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.     

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.     

Site-specific OsO4 modification of the B-Z junctions formed at the (dA-dC)32 region in supercoiled DNA

OsO4 in the presence of pyridine specifically modifies the structural distortions of the primary helix of supercoiled pRW777 near the (dA-dC)32 sequence. Modification occurs at the same negative superhelix density value as required for formation of the Z-helix within the polymer block. Fine mapping of the distorted regions, which are probably the B-Z junctions, is presented. OsO4 reactions provide a powerful and sensitive chemical approach to study DNA polymorphism in solution.     

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.     

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.     

Effect of DNA supercoiling on the geometry of holliday junctions

Unusual DNA conformations including cruciforms play an important role in gene regulation and various DNA transactions. Cruciforms are also the models for Holliday junctions, the transient DNA conformations critically involved in DNA homologous and site-specific recombination, repair, and replication. Although the conformations of immobile Holliday junctions in linear DNA molecules have been analyzed with the use of various techniques, the role of DNA supercoiling has not been studied systematically. We utilized atomic force microscopy (AFM) to visualize cruciform geometry in plasmid DNA with different superhelical densities at various ionic conditions. Both folded and unfolded conformations of the cruciform were identified, and the data showed that DNA supercoiling shifts the equilibrium between folded and unfolded conformations of the cruciform toward the folded one. In topoisomers with low superhelical density, the population of the folded conformation is 50-80%, depending upon the ionic strength of the buffer and a type of cation added, whereas in the sample with high superhelical density, this population is as high as 98-100%. The time-lapse studies in aqueous solutions allowed us to observe the conformational transition of the cruciform directly. The time-dependent dynamics of the cruciform correlates with the structural changes revealed by the ensemble-averaged analysis of dry samples. Altogether, the data obtained show directly that DNA supercoiling is the major factor determining the Holliday junction conformation.     

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.     

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.     

The structure of intramolecular triplex DNA: atomic force microscopy study

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.     

S1 nuclease recognizes DNA conformational junctions between left-handed helical (dT-dG n. dC-dA)n and contiguous right-handed sequences

The ability of negative supercoiling to induce a left-handed helix in the recombinant plasmid pRW777, which contains a tract of 64 base pairs of almost perfect (dT-dG) . (dC-dA) from the mouse kappa immunoglobin gene, was studied. S1 nuclease recognizes and cleaves within the junction region which must exist adjacent to the (dT-dG)n . (dC-dA)n tract when in a left-handed state. The cleavage pattern indicates conformational flexibility and structural differences between the two existing junctions. The 64-base pair alternating copolymer undergoes the supercoil-induced formation of a left-handed state over the superhelical density range of -0.04 to -0.06, indicating that (dT-dG)n . (dC-dA) sequences form a left-handed helix less readily than (dC-dG)n . (dC-dG)n sequences of equivalent length. However, these supercoil densities are within the range found in vivo. Supercoil relaxation and antibody binding studies confirmed that the (dT-dG)n . (dC-dA)n tract in supercoiled pRW777 was in a left-handed helix.     

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.     

Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA.

The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling. We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification. We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength. The plasmids fall into a number of different classes, in terms of the conformational behavior. As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence. The two transitions are coupled through the topology of the molecule but are otherwise independent. When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment. Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions. Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences. These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules.     

Inverted repeats,stem-loops,and cruciforms: Significance for initiation of DNA replication

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.     

Z DNA and loop structures by immunoelectronmicroscopy of supercoiled pRW751, a plasmid containing left-handed helices.

Single and multiple loops were seen when the plasmid pRW751 was allowed to react with anti-Z-DNA or with a Z-specific cross-linking agent. Loop formation was dependent upon negative supercoiling and the presence of Z-specific antibody or cross-linking agent. Restriction enzyme mapping located 18 sites at the bottoms of loops, in addition to the two (dG-dC)n inserts of pRW751. No more than 5 loops were seen in any of the measured molecules; thus, not all potential Z-sites assume the Z conformation at any particular time. Stretches of alternating purine-pyrimidine sequences occur at all 20 sites. Almost all of the Z sites could be mapped to regions located at the beginnings or ends of reading frames or at various regulatory sites. Our findings support the concept that supercoiling brings distant sequences to within 5A of one another, allowing joint participation in regulatory processes controlled by DNA-binding proteins.     

Formation of Z-DNA in negatively supercoiled plasmids is sensitive to small changes in salt concentration within the physiological range.

Negative supercoiling of the plasmid pBR322 with or without an insert of (dG-dC)n induces the formation of Z-DNA as measured by the binding of antibodies specific for Z-DNA. Increasing the concentration of Na+ (or K+) is shown to inhibit the B to Z-DNA conversion. This may be due to the effect of the cation on the B-Z junction. Using the data for B to Z-DNA conversion of the (dG-dC)n inserts, we have estimated the free energy change per base pair as well as the energy of the B-Z junction. In pBR322, a 14-bp segment [CACGGGTGCGCATG] is believed to form Z-DNA at bacterial negative superhelical densities under salt conditions which are similar to those found in vivo.     

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.     

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strand-cutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.     

Influence of DNA sequence on the formation of non-B right-handed helices in oligopurine.oligopyrimidine inserts in plasmids

A systematic study was conducted on seven recombinant plasmids harboring synthetic inserts which had all purines on one strand and all pyrimidines on the complementary strand (Pur.Pyr). The inserts ranged in G+C content from 100% [G19.C19] to 0% [A20.T20] with intermediate contents at 66% [(TCC)8.(GGA)8], 50% [(CT)12.(AG)12 and (TTCC)6.(GGAA)6], 33% [(TTC)8.(GAA)8], and 25% [(GAAA)6.(TTTC)6]. The specific reactions at the base pair level of these inserts with enzymatic (S1 and P1 nucleases) and chemical (bromoacetaldehyde, OsO4, diethyl pyrocarbonate, and dimethyl sulfate) probes were evaluated as influenced by pH, negative supercoiling, and ionic strength (NaCl). Supercoil-induced relaxation studies using two-dimensional gels also provided important conformational information. We conclude that the five inserts with 66-25% G+C adopt a non-B right-handed conformation which is stabilized by negative supercoiling. Low pH (pH values 4.5-5.0) tends to stabilize this structure but is not essential for its formation. Surprisingly, an end bias of reactivity from the center toward the 5'-end of the purine strand of these inserts was generally found for the enzymatic and chemical probes which was irrespective of the orientation of the insert in the pRW790 vector. An intramolecular triple-stranded model for the unusual structure of the insert accounts most favorably for these observations. Unexpectedly, the A20.T20 insert seems to remain in an orthodox right-handed B-conformation under all conditions tested. The G19.C19 insert does adopt a non-B right-handed structure as for the five inserts with 66-25% G+C, but the pattern of reactivities and hence its conformation is different.     

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.