NMR and CD studies on an oligonucleotide containing N4-methylcytosine.

The hexamer d(CGm4CGCG) exists predominantly as a right handed B form helix at 20 degrees C in 150 mM NaCl, as shown by 2D NOE spectra. Under these conditions a minor species is also observed which corresponds to the single strand in slow exchange on a proton NMR time scale with the double strand. This exchange is unusually slow and separate resonances for the two species are seen up to 65 degrees C. At 50 degrees C the lifetime of the single strand species is 0.85 s. Under high salt conditions the hexamer is partly converted into the Z form, but the complete transition is only observed at 5M NaCl at -6 degrees C.     

The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2+ but only when the extent of the reaction is limited. Neither the complementary strands nor sequences representing the termini of human immunodeficiency virus type 1 DNA were cleaved at analogous positions. Analysis of a series of substrates containing U5 base substitutions has defined the sequence requirements for site-selective nicking; nucleotides near the cleavage site are most critical for activity. The minimum substrate size required to demonstrate significant activity corresponds to the nearly perfect 15-base terminal inverted repeat. This in vitro activity of IN thus produces viral DNA ends that are joined to host DNA in vivo and corresponds to an expected early step in the integrative recombination reaction. These results provide the first enzymatic support using purified retroviral proteins for a linear DNA precursor to the integrated provirus.     

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.     

Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (-) strand viral DNA and reverse transcription of primed RNA templates

Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (-) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (-) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (-) strand DNA. These are discussed in relation to their role as possible replicative intermediates.     

Transcriptional organization of the Drosophila melanogaster ribosomal RNA genes.

Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis.     

Underwinding of DNA associated with duplex-duplex pairing by RecA protein

Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.     

Resveratrol Induces Premature Senescence in Lung Cancer Cells via ROS-Mediated DNA Damage

Resveratrol (RV) is a natural component of red wine and grapes that has been shown to be a potential chemopreventive and anticancer agent. However, the molecular mechanisms underlying RV''s anticancer and chemopreventive effects are incompletely understood. Here we show that RV treatment inhibits the clonogenic growth of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Interestingly, the tumor-suppressive effect of low dose RV was not associated with any significant changes in the expression of cleaved PARP and activated caspase-3, suggesting that low dose RV treatment may suppress tumor cell growth via an apoptosis-independent mechanism. Subsequent studies reveal that low dose RV treatment induces a significant increase in senescence-associated β–galactosidase (SA-β-gal) staining and elevated expression of p53 and p21 in NSCLC cells. Furthermore, we show that RV-induced suppression of lung cancer cell growth is associated with a decrease in the expression of EF1A. These results suggest that RV may exert its anticancer and chemopreventive effects through the induction of premature senescence. Mechanistically, RV-induced premature senescence correlates with increased DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production in lung cancer cells. Inhibition of ROS production by N-acetylcysteine (NAC) attenuates RV-induced DNA DSBs and premature senescence. Furthermore, we show that RV treatment markedly induces NAPDH oxidase-5 (Nox5) expression in both A549 and H460 cells, suggesting that RV may increase ROS generation in lung cancer cells through upregulating Nox5 expression. Together, these findings demonstrate that low dose RV treatment inhibits lung cancer cell growth via a previously unappreciated mechanism, namely the induction of premature senescence through ROS-mediated DNA damage.     

Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles.

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.     

Mouse mammary tumor virus DNA in infected rat cells: characterization of unintegrated forms.

Mouse mammary tumor virus (MMTV) DNA in chronically infected rat hepatoma cells is maintained in both the integrated and unintegrated state. Fractionation of DNA by the procedure of Hirt (1967) as well as by sedimentation through alkaline sucrose suggests that about two thirds of the viral DNA is associated with high molecular weight cell DNA. The remainder of the viral DNA is unintegrated and is present primarily as linear or open circular duplexes consisting of a genome-length strand complementary to the viral RNA ("minus" strand) and "plus" strands of subgenomic length. Approximately 20% of the unintegrated MMTV DNA is present as double-stranded, covently closed circles (form I) with a molecular weight of 6 X 10(6) daltons. Form I viral DNA is found primarily in the nucleus, whereas the open forms are both nuclear and cytoplasmic.     

Regenerating rat liver DNA polymerases: disimilitude or relationship between nuclear and cytoplasmic enzymes?

The possible relationship between the nuclear and cytoplasmic DNA polymerases of regenerating rat liver was studied by sucrose gradient analysis, salt dissociation, and with specific inhibitors. After aqueous subcellular fractionation and removal of the nuclear membranes, three species of DNA-dependent DNA polymerases were characterized: 1) a DNA polymerase-beta in the nuclei. 2) a DNA polymerase-alpha in the cytosol which was not dissociated at high salt concentrations; and 3) an intermediate form in the cytosol and in the Triton wash containing the nuclear membranes. The latter form behaved like DNA polymerase-alpha et low salt concentration but was dissociated at high salt concentrations to a low molecular weight species with properties like DNA polymerase-beta (resistance to inhibition by N-ethylmaleimide, heparin and KCL). In vitro reassociation experiments suggest that this intermediate form corresponds to the association of DNA polymerase-beta with a membrane component or cytoplasmic protein(s) which appear(s) in regenerating rat liver.     

Free radical formation and DNA strand breakage during metabolism of diaziquone by NAD(P)H quinone-acceptor oxidoreductase (DT-diaphorase) and NADPH cytochrome c reductase.

One-electron reduction of diaziquone (AZQ) by purified rat liver NADPH cytochrome c reductase was associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as indicated by ESR spin-trapping studies. Reactive oxygen formation correlated with AZQ-dependent production of single and double PM2 plasmid DNA strand breaks mediated by this system as detected by gel electrophoresis. Direct two-electron reduction of AZQ by purified rat liver NAD(P)H (quinone acceptor) oxidoreductase (QAO) was also associated with formation of AZQ semiquinone, superoxide anions, hydrogen peroxide, and hydroxyl radicals as detected by ESR spin trapping. Furthermore, PM2 plasmid DNA strand breaks were detected in the presence of this system. Plasmid DNA strand breakage was inhibited by dicumarol (49 +/- 5%), catalase (57 +/- 2.3%), SOD (42.2 +/- 3.6%) and ethanol (41.1 +/- 3.9%) showing QAO and reactive oxygen formation was involved in the PM2 plasmid DNA strand breaks observed. These results show that both one- and two-electron enzymatic reduction of AZQ give rise to formation of reactive oxygen species and DNA strand breaks. Autoxidation of the AZQ semiquinone and hydroquinone in the presence of molecular oxygen appears to be responsible for these processes. QAO appears to be involved in the metabolic activation of AZQ to free radical species. The cellular levels and distribution of this enzyme may play an important role in the response of tumor and normal cells to this antitumor agent.     

Formation of nucleoprotein filaments by mammalian DNA methyltransferase Dnmt3a in complex with regulator Dnmt3L

The C-terminal domains of Dnmt3a and Dnmt3L form elongated heterotetramers (3L-3a-3a-3L). Analytical ultracentrifugation confirmed the Dnmt3a-C/3L-C complex exists as a 2:2 heterotetramer in solution. The 3a–3a interface is the DNA-binding site, while both interfaces are essential for AdoMet binding and catalytic activity. Hairpin bisulfite analysis shows correlated methylation of two CG sites in a distance of ~8-10 bp in the opposite DNA strands, which corresponds to the geometry of the two active sites in one Dnmt3a-C/3L-C tetramer. Correlated methylation was also observed for two CG sites at similar distances in the same DNA strand, which can be attributed to the binding of two tetramers next to each other. DNA-binding experiments show that Dnmt3a-C/3L-C complexes multimerize on the DNA. Scanning force microscopy demonstrates filament formation rather than binding of single tetramers and shows that protein–DNA filament formation leads to a 1.5-fold shortening of the DNA length.     

Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments.     

Structure of F-actin needles from extracts of sea urchin oocytes

The mouse L-cell line LD maintains its mitochondrial DNA genome in the form of a head-to-tail unicircular dimer of the monomeric 16,000 base-pair species. This situation permits a comparison of the mechanism of replication of this dimeric molecule with our previous studies of replication of monomeric mouse L-cell mitochondrial DNA. Whereas monomeric mitochondrial DNA requires about one hour for a round of replication, the dimeric molecule requires almost three hours. Denaturing agarose gel electrophoretic analyses of replicative intermediates reveals several discrete size classes of partially replicated daughter strands of dimeric mitochondrial DNA. This suggests that replication occurs with specific discontinuities in the rate of daughter strand synthesis. The strand specificity of these daughter strands was determined by hybridization with ³²P-labeled DNA representing either the heavy or light strand mitochondrial DNA sequence. The sizes and strand specificities of these discrete daughter strands indicate that the same set of control sequences is functional in both dimer and monomer mitochondrial DNA replication.Immediately following a round of replication, the majority of dimeric mitochondrial DNA molecules contain displacement loops, as assessed by their sensitivity to nicking within the displaced DNA strand by single-strand DNA specific S₁ nuclease under conditions which leave supercoiled DNA intact. This result is in contrast with the conformation of newly replicated monomeric mitochondrial DNA molecules, which lack both superhelical turns and displacement loops. This indicates that dimeric mitochondrial DNA proceeds through a different series of post-replicative processing steps than does monomeric mitochondrial DNA. We postulate that intermediates at late stages of dimeric mitochondrial DNA replication contain displacement loops which remain intact following closure of the full-length daughter strands.     

Novobiocin induces accumulation of a single strand of plasmid pGRB-1 in the archaebacterium Halobacterium GRB.

Treatment of Halobacterium GRB cells with the DNA topoisomerase II inhibitor novobiocin induces the accumulation of a circular single-stranded DNA form of the plasmid pGRB-1. This form corresponds to the transcribed strand of pGRB-1. A tiny amount of this form is detectable in untreated cells. The induction of single-stranded pGRB-1 molecules by novobiocin is abolished when cells are pretreated with aphidicolin or anisomycin, which inhibit halobacterial DNA replication and protein synthesis, respectively. These results suggest that the single-stranded form of pGRB-1 is generated in the course of plasmid replication.     

Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition.

Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.     

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94- and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA liked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent DNA synthesis with dexamethasone. In this case, multiple, discrete subgenomic DNA plus strands were not found when the same methods applied to avian sarcoma virus DNA were used; instead, the plus strands present in the linear DNA of mouse mammary tumor virus fell mainly into two classes: (i) strands of ca. 1.3 kb which appeared early in synthesis and were similar in size and genetic content to the terminally repeated sequence in linear DNA; and (ii) plus strands of the same length as linear DNA. A heterogeneous population of other strands diminished with time, was not found in completed molecules, and was probably composed of strands undergoing elongation. These two retroviruses thus appear to differ with respect to both the number of priming sites used for the synthesis of plus strands and the abundance of full-length plus strands. On the other hand the major subgenomic plus strand of mouse mammary tumor virus DNA (1.3 kb) is probably the functional homolog of a major subgenomic plus strand of avian sarcoma virus DNA (0.35 kb). The significance of this plus strand species is discussed in the context of current models which hold that it is used as a template for the completion of the minus strand, thereby generating the long terminal redundancy.