RESUMEN
Complex of osmium tetroxide with 1,10-phenanthroline (Os,phen) reacts with double-stranded B-DNA in contrast to osmium tetroxide, pyridine and other osmium structural probes which show a strong preference for single-stranded DNA (ssDNA) (Palecek, E. in Abelson, J.N., and Simon, M.I. (eds), Lilley, D.M.J., and Dahlberg, J.E., (volume eds.), Methods in Enzymology, Vol. 212, DNA Structures, part B., Academic Press, 139-155 (1992)). Modification of negatively supercoiled DNA (scDNA) with Os,phen changes the DNA electrophoretic mobility inducing the DNA relaxation at lower degrees of modification followed by formation of positive supercoils at higher modification extents. Electrophoretic mobility of the Os,phen-modified DNA fragments in agarose gel is almost unchanged while a strong retardation of the same fragments is observed in polyacrylamide gels. Os,phen-modified DNA is hypersensitive to nuclease S1. Cleavage of this DNA by restriction enzymes is selectively inhibited showing a preference of Os,phen for TA and AT dinucleotide steps. DNA modification by Os,phen is inhibited by low and moderate concentrations of MgCl2. The covalent binding of Os,phen to double-stranded DNA (dsDNA) is preceded by noncovalent interactions (probably intercalation) inducing DNA structural changes; the shape of the Os,phen-modified DNA molecule appears to be severely deformed.
Asunto(s)
ADN/metabolismo , Tetróxido de Osmio/metabolismo , Fenantrolinas/metabolismo , Composición de Base , ADN Superhelicoidal , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Etidio/metabolismo , Estructura Molecular , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Relación Estructura-ActividadRESUMEN
Superhelical tension of DNA in living bacteria is believed to be partially constrained by interaction with proteins. Yet DNA topology is a significant factor in a number of genetic functions and is apparently affected by both genetic and environmental influences. We have employed a technique that allows us to estimate the level of unconstrained superhelical tension inside the cell. We study the formation of cruciform structures by alternating adenine-thymine sequences in plasmid DNA by in situ chemical probing. This structural transition is driven by superhelical torsion in the DNA and thus reports directly on the level of such tension in the cellular DNA. We observe that the effect of osmotic shock is an elevation of superhelical tension; quantitative comparison with changes in plasmid linking number indicates that the alteration in DNA topology is all unconstrained. We also show that the synthesis of defective topoisomerase leads to increased superhelical tension in plasmid DNA. These experiments demonstrate that the effect of environmental and genetic influences is felt directly at the level of torsional stress in the cellular DNA.
Asunto(s)
ADN Superhelicoidal/metabolismo , Escherichia coli/genética , Conformación de Ácido Nucleico , Plásmidos , Composición de Base , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Mutagénesis , Mapeo RestrictivoRESUMEN
It was shown that the complex of osmium tetroxide with N,N,N',N'-tetramethylethylendiamine can be applied as a probe of DNA structure in the cell. This probe site-specifically recognized structural distortions at the B-Z junctions in plasmids pRW751 and pPK1 (containing (dC-dG)n segments) in E. coli cells.
Asunto(s)
Sondas de ADN , Etilenodiaminas , ADN Bacteriano , Escherichia coli/genética , Técnicas Genéticas , Tetróxido de Osmio , Plásmidos , Mapeo RestrictivoRESUMEN
Complexes of OsO4 with 2,2'-bipyridine (Os,2,2'-bipy),4,4'-bipyridine (Os,4,4'-bipy), 1,10-phenanthroline (Os,phe), bathophenanthroline disulfonic acid (Os,bpds) and OsO4, pyridine reagent (Os,py) were used to probe structural distortions at the junctions between right-handed B and left-handed Z DNA in supercoiled plasmids pRW751 and pPK1 (both containing (dC-dG)13 and (dC-dG)16 segments). With all five complexes the site-specific modification at the B-Z junctions was detected in vitro but only Os,2,2'-bipy and Os,bpds produced strong site specific modification at submillimolar concentrations. In addition to the B-Z junctions. Os,phe also reacted at other sites. With the exception of Os,2,2'-bipy no one of the tested OsO4 complexes has proved to be suitable for probing structural distortions at the B-Z junctions in E. coli cells.
Asunto(s)
ADN Recombinante , Conformación de Ácido Nucleico , Plásmidos , ADN Bacteriano , ADN Superhelicoidal , Escherichia coli/genética , Sondas Moleculares , Tetróxido de OsmioRESUMEN
Fourteen OsO4 complexes with different ligands were tested as probes of DNA structure. Of these complexes, only OsO4-2,2'-bipyridine (Os-bipy), OsO4-bathophenanthrolinedisulfonic acid (Os-bpds) and OsO4-N,N,N',N'-tetramethylenediamine (Os-TMEN) site-specifically modified the ColE1 cruciform in a supercoiled plasmid pColIR215 at millimolar concentrations. Os-bipy, Os-bpds and Os-TMEN also displayed site-specific modification of the B-Z junctions in the supercoiled plasmid pRW751 containing (dC-dG)n inserts.
Asunto(s)
Plásmidos de Bacteriocinas , ADN Bacteriano , Tetróxido de Osmio , Osmio , Plásmidos , ADN Bacteriano/análisis , ADN Bacteriano/metabolismo , ADN Superhelicoidal , Desoxirribonucleasa BamHI , Electroforesis , Ligandos , Estructura Molecular , Conformación de Ácido NucleicoRESUMEN
It is shown that osmium tetroxide, 2,2'-bipyridine can be applied as a probe of DNA structure in a bacterial cell. Using this probe we demonstrate (a) presence of structural distortions at the junctions between the right-handed B and left-handed Z DNA in a recombinant plasmid pRW751 and (b) unusual structure of the d(A-T)16 insert in pAT32 plasmid in E. coli cells and in in vitro.
Asunto(s)
ADN Bacteriano/ultraestructura , Tetróxido de Osmio/farmacología , Osmio/farmacología , Plásmidos , Enzimas de Restricción del ADN , ADN Bacteriano/efectos de los fármacos , Escherichia coli/genética , Conformación de Ácido Nucleico , Plásmidos/efectos de los fármacosRESUMEN
It was shown for the first time that the structural distortions at the junctions between contiguous right-handed and left-handed Z-DNA segments can be recognized in bacterial cells. E. coli containing recombinant plasmid pPK1 (a derivative of pUC19 containing (dC-dG)13 and (dC-dG)16 blocks) were treated with osmium tetroxide, 2.2'-bipyridine (Os,bipy); after this treatment pPK1 DNA was isolated by the boiling method. pPK1 DNA was then cleaved with BglI, and inhibition of BamHI (with its recognition sequence GGATCC lying on the boundary between the (dC-dG)n segments and the pUC19 nucleotide sequence) cleavage was tested. Treatment of cells with 2 mmol/l Os,bipy resulted in a strong inhibition of BamHI cleavage at both restriction sites showing a site-specific osmium modification at the B--Z junction. About the same inhibition of BamHI cleavage was observed after treatment of isolated pPK1 DNA with 0.2 mmol/l Os,bipy.
Asunto(s)
ADN Bacteriano/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II , Conformación de Ácido Nucleico , Tetróxido de Osmio/metabolismo , Osmio/metabolismo , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasa BamHI , Escherichia coli/genética , Plásmidos , Relación Estructura-ActividadRESUMEN
Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.
Asunto(s)
Composición de Base , ADN Superhelicoidal/ultraestructura , ADN/ultraestructura , Plásmidos , Secuencia de Bases , ADN Recombinante , ADN Superhelicoidal/efectos de los fármacos , Desoxirribonucleasa BamHI/antagonistas & inhibidores , Glioxal/farmacología , Conformación de Ácido Nucleico/efectos de los fármacos , Tetróxido de Osmio/farmacología , Piridinas/farmacología , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismoRESUMEN
Structural distortions on the boundary between right-handed B and left-handed Z DNA segments in plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of chemical probes. Samples of supercoiled DNA were treated with the respective chemical probe, linearized with EcoRI and inhibition of BamHI (whose recognition sequence GGATCC lies on the boundary between the (dC-dG)n segments and the pBR322 nucleotide sequence) cleavage was tested. Treatment with osmium tetroxide in the presence of pyridine or 2,2'-bipyridine, respectively, resulted in a strong inhibition of the BamHI cleavage at both restriction sites, provided the (dC-dG)n segments were in the left-handed form. In the presence of 2,2'-bipyridine submillimolar concentrations of OsO4 (at 26 degrees C) were sufficient to induce the inhibition of BamHI. Chloroacetaldehyde was used as a probe reacting selectively with atoms involved in the Watson-Crick hydrogen bonding. Similarly as in the case of osmium tetroxide treatment of pRW751 with this agent resulted in the inhibition of BamHI cleavage. It was concluded that the B-Z junction regions in pRW751 contain few solitary bases with disturbed hydrogen bonding or non-Watson-Crick base pairs.