules, 52-4 +/- 3-3 micronm in length, (b) DNA intermediates, (c) virus DNA molecules having one or more single-stranded filaments attached to them and (d) molecules with collapsed regions or with branches. A few circular molecules as well as linear DNA molecules longer than unit length were also observed. The virus DNA molecules resembling replicative intermediates gradually increased in number and reached a maximal amount of about 5% of the virus DNA population at 12 h after infection. The other forms of virus DNA were found to persist after the number of replicating DNA molecules decreased.