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At least 469 records · Page 26Linked to original sources
PubMed2001

DNA topoisomerase VI generates ATP-dependent double-strand breaks with two-nucleotide overhangs.

A key step in the DNA transport by type II DNA topoisomerase is the formation of a double-strand break with the enzyme being covalently linked to the broken DNA ends (referred to as the cleavage complex). In the present study, we have analyzed the formation and structure of the cleavage complex catalyzed by Sufolobus shibatae DNA topoisomerase VI (topoVI), a member of the recently described type IIB DNA topoisomerase family. A purification procedure of a fully soluble recombinant topoVI was developed by expressing both subunits simultaneously in Escherichia coli. Using this recombinant enzyme, we observed that the formation of the double-strand breaks on supercoiled or linear DNA is strictly dependent on the presence of ATP or AMP-PNP. This result suggests that ATP binding is required to stabilize an enzyme conformation able to cleave the DNA backbone. The structure of cleavage complexes on a linear DNA fragment have been analyzed at the nucleotide level. Similarly to other type II DNA topoisomerases, topoVI is covalently attached to the 5'-ends of the broken DNA. However, sequence analysis of the double-strand breaks revealed that they are all characterized by staggered two-nucleotide long 5' overhangs, contrasting with the four-base staggered double-strand breaks catalyzed by type IIA DNA topoisomerases. While no clear consensus sequences surrounding the cleavage sites could be described, interestingly A and T nucleotides are highly represented on the 5' extensions, giving a first insight on the preferred sequences recognized by this type II DNA topoisomerase.

Adenosine Triphosphate↗
PubMed1992

Synthesis of human immunodeficiency virus DNA in a cell-to-cell transmission model.

Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) was modelled by coculturing virus-infected cells with uninfected target cells at a ratio of 1:4. While H9 cells persistently infected with HTLV-IIIB did not contain unintegrated viral DNA detectable by Southern blotting, when cocultured with uninfected HUT-78 cells the mixed culture effectively underwent a new round one-step virus replication which began de novo synthesis of free viral DNA within 4 hours. Linear DNA was synthesized before the accumulation of circular DNA, and two seemingly distinct phases of viral DNA synthesis were involved. When both virus donor cells and recipient cells were arrested in the G0/G1 phase of the cell cycle, accumulation of circular viral DNA was inhibited. In contrast to cell-free virus infection of resting human peripheral blood mononuclear cells (PBMC), where no free viral DNA of discrete sizes could be detected by Southern blot, cell-to-cell transmission infection of resting PBMC resulted in the synthesis of full-length linear as well as circular viral DNA. The efficiency with which cell-to-cell transmission of HIV initiates virus replication underlines the importance of this mode of transmission in virus dissemination in vivo.

Cell Cycle↗
PubMed2000

PCR-mediated gene replacement in Escherichia coli.

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E. coli. Three of these replacements were performed without prior cloning of the genes.

Bacteriophage lambda↗
PubMed1997

DNA bending and the initiation of transcription at sigma54-dependent bacterial promoters.

We have examined the effects on transcription initiation of promoter and enhancer strength and of the curvature of the DNA separating these entities on wild-type and mutated enhancer-promoter regions at the Escherichia coli sigma54-dependent promoters glnAp2 and glnHp2 on supercoiled and linear DNA. Our results, together with previously reported observations by other investigators, show that the initiation of transcription on linear DNA requires a single intrinsic or induced bend in the DNA, as well as a promoter with high affinity for sigma54-RNA polymerase, but on supercoiled DNA requires either such a bend or a high affinity promoter but not both. The examination of the DNA sequence of all nif gene activator- or nitrogen regulator I-sigma54 promoters reveals that those lacking a binding site for the integration host factor have an intrinsic single bend in the DNA separating enhancer from promoter.

Bacterial Proteins↗
PubMed2004

Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration.

Integrated hepadnaviral DNA in livers and tumors of chronic hepatitis B patients has been reported for many years. In this study, we investigated whether hepatitis B virus DNA integration occurs preferentially at sites of cell DNA damage. A single I-SceI homing endonuclease recognition site was introduced into the DNA of the chicken hepatoma cell line LMH by stable DNA transfection, and double-strand breaks were induced by transient expression of I-SceI after transfection of an I-SceI expression vector. Alteration of the target cleavage site by imprecise nonhomologous end joining occurred at a frequency of approximately 10(-3) per transfected cell. When replication of an avian hepadnavirus, duck hepatitis B virus, occurred at the time of double-strand break repair, we observed integration of viral DNA at the site of the break with a frequency of approximately 10(-4) per transfected cell. Integration depended on the production of viral double-stranded linear DNA and the expression of I-SceI, and integrated DNA was stable through at least 17 cell divisions. Integration appeared to occur through nonhomologous end joining between the viral linear DNA ends and the I-SceI-induced break, because small deletions or insertions were observed at the sites of end joining. The results suggest that integration of hepadnaviral DNA in infected livers occurs at sites of DNA damage and may indicate the presence of more widespread genetic changes beyond that caused by viral DNA integration itself [corrected].

Animals↗
PubMed2005

Replication of hepatitis B virus in primary duck hepatocytes transfected with linear viral DNA.

AIM: To explore the expression and replication of hepatitis B virus (HBV) DNA in primary duck hepatocytes (PDHs). METHODS: Complete HBV genome was transfected into PDHs by electroporation (transfected group, 1.19 x 10(12) copies of linear HBV DNA/1 x 10(7) PDHs). After 1-5 d of transfection, HBsAg and HBeAg in the supernatant and lysate of PDHs were measured with the IMX System. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PDHs electroporated were used as control group. RESULTS: HBsAg in the hepatocyte lysates of transfected group was 15.24 (1 d), 14.55 (3 d) and 5.13 (5 d; P/N values, positive > or =2.1) respectively. HBeAg was negative (<2.1). Both HBsAg and HBeAg were negative in the supernatant of transfected group. Dot blotting revealed that HBV DNA was strongly positive in the transfected group and negative in the control group. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (rc DNA), covalently closed circular (ccc DNA), and single-stranded (ss DNA) HBV DNA replicative intermediates in the transfected group, there was no integrated HBV DNA in the cellular genome. These parameters were negative in control group. CONCLUSION: Expression and replication of HBV genes can occur in hepatocytes from non-mammalian species. HBV replication has no critical species-specificity, and yet hepatic-specific regulating factors in hepatocytes may be essential for viral replication.

Animals↗
PubMed2002

Phage N15 telomere resolution. Target requirements for recognition and processing by the protelomerase.

The Escherichia coli prophage N15 exists as a linear DNA molecule with covalently closed ends. Purified N15 protelomerase TelN is the only protein required to convert circular DNA substrates to the linear form with hairpin termini. Within the center of the telomerase occupancy site tos, the target for TelN is the 56-bp telRL consisting of the central 22-bp palindrome telO and two 14-bp flanking inverted sequence repetitions. DNase I footprinting of TelN-telRL complexes shows a segment of approximately 50 bp protected by TelN. Surface plasmon resonance studies demonstrate that this extended footprint is caused by two TelN molecules bound to telRL. Stable TelN-target DNA complexes are achieved with telRL; however, the additional sequences of tos stabilize the TelN-target complexes. TelO alone is not sufficient for specific stable complex formation. However, processing can occur, i.e. generation of the linear covalently closed DNA. Within the context of telRL, sequences of telO are involved in specific TelN-telRL complex formation, in processing itself, and/or in recognition of the processing site. The sequence of the central (CG)(3) within telO that is part of a 14-bp stretch proposed to have Z-DNA conformation is essential for processing but not for formation of specific TelN-telRL complexes. The concerted action of both TelN molecules at the target site is the basis for telomere resolution. Capturing of reaction intermediates demonstrates that TelN binds covalently to the 3'-phosphoryl of the cleaved strands.

Base Sequence↗
PubMed1985

A study of interactions of platinum (II) compounds with DNA by means of CD spectra of solutions and liquid crystalline microphases of DNA.

The optical properties of the DNA complexes with divalent platinum compounds of the cis-diamine type differing both in the nature of anionic and neutral ligands and in the spatial arrangement about the platinum atom were studied. The platinum compounds cis-[Pt(NH3)2Cl2], [Pt(en)Cl2], [Pt(tetrameen)Cl2], cis-[Pt(NH3)2NO2Cl], and cis-[PtNH3(Bz)Cl2] at small values of r (r is the molar ratio of a platinum compound to DNA nucleotides in the reaction mixture) were found to induce an increase in the amplitude of the positive band in the circular dichroic (CD) spectrum of linear DNA. All the compounds listed except cis-[Pt(NH3)2NO2Cl] caused a sharp decrease of the amplitude of the negative band in the CD spectrum of a liquid crystalline microphase of DNA formed in solution in the presence of poly(ethylene glycol). All these platinum compounds (except [Pt(tetrameen)Cl2]) exhibit biological (antimitotic, antitumour, etc.) activity. The platinum compounds trans-[Pt(NH3)Cl2], trans-[Pt(NH3)2NO2Cl], cis-[PtNH3PyCl2], cis-[Pt(NH3)2(NO2)2], and [Pt(NH3)3Cl]Cl exhibiting a low (if any) biological activity, either induced a decrease of the amplitude of the positive band in the CD spectrum of linear DNA, or did not affect the CD spectrum at all. The effect of these platinum compounds on the CD spectrum of the liquid crystalline microphase of DNA was either weak or absent. It is assumed that the specific biological action of platinum compounds of the cis-diamine type is determined by the polydentate binding to DNA: in addition to the cis-bidentate covalent binding of platinum to DNA nitrogen bases, a hydrogen bond formation between the DNA and cis-amino ligands occurs by means of protons at nitrogen atoms.

Circular Dichroism↗
PubMed2006

Cleavage of double-strand DNA by linear and triangular trinuclear copper complexes.

Two trinuclear copper(II) complexes with linear or triangular metal-binding pendants, [Cu(3)(L(1))](6+) (1) and [Cu(3)(L(2))](6+) (2), have been synthesized and characterized, where L(1)=N,N,N',N'-tetra[(2-pyridyl)methyl]-5,5'-bis(aminomethyl)-2,2'-dipyridyl, L(2)=N,N,N',N',N'',N''-hexa[(2-pyridyl)methyl]-1,3,5-tris(aminomethyl)-benzene. Interactions of them with calf thymus DNA have been investigated by measuring the changes in the melting temperature. The obtained DeltaT(m) values indicated that both 1 and 2 exhibited very high affinities towards DNA and strong destabilization of DNA, and were far better than their mononuclear analogues. In the absence of any reducing agent, 1 showed markedly higher nuclease activity than 2, and its hydrolytic process was further clarified in the presence of a few of radical scavengers. The pseudo Michaelis-Menten kinetic parameters (k(cat)) were determined through DNA cleavage versus the concentration of the complexes, to be 6.05 and 0.19 h(-1) for 1 and 2 respectively. Much higher nuclease activity of 1 is probably attributed to its linear multiple metal sites that fit well to the phosphodiester backbone of nucleic acid. DNA cleavage versus the concentration of DNA shows that rate constants rapidly increase at beginning and then markedly decrease with increasing DNA concentration. It is likely that excess substrates would promote intermolecular interaction between a complex and more plasmid DNA(s) and weaken its intramolecular cooperative effect that is propitious to DNA cleavage.

Copper↗
PubMed1990

Encapsulation of cells in agarose beads for use in the construction of human DNA libraries as yeast artificial chromosomes (YAC).

A simple and general method for the molecular cloning of fragments of over one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was developed for the construction of a human genomic DNA library in a yeast artificial YAC chromosome vector (in situ YAC construction). The main advantages of this method for use in the construction of a human genome library are as follows. First, linear DNA molecules of up to several hundred kilobase pairs in size can easily be prepared by the partial restriction enzyme digestion of the DNA encapsulated in agarose beads in vitro. Second, less than 2 x 10(6) cells scraped from tissue culture plates are sufficient for preparation of the linear DNA molecule for construction of the genome library. The technical manipulations involved in construction of clones of very large segments of DNA, including encapsulation of cells in agarose beads, restriction enzyme digestion, ligation with the YAC vector, transformation into host yeast cells, and stable propagation are discussed.

Blotting, Southern↗
PubMed1996

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

DNA bending induced by cruciform formation.

Cruciform structures in DNA are of considerable interest, both as extreme examples of sequence-dependent structural heterogeneity and as models for four-way junctions such as the Holliday junction of homologous genetic recombination. Cruciforms are of lower thermodynamic stability than regular duplex DNA, and have been observed only in negatively supercoiled molecules, where the unfavourable free energy of formation is offset by the topological relaxation of the torsionally stressed molecule. From an experimental viewpoint this can be a disadvantage, as cruciform structures can be studied only in relatively large supercoiled DNA circles, and are destabilized when a break is introduced at any point. We therefore set out to construct a pseudo-cruciform junction--by generating hereroduplex formation between two inverted repeat sequences. Stereochemically, this should closely resemble a true cruciform but remain stable in a linear DNA fragment. We have now created such a junction and find that it has the expected sensitivities to endonucleases. These DNA fragments exhibit extremely anomalous gel electrophoretic mobility, the extent of which depends on the relative position of the pseudo-cruciform along the length of the molecule. Our results are very similar to those obtained by Wu and Crothers using kinetoplast DNA, and we conclude that the pseudo-cruciform junction introduces a bend in the linear DNA molecule.

DNA↗
PubMedDate not supplied

[Influence of chemical analogues of microbial autoregulators on the sensitivity of DNA to UV radiation].

We established that chemical analogues of alkylhydroxybenzenes (AHB), belonging to alkylresorcinols and functioning as microbial autoregulatory d1 factors, enhance the UV resistance of various DNA molecules of different origin and conformation. These include the linear DNA of the lambda phage, bovine spleen DNA, and the DNA of the pUC19 plasmid that is composed of a number of annular (supercoiled and relaxed) and linearized molecules. Irradiating DNA with UV light (lambda = 254 nm) in the presence of methylresorcinol (MR) or hexylresorcinol (HR) results in comparatively insignificant DNA destruction as evidenced by our data on the electrophoretic mobility pattern in agarose gel. Using the linear Hind III restricts of the lambda phage DNA, we revealed that the protective effect of AHB varies depending on their chemical structure (it is more manifest with HR than MR) and concentration. Importantly, the effect of HR on bovine spleen DNA was based on its protective activity and manifested itself after a long incubation period. Studies using the pUC19 plasmid demonstrated that AHB, apart from increasing the resistance of linearized DNA molecules to UV irradiation, prevented both the supercoiled annular-supercoiled relaxed and the supercoiled relaxed-linearized transitions. The possible mechanisms of the UV-protective effect of AHB on DNA and their contributions to the resistance of dormant microbial forms to environmental factors are discussed.

Animals↗
PubMed1989

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA.

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.

Bacterial Proteins↗
PubMed1998

DNA looping by the Sfi I restriction endonuclease.

The SfiI endonuclease has to interact with two copies of its recognition sequence before it can cleave DNA. To demonstrate that the reaction of SfiI on a DNA with two sites involves the formation of a DNA loop, and to characterise the looping interactions on supercoiled and linear DNA, a series of plasmids was constructed with lengths of DNA between two SfiI sites varying from 104 to 211 bp. Both supercoiled and linear forms of each DNA were tested as substrates for SfiI. The reactions were monitored from the rates of DNA cleavage and from the generation of partially cleaved products, the latter indicating loop disruption before cleavage of both sites. On both supercoiled and linear DNA, the stabilities of the complexes spanning two SfiI sites varied in sinusoidal fashion with the distance between the sites, in the manner characteristic of a process governed by the helical periodicity of DNA. In all cases, the looping interaction was stabilised by DNA supercoiling. The sinusoidal variation from SfiI reactions on supercoiled DNA at 50 degreesC yielded a helical repeat of about 11.5 base-pairs per turn.

DNA↗
PubMedDate not supplied

Migration properties of circular DNAs using orthogonal-field-alternation gel electrophoresis.

The migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.

Animals↗
PubMed1993

Genome structure of a virus infecting the marine brown alga Ectocarpus siliculosus.

We describe a procedure for the isolation of virus particles from the marine brown alga Ectocarpus siliculosus. Virus particles are composed of at least 13 different polypeptides, including two glycoproteins, and double-stranded DNA. A typical virus DNA preparation contains three fractions, namely linear DNA and circular DNA, each composed of about 320 kilobase pairs, as well as DNA fragments, 10 to 60 kilobase pairs in size. The large linear and the circular DNA contain single-stranded regions (average length: 2.9 kilobase). We propose that the native Ectocarpus virus genome is a circular DNA molecule whose double strand is interrupted by single-stranded regions. During the preparation procedure, the DNA circles tend to break at the single-stranded sites producing large linear as well as fragmented DNA.

DNA, Viral↗