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PubMed1994

Thermal energy requirement for strand separation during transcription initiation: the effect of supercoiling and extended protein DNA contacts.

We have studied the role of extended protein DNA contacts and DNA topology on the ability of Escherichia coli RNA polymerase to form open complexes at several related promoters. The -35 region of several Escherichia coli promoters do not have homology with the consensus sequence, but still drive activator independent transcription initiation. This is due to the presence of a TG motif upstream from the -10 hexamer creating an 'extended -10' promoter. We have previously shown that two 'extended -10' promoters, galP1 and pBla, can form open complexes at lower temperatures than the galP1 derivative, galPcon6, which has a consensus -35 hexamer. Here we report further investigations into the mechanism of open complex formation by RNA polymerase, in particular the thermal energy requirement. A single base pair change in galPcon6 creating an 'extended -10' sequence, results in a 20 degrees C reduction in the temperature requirement for open complex formation. The DNA topology has also been shown to effect the thermal energy requirement for strand separation. Promoters carried on supercoiled plasmids form open complexes at lower temperatures than when present on linear DNA templates. We have also shown that in vivo, RNA polymerase can form open complexes at lower temperatures than those observed for linear templates in vitro, but requires slightly higher temperatures than supercoiled templates in vitro, however the promoter hierachy remains the same.

Base Composition↗

PubMed2005

The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli.

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.

3' Untranslated Regions↗

PubMed2000

Evaluation of degradation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies.

The stability of highly purified supercoiled plasmid DNA formulated in simple phosphate or Tris-buffered saline solutions has been characterized to establish the overall degradation processes that occur during storage in aqueous solution. Plasmid DNA stability was monitored during accelerated stability studies (at 50 degrees C) by measurements of supercoiled, open-circle, and linear DNA content, as well as the accumulation of apurinic sites and 8-hydroxydeoxyguanosine residues over time. The effects of formulation pH, demetalation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on the supercoiled content of plasmid DNA during storage at 50 degrees C were also determined. The results indicate that free radical oxidation may be a major degradative process for plasmid DNA in pharmaceutical formulations unless specific measures are taken to control it by the addition of free radical scavengers, specific metal ion chelators, or both. The generation of hydroxyl radicals in phosphate-buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high-performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability and to inhibit the hydroxylation of phenylalanine; both observations are consistent with the known ability of ethanol to serve as a hydroxyl radical scavenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDTA) and ethanol had a synergistic enhancing effect on DNA stability. However, the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as potent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and beta-elimination were then determined, allowing accurate predictions of DNA storage stability as a function of formulation pH and temperature. The ability to predict plasmid DNA storage stability in the absence of free radical oxidation should prove to be a valuable tool for the design of stable pharmaceutical formulations of plasmid DNA.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1991

Efficient cloning of hepatitis B virus DNA from single-stranded replicative intermediates and its application to S1 mapping of viral RNA in human liver tissue.

An efficient method for cloning subgenomic fragments of the hepatitis B virus (HBV) was developed, utilizing its abundant single-stranded replicative intermediates. The total genomic DNA obtained from the liver tissue of patients with chronic HBV infection was treated by using the Klenow fragment of E. coli DNA polymerase I without adding any exogenous primers. Single-stranded replicative intermediates were efficiently converted to double-stranded linear DNA, one end of which terminated at (or near to) the direct repeat 1 (DR1) sequence of the HBV genome. By screening less than 1,000 recombinants from a DNA library after this treatment, we obtained a subgenomic HBV fragment of 2.0 kilobases. We then analysed HBV RNA in human liver tissue by S1 mapping. It was possible to map HBV RNA only when a DNA probe from the same tissue was used.

Cloning, Molecular↗

PubMed1995

Outer membrane-associated deoxyribonuclease activity of Porphyromonas gingivalis.

Extracellular outer membrane vesicles which are produced by Gram-negative bacteria may enclose deoxyribonucleic acid (DNA). While characterizing vesicles of Porphyromonas gingivalis, it was found that they do not contain detectable amount of DNA. It was also shown that the presence of deoxyribonuclease activity on whole cells and vesicles can degrade plasmidic and linear DNA. Deoxyribonuclease activity was also demonstrated in several other Gram-negative oral bacterial species. The nuclease activity of P. gingivalis was further characterized. When deoxyribonuclease activity was analyzed by zymography, only one active band was detected under the conditions tested. This nuclease enzyme showed a molecular weight of approximately 50 kDa. The activity was inhibited by 5 mM ZnCl2 or 100 mM EDTA whereas Mg2+ or Ca2+ ions were not required for activity. Activity was totally destroyed by treatment at 70 degrees C for 15 min. Although the enzyme may participate in virulence or provide nucleic acid precursors for bacterial growth, its exact role is still unknown.

Journal Article↗

PubMed2000

Alternative geometries of DNA looping: an analysis using the SfiI endonuclease.

Many processes are governed by proteins that bind to separate sites in DNA and loop out the intervening DNA, but the geometries of the loops have seldom been determined. The SfiI endonuclease cleaves DNA after interacting with two recognition sites, and is a favourable system for the analysis of DNA looping. A gel-shift assay was used here to examine the binding of SfiI to a series of linear DNA molecules containing two SfiI sites separated by 109-170 base-pairs. The complexes in which SfiI trapped a loop by binding to two sites in the same DNA were separated from the complexes containing SfiI bound to separate DNA molecules. Step-wise changes in the inter-site spacing generated two forms of the looped complex with different electrophoretic mobilities. The yields of each looped complex and the complexes from intermolecular synapses all varied cyclically with the inter-site spacing, with similar periodicities ( approximately 10.5 base-pairs) but with different phases. One looped complex predominated whenever the DNA between the sites needed to be underwound in order to produce the correct helical orientation of the binding sites. The other looped complex predominated whenever the intervening DNA needed to be overwound. We conclude that the former has trapped a right-handed loop with a negative node and the latter a left-handed loop with a positive node.

Base Sequence↗

PubMed1995

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures.

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.

Animals↗

PubMed2003

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer.

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.

Animals↗

PubMed1994

Supercoiling-regulated liquid-crystalline packaging of topologically-constrained, nucleosome-free DNA molecules.

Electron microscopy and circular dichroism studies of cholesteric aggregates derived from topologically-constrained DNA molecules indicate that the overall morphology and structural properties of these aggregates are fundamentally different from those characterizing condensed structures of nonconstrained DNA species. Specifically, the cholesteric pitch and twist of all hitherto characterized lyotropic mesophases of biopolymers--including those obtained from linear DNA--depend predominantly upon environmental parameters such as the dielectric constant of the solvent. In contrast, the properties of aggregates derived from closed circular supercoiled DNA are found to be solely and directly dictated by the superhelical density and handedness. On the basis of these results, as well as on the demonstrated ubiquity of liquid-crystalline DNA organizations in vivo, we suggest that supercoiling-regulated liquid crystallinity represents an effective packaging mode of nucleosome-free, topologically-constrained DNA molecules in living systems.

Circular Dichroism↗

PubMed2002

DNA secondary structure and Raman markers of supercoiling in Escherichia coli plasmid pUC19.

Negative supercoiling in the 2686 bp Escherichia coli plasmid pUC19 is comparable in linking number (Lk(0) = 258) and superhelical density (sigma = -0.05) to the moderate supercoiling exhibited by many eukaryotic chromosomal DNAs in vivo. Supercoiled and relaxed forms of purified pUC19 in aqueous solution (0.1 M NaCl, pH 8.3, 20 degrees C) have been investigated by Raman spectroscopy to assess changes in B-DNA secondary structure induced by superhelical stress and to identify putative Raman markers of DNA supercoiling. We find that supercoiling leads to small but significant changes to the B-form Raman signature of linear DNA. Spectral band shifts in the 780-850 cm(-1) interval are interpreted as resulting from a small net change in the average phosphodiester torsions alpha (O3'-P->-O5'-C5') and zeta (C3'-O3'->-P-O5') from the gauche(-)/gauche(-) range to the gauche(-)/trans range with supercoiling. The magnitude of the spectral intensity change implies that approximately 5% of the nucleotide moieties are affected. Supercoiling also introduces small redistributions of Raman intensity within the 1460-1490 and 1660-1670 cm(-1) intervals, consistent with small structural perturbations. Importantly, no Raman markers of Watson-Crick base pairing, base stacking, or C2'-endo/anti deoxynucleoside conformations are perturbed significantly by supercoiling of pUC19, indicating that the B-DNA structure is largely conserved under moderate superhelical stress. Peak and trough features at 814 and 783 cm(-1), and at 1462 and 1489 cm(-1), respectively, in the Raman difference spectrum between superhelical and relaxed DNA are proposed as markers of moderate negative supercoiling. We also show that in Tris-buffered solutions the Raman signature of supercoiled DNA can be obscured by Raman bands of Tris counterions. The subtle structural perturbations to B-DNA induced by moderate supercoiling are consistent with proposed mechanisms of transcriptional activation.

Base Sequence↗

PubMed1990

Telomeres shorten during ageing of human fibroblasts.

The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.

Aging↗

PubMed1979

Replication in vitro of the 2-micrometer DNA plasmid of yeast.

Cell-free extracts prepared from growing cells of the budding yeast Saccharomyces stimulated DNA synthesis directed by the supercoiled 2-micrometer yeast DNA plasmid. The major products of the reaction were open-circular daughter molecules possessing newly synthesized full-length linear DNA strands. Some of these were ligated and supertwisted by the extracts to yield a supercoiled DNA product. Both of the complementary DNA strands of the template were replicated. In addition, the extracts induced the appearance of theta-forms of the plasmid DNA, which are presumed to be replicative intermediates. The results of experiments utilizing BrdUTP incorporation indicated that DNA repair did not contribute significantly to the overall reaction. Extracts prepared from the cell division cycle mutants cdc7 and cdc8, held in culture at the nonpermissive temperature, possessed diminished activity. Because these mutants define a dependent sequence of events leading from the start of the cell cycle through G1 to S phase, this result suggests that the activity that stimulates 2-micrometer DNA replication in vitro is subject to control in the yeast cell cycle.

DNA↗

PubMed1996

YY1 transcriptional initiator: protein interactions and association with a DNA site containing unpaired strands.

The Ying-Yang 1 protein (YY1) DNA-binding site functions as an initiator element at which YY1, transcription factor IIB (TFIIB), and RNA polymerase II sponsor basal transcription from a supercoiled DNA template. We show that TFIIB binds to YY1, stabilizing its interaction with DNA, and YY1 contacts the large subunit of polymerase II, directing it to the initiation site. YY1 directs initiation from linear DNA containing mismatched sequences within its binding site, leading us to infer that supercoiling facilitates the separation of DNA strands and to suggest that YY1 likely remains bound to the start site as DNA strands separate during initiation. These results provide a mechanistic basis for transcriptional initiation directed by YY1 in the absence of the TATA box-binding protein.

Base Sequence↗

PubMed2006

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA.

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.

Amino Acid Sequence↗

PubMed1997

DNA interactions of bifunctional dinuclear platinum(II) antitumor agents.

Modifications of natural DNA in a cell-free medium by dinuclear bisplatinum complexes with equivalent coordination spheres, represented by the general formula [¿trans-PtCl(NH3)2¿2(H2N-R-NH2)]2+, where R is a propane or hexane, were studied by various methods of biochemical analysis or molecular biophysics. These methods include binding studies by means of differential-pulse polarography, measurements of melting curves with the aid of absorption spectrophotometry, measurements of CD spectra, ELISA with specific antibodies that recognize DNA modified by platinum complexes, interstrand cross-linking assay employing gel electrophoresis under denaturing conditions and mapping of DNA adducts by means of transcription assays. The results indicated that the major adduct of [¿trans-PtCl(NH3)2¿2(H2N-R-NH2)]2+ in DNA was an interstrand cross-link which was formed with a relatively short half-time (approximately 1 h). At least some types of these interstrand cross-links induced local denaturational changes in the DNA. The results of analyses of interactions of [¿trans-PtCl(NH3)2¿2(H2N-R-NH2)]2+ with linear DNA at relatively higher levels of the modification could be interpreted to mean that these dinuclear platinum complexes were also capable of intrastrand-cross-link formation between adjacent base residues in DNA. However, these intrastrand adducts of [¿trans-PtCl(NH3)2¿2(H2N-R-NH2)]2+ distorted DNA conformation in a way different from the DNA intrastrand adducts of cisplatin. In addition, the DNA adducts of the dinuclear platinum complexes inhibited DNA transcription in vitro. The length of the aliphatic linker chain affected the DNA-binding mode of [¿trans-PtCl(NH3)2¿2(H2N-R-NH2)]2+ and the resulting conformational changes in DNA. The extensive analysis of DNA interactions with [¿trans-PtCl(NH3)2¿2(H2N-R-NH2)]2+ described in this communication has provided further experimental support for previous suggestions [Farrell, N. (1991) in Platinum and other metal coordination compounds in cancer chemotherapy (Howell, S. B., ed.) pp. 81-91, Plenum Press, New York] that the binding of the dinuclear platinum complexes modifies DNA in a way that is different from the modification by antitumor cisplatin. Thus, the results of this work are consistent with the hypothesis that platinum drugs that bind to DNA in a manner fundamentally different from that of cisplatin can exhibit altered biological properties, including a different spectrum and intensity of antitumor activity.

Animals↗

PubMed1994

The RNA polymerase I transactivator upstream binding factor requires its dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA.

Upstream binding factor (UBF) is an important transactivator of RNA polymerase I and is a member of a family of proteins that contain nucleic acid binding domains named high-mobility-group (HMG) boxes because of their similarity to HMG chromosomal proteins. UBF is a highly sequence-tolerant DNA-binding protein for which no binding consensus sequence has been identified. Therefore, it has been suggested that UBF may recognize preformed structural features of DNA, a hypothesis supported by UBF's ability to bind synthetic DNA cruciforms, four-way junctions, and even tRNA. We show here that full-length UBF can also bend linear DNA to mediate circularization of probes as small as 102 bp in the presence of DNA ligase. Longer probes in the presence of UBF become positively supercoiled when ligated, suggesting that UBF wraps the DNA in a right-handed direction, opposite the direction of DNA wrapping around a nucleosome. The dimerization domain and HMG box 1 are necessary and sufficient to circularize short probes and supercoil longer probes in the presence of DNA ligase. UBF's sequence tolerance coupled with its ability to bend and wrap DNA makes UBF an unusual eukaryotic transcription factor. However, UBF's ability to bend DNA might explain how upstream and downstream rRNA gene promoter domains interact. UBF-induced DNA wrapping could also be a mechanism by which UBF counteracts histone-mediated gene repression.

Animals↗

PubMed1990

Acid depurination after field inversion agarose gel electrophoresis reduces transfer of large DNA molecules.

Field-inversion gel electrophoresis (FIGE) is an economical method for the resolution of DNA fragments 20 to 1000 kilobase pairs (kbp) in length, sizes beyond the resolving capabilities of normal agarose gel electrophoresis. A theoretical limitation to FIGE is the subsequent transfer of large DNA molecules to membrane supports for hybridization. After normal electrophoresis, uniform and rapid capillary transfer of DNA fragments from agarose gels has been previously reported to be facilitated by brief depurination of DNA with 0.25 N HCl. However, after FIGE we have found that brief treatment of large (greater than 200 kbp) linear DNA fragments with 0.25 N HCl reduces the extent of subsequent transfer by capillary methods. After FIGE and transfer, ethidium bromide staining of DNA suggests that acid treatment causes trapping of DNA within the agarose matrix.

DNA↗

PubMed1989

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids.

Wild-type Escherichia coli are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.

Bacterial Proteins↗