B-Z junctions in supercoiled pRW751 DNA contain unpaired bases or non-Watson-Crick base pairs

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.     

Recognition of the structural distortions at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide

It has been shown for the first time that conformational junction between contiguous right-handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)13 and (dC-dG)16 blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease S1 cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)13 segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Osmium tetroxide recognized structural distortions at junctions between right- and left-handed DNA in a bacterial cell

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.     

B-Z Junctions in Supercoiled pRW751 DNA Contain Unpaired Bases or Non-Watson-Crick Base Pairs

Abstract

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 SI 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 SI 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.     

Recognition of the Structural Distortions at the Junctions Between B and Z Segments in Negatively Supercoiled DNA by Osmium Tetroxide

Abstract

It has been shown for the first time that conformational junction between contiguous right- handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)₁₃ and (dC-dG)₁₆ blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease SI cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)_n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)₁₃ segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)_n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Probing of B-Z junctions in recombinant plasmids in vitro and in the cell with different osmium tetroxide complexes

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.     

Inhibition of restriction endonuclease cleavage due to site-specific chemical modification of the B-Z junction in supercoiled DNA

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.     

B-Z DNA junctions contain few, if any, nonpaired bases at physiological superhelical densities

The reactions of bromoacetaldehyde (BAA) with recombinant plasmids that contain sequences which can adopt left-handed Z structures or, at other locations, cruciforms were studied as a function of supercoil density. The sequence in pRW756 that undergoes a supercoil induced transition from a right to left-handed helix was (dC-dG)16 and regions near the replication origin of the pBR322 vector were converted from linearforms to cruciforms. The locations of the most nonpaired structural features were mapped by S1 nuclease cleavage of the "wedged open" duplexes after linearization of the DNAs. Three cruciforms in the pBR322 portions of the plasmids were specifically detected by BAA reaction at physiological supercoil densities (sigma = -0.067). However, the B-Z junctions did not react with BAA under these conditions although the junctions were present since the (dC-dG)16 was shown to be left-handed. Thus, the B-Z junctions have less single-stranded character than the pBR322 cruciforms (3-6 nonpaired bases) and may be fully paired. At much higher superhelical densities (sigma = -0.11-0.12), the B-Z junctions as well as the cruciforms react with BAA indicating a change in the nature of the junctions. Studies were also performed with pRW777 which harbors the mouse kappa immunoglobin sequence (dT-dG)32 . (dC-dA)32 that adopts a left-handed helix under appropriate conditions; the results were similar to those found with pRW756.     

Activation of DNA cleavage by dynemicin A in a B-Z conformational junction.

We report here that the DNA strand scission by dynemicin A is not only sequence-specific but also conformation-specific. The salt-induced B----Z conformational transition dramatically enhanced the cleavage by dynemicin A in a B-Z junction region. By contrast, the bleomycin-Fe(II) complex, the elsamicin A-Fe(II) complex, and esperamicin A1 did not induce any preferential DNA cutting in such a DNA structure. The characteristic hyperreactivity of dynemicin A is observed in (dC-dG)8- and (dC-dG)12-inserted DNAs, but not in (dC-dG)5-inserted DNA. These results suggest value in the use of dynemicin A as proof of the existence of a B-Z junction in vivo and also may aid in understanding the structure of B-Z junctions.     

The effect of Z-conformation on enzymatic activity of restriction endonucleases

Recombinant plasmid pGC20 containing (GC)9-insert into SmaI site of pUC19 has been used to study the inhibition of cleavage by six restriction endonucleases; KpnI, SacI, EcoRI and also BamHI, XbaI and SalI, due to Z-DNA formation in negatively supercoiled plasmid. The recognition sites of these enzymes were located at different distances on both sides of the (CG)10-sequence. It was shown that the inhibition of the cleavage by KpnI, SacI and EcoRI was decreased in this series as fast as the distance between recognition site and B-Z junction was increased, and no inhibition of cleavage by EcoRI was found. However, such a correlation was not found in the series of BamHI, XbaI and SalI. In contrast with EcoRI the cleavage by SalI was inhibited completely. These results indicate the difference for "sensitivity" of restriction endonucleases to the structural perturbations of DNA associated with B-Z junctions. It seems to depend on features of the enzyme-substrate interaction mechanisms and also on recognition and flanking sequences of DNA. Consequently, experiments with the inhibition of the cleavage by any enzyme can not help to determine the dimension of the region of DNA with altered structure.     

Left-handed Z-DNA helices in polymers, restriction fragments, and recombinant plasmids

Studies on DNA polymers, restriction fragments, and recombinant plasmids have revealed the following: A) A family of left-handed DNA conformations exists for (dC-dG)n.(dC-dG)n. The observation of a particular conformation is dependent on the salt, the salt concentration and dehydrating agent. B) In sodium acetate solutions, (dC-dG)n.(dC-dG)n forms left-handed, psi(+)-condensed structures as detected by Raman spectroscopy and circular dichroism. C) (dT-dG)n.(dC-dA)n undergoes a right-to-left-handed transition only when reacted with AAF and at high salt concentrations. D) Transitions observed for polymer DNAs also are observed for restriction fragments containing both (dC-dG).(dC-dG) and (dT-dG).(dC-dA) sequences, but the transitions in the fragments generally require higher salt concentrations than observed for the polymers. E) Studies with recombinant plasmids containing (dC-dG) sequences from 10 to 58 bp in length demonstrate that left-handed Z-DNA segments can exist contiguous to B-DNA segments. F) Negative supercoil density (sigma less than or equal to -0.072) is sufficient to convert the (dC-dG) regions in those plasmids into left-handed structures under physiological ionic conditions (200 mM NaCl). G) The favorable free energy contribution of methylation in stabilizing the Z form in fragments and plasmids is approximately offset by the unfavorable free energy contributions of the B/Z junctions. H) Sl and BAL 31 nucleases recognize aberrant structural features at the confluence of the B and Z regions. I) Detailed mapping of Sl nuclease cleavage on supercoiled plasmids shows that the nuclease sensitive regions extend over at least five to ten bp. J) Even though the (dT-dG)n.(dC-dA)n polymer requires base modification and high salt conditions to undergo the R----L transition, supercoiling (sigma less than or equal to -0.07) can supply enough energy to allow a plasmid containing the intervening sequence of a human fetal globin gene with (dT-dG).(dC-dA) sequences to undergo a R----L transition.     

The ultimate carcinogen of 4-nitroquinoline 1-oxide does not react with Z-DNA and hyperreacts with B-Z junctions.

DNA secondary and tertiary structures are known to affect the reaction between the double helix and several damaging agents. We have previously shown that the tertiary structure of DNA influences the reactivity of 4-acetoxyaminoquinoline 1-oxide (Ac-4-HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide (4-NQO), being more reactive with naturally supercoiled DNA than with relaxed DNA. The relative proportion of the three main stable adducts and of an unstable adduct, that resulted in strand scission and/or AP sites, was also affected by the degree of supercoiling of plasmid DNA. In this study we examined the influence of Z-DNA structure on the reactivity of Ac-4-HAQO by mapping the distribution of the two main Ac-4-HAQO adducts, C8-guanine and N2-guanine, along a (dC-dG)16 sequence inserted at the BamHI site of pBR322 plasmid DNA. This insert adopted the left-handed Z and right-handed B structure depending on the superhelical density of the plasmid. Sites of C8-guanine adduct formation were determined by hot piperidine cleavage of Ac-4-HAQO modified DNA, while N2-guanine adducts were mapped by the arrest of the 3'-5' exonuclease activity of T4 DNA polymerase. The results showed that Ac-4-HAQO did not react with guanine residues when the (dC-dG)16 sequence was in Z conformation, while hyperreactivity at the B-Z junction was observed. These results indicate that Ac-4-HAQO can probe the polymorphism of DNA at the nucleotide level.     

Antibodies to DNAs chemically modified with osmium structural probes

It has previously been shown that osmium tetroxide, pyridine (Os,py) and osmium tetroxide, 2,2'-bipyridine (Os,bipy) are powerful probes of the DNA structure. To increase the possibilities of the detection of osmium-modified DNAs polyclonal antibodies against DNA modified with Os,py and Os,bipy were elicited in rabbits. Specificity of these sera or purified IgG was tested by ELISA and retardation of the DNA electrophoretic mobility in agarose gels. Antibodies against DNA-Os,py (anti-DNA-Os,py) reacted with single-stranded and double-stranded DNA-Os,py but they did not react with unmodified DNA; with DNA-Os,bipy only a weak reaction was observed. The specificity of the anti-DNA-Os,bipy was similar. Competition experiments with anti-DNA-Os,py showed a weak reaction with RNA-Os,py but no reaction with osmium-modified proteins and unmodified proteins and RNA. The results suggest that anti-DNA-Os,py may become an important tool in studies of DNA structure in situ.     

Probing of DNA structure with osmium tetroxide. Effect of ligands

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.     

Effects of 5 cytosine methylation on the B-Z transition in DNA restriction fragments and recombinant plasmids

Alternating (dC-dG)n regions in DNA restriction fragments and recombinant plasmids were methylated at the 5 position of the cytosine residues by the HhaI methylase. Methylation lowers the concentration of NaCl or MgCl2 necessary to cause the B-Z conformational transition in these sequences. Ionic strengths higher than physiological conditions are required to form the Z conformation when the methylated (dC-dG)n tract is contiguous with regions that do not form Z structures, in contrast to the results with the DNA polymer poly(m5dC-dG) . poly(m5dC-dG). In supercoiled plasmids containing (dC-dG)n sequences, methylation reduces the number of negative supercoils necessary to stabilize the Z conformation. Calculations of the observed free energy contributions of the B-Z junction and cytosine methylation suggest that two junctions offset the favorable effect of methylation on the Z conformation in (dC-dG)n sequences (about 29 base-pairs in length). Studies with individual methylated topoisomers demonstrate that increasing Na+ concentration up to approximately 0.2 M inhibits the formation of the Z conformation in the (m5dC-dG)n region of supercoiled plasmids. The results suggest that methylation may serve as a triggering mechanism for Z DNA formation in supercoiled DNAs.     

Osmium tetroxide probing of local DNA structure in linear and supercoiled plasmids containing curvature-inducing sequences

Recombinant plasmids pK1A108, pK3A108, pK4A108 and pK5/6T217 containing 80 +/- 1 base pair inserts with different curvature-inducing sequences were studied using the DNA structure probe osmium tetroxide in the presence of pyridine (Os, py). The insertion sequences of the plasmids pK1A108, pK3A108, and pK4A108 are strongly related while the degree of curvature increases from pK1A108 (no curvature) less than pK3A108 less than pK4A108 less than pK5/6T217. The Os, py probe reacts selectively with single-stranded and distorted double-stranded regions in the DNA double helix. Nuclease S1 was used to recognize and cleave regions made permanently single-stranded due to osmium recognize and cleave regions made permanently single-stranded due to osmium modification. In linearized plasmids treatment with Os, py produced no S1-detectable site-specific modification. This result is in agreement with models suggested for DNA curvature; in general, continuous base pairing and base stacking is considered through different sequence blocks as well as through structural junctions. Os, py-probing of the plasmids in the supercoiled state also resulted in no S1-detectable site-specific modification within the inserts of pK1A108, pK3A108, and pK4A108 plasmids (while the regions containing inverted repeat nucleotide sequences in these plasmids were site-specifically modified). In contrast, supercoiled pK5/6T217 DNA was site-specifically modified within the curvature-inducing insert sequence. The nucleotide sequence of the insert of this plasmid strongly differs from the insertion sequences of the other three plasmids; it is extremely AT-rich and contains regularly arranged dAGAGA and dATATA sequences. The structural distortion observed in supercoiled pK5/6T217 is most probably due to the presence of these sequences in a particular arrangement in the insertion sequence.     

Chemical probing of the homopurine.homopyrimidine tract in supercoiled DNA at single-nucleotide resolution

Local structure of the homopurine.homopyrimidine tract in a supercoiled plasmid pEJ4 was studied using chemical probes at single-nucleotide resolution. The conformation of the homopyrimidine strand was probed by osmium tetroxide, pyridine (Os,py) while that of the homopurine strand was tested by diethyl pyrocarbonate (DEPC), i.e. by probes reacting preferentially with single-stranded DNA. At weakly acidic pH values, a strong Os,py attack on three nucleotides at the centre of the (dC-dT)16 block and a weaker attack on two nucleotides at the end of the block were observed. DEPC modified adenines in the 5'-half of the homopurine strand. Os,py modification at the centre of the block corresponded to the loop of the hairpin formed by the homopyrimidine tract, while DEPC modification corresponded to the unstructured half of the homopurine strand in the model of protonated triplex H form of DNA.     

Cytosine methylation enhances Z-DNA formation in vivo.

The influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in Escherichia coli was analyzed by two independent assays. Both the M.EcoRI inhibition assay and the linking-number assay have been used previously to establish that dC-dG segments of sufficient lengths can exist as left-handed helices in vivo. A series of dC-dG plasmid inserts with Z-form potential, ranging in length from 14 to 74 base pairs, was investigated. Complete methylation of cytosine at all HhaI sites, including the inserts, was obtained by coexpression of the HhaI methyltransferase (M.HhaI) in cells also carrying a dC-dG-containing plasmid. Both assays showed that for all lengths of dC-dG inserts, the relative amounts of B and Z helices were shifted to more Z-DNA in the presence of M.HhaI than in the absence of M.HhaI. These results indicate that cytosine methylation enhances the formation of Z-DNA helices at the superhelix density present in E. coli. The B-Z equilibrium, in combination with site-specific base methylation, may constitute a concerted mechanism for the modulation of DNA topology and DNA-protein interactions.     

Stochastic distribution of a short region of Z-DNA within a long repeated sequence in negatively supercoiled plasmids

We have analyzed, at nucleotide resolution, the progress of the B-to-Z transition as a function of superhelical density in a 2.2-kilobase plasmid containing the sequence d(C-A)31.d(T-G)31. The transition was monitored by means of reactivity to two chemical probes: diethyl pyrocarbonate, which is sensitive to the presence of Z-DNA, and hydroxylamine, which detects B-Z junctions. At a threshold negative superhelical density between about 0.048 and 0.056, hyper-reactivity to diethyl pyrocarbonate appears throughout the CA/TG repeat and remains as the superhelical density is further increased. However, there is no reactivity characteristic of B-Z junctions until the superhelical density reaches 0.084, when single cytosines at each end of the repeat become hyper-reactive to hydroxylamine. A two-dimensional gel analysis of this system by others (Haniford, D. B., and Pulleyblank, D. E. (1983) Nature 302, 632-634) indicates that only about half of the 62 base pairs of the CA/TG repeat undergo the initial transition at omega = 0.056. Our results indicate that this region of Z-DNA is free to exist anywhere along the CA/TG repeat and is probably constantly in motion. Well defined B-Z junctions are seen only when there is sufficient supercoiling to convert the entire CA/TG sequence to Z-DNA. The implications for possible B-Z transitions in chromosomal domains of different sizes are discussed.     

Comparison of the reactivity of B-DNA and Z-DNA with two isosteric chemical carcinogens: 2-N,N-acetoxyacetylaminofluorene and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido-[1,2-a:3'',2'' -d] imidazole.

The reactivity of nucleic acids in various conformations and two isosteric chemical carcinogens 2-N,N-acetoxyacetylaminofluorene (N-AcO-AAF) and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido [1,2-a:3',2'-d] imidazole (N-AcO-AGlu-P-3) have been studied. Both carcinogens bind covalently to poly(dG-dC).poly(dG-dC) (B form) and to poly(dG-br5C).poly(dG-br5dC) (Z form). They also bind covalently to (dC-dG)16 and to (dG-dT)15 sequences inserted in plasmids when the inserts are in the B form but they do not bind to the inserts in the Z form. The reactivity of guanine residues at the B-Z junctions depends upon the superhelical density of the plasmids and upon the base sequences at the junction. The distribution of AGlu-P-3 modified guanines in a restriction fragment of pBR322 is not uniform and is different from that of AAF-modified guanines. The conclusion is that N-AcO-Glu-P-3 as N-AcO-AAF can probe at the nucleotide level the polymorphism of DNA. On the other hand, the non-reactivity of both chemical carcinogens and Z-DNA and the hyperreactivity of some junctions might have some importance in the understanding of chemical carcinogenesis.