Length-dependent cruciform extrusion in d(GTAC)n sequences

pBR322-derived plasmids have been constructed carrying d(GTAC)n.d(GTAC)n inserts of different lengths, in order to investigate the effect of insert size on cruciform extrusion and/or the B-Z transition. Plasmids with n ranging from 4 to 12 are hypersensitive to cleavage by the single-strand specific nucleases, S1 nuclease and Bal31 nuclease. Hypersensitive sites associated with the smaller alternating purine-pyrimidine tracts, however, coexist with the major pBR322 sites. Site-selective cleavage of these plasmids with the resolvase, T7 endonuclease I, demonstrates that all the inserts form cruciform structures when stably integrated into negatively supercoiled plasmids. An increase in the negative superhelical density of the DNA's induces cruciform formation within the insert region, resulting in a reduction in torsional stress consistent with the size of the insert. Moreover, as n decreases, the superhelical density required to stabilise the cruciform state increases. Therefore, the cruciform geometry is the favoured conformation of these d(GTAC)n.d(GTAC)n sequences under torsional stress. The stability of these cruciforms increases as n increases, with cruciformation occurring at lower superhelical densities and to the exclusion of the other pBR322 cruciforms.     

Supercoil-induced Z-DNA formation within 5'-end of chicken myb proto-oncogene

We have analyzed the recently sequenced and characterized 2.9 kb fragment derived from the 5'-end of chicken myb proto-oncogene with respect to structural perturbations induced by DNA supercoiling. Within the first intron a 50 bp sequence stretch was localized, starting approximately 450 nucleotides downstream from putative ATG initiation codon, which forms a non-B-DNA structure. Fine mapping with structural probes revealed the three adjacent regions with imperfect purine-pyrimidine alternation creating together relatively long Z-forming tract, parts of which may undergo a B-Z DNA transition at different superhelical densities.     

Length-Dependent Cruciform Extrusion in d(GTAC)n Sequences

Abstract

pBR322-derived plasmids have been constructed carrying d(GTAC)_n·d(GTAC)_n inserts of different lengths, in order to investigate the effect of insert size on cruciform extrusion and/or the B-Z transition. Plasmids with n ranging from 4 to 12 are hypersensitive to cleavage by the single-strand specific nucleases, S1 nuclease and Bal31 nuclease. Hypersensitive sites associated with the smaller alternating purine-pyrimidine tracts, however, coexist with the major pBR322 sites. Site-selective cleavage of these plasmids with the resolvase, T7 endonuclease I, demonstrates that all the inserts form cruciform structures when stably integrated into negatively supercoiled plasmids. An increase in the negative superhelical density of the DNA's induces cruciform formation within the insert region, resulting in a reduction in torsional stress consistent with the size of the insert. Moreover, as n decreases, the superhelical density required to stabilise the cruciform state increases. Therefore, the cruciform geometry is the favoured conformation of these d(GTAC)_n·d(GTAC)_n sequences under torsional stress. The stability of these cruciforms increases as n increases, with cruciformation occurring at lower superhelical densities and to the exclusion of the other pBR322 cruciforms.     

Structural changes in positively and negatively supercoiled DNA

The effect of superhelical constraint on the structure of covalently closed circular DNA (cccDNA; pBR322) with positive and negative writhe (superturn) has been investigated as a function of decreasing and increasing specific linking difference (mean superhelical density sigma). At low and moderate negative superhelical densities sigma, the overall average structure is maintained in an unwound B-form slightly modified. The overwound cccDNAs with positive writhe differ from those with negative writhe by an absence of cruciform structure. At high negative densities of supercoiling different changes involving the reversal of twist handedness are shown to lead to the formation of DNA segments in a conformation identical to the left-handed component of form V DNA.     

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.     

Chemical Probing of the B-Z Transition in Negatively Supercoiled DNA

Abstract

An analysis of the B-to-Z transition as a function of supercoiling for a natural Z-DNA- forming sequence found in plasmid pBR322 is presented at nucleotide resolution. The analysis is based on reactivity to four chemical probes which exhibit hyperreactivity in the presence of Z-DNA: hydroxylamine, osmium tetroxide, diethyl pyrocarbonate and dimethyl sulfate. We find that the initial transition occurs largely within a 14 base pair region which is mostly alternating purines and pyrimidines. With increasing negative supercoiling, Z-DNA extends into flanking regions having less and less alternating character, first in one direction and then in the other. Evidence of B-Z junctions is seen at four sites bracketing these three adjacent regions. One of these Z-forming regions contains the non-alternating sequence CTCCT, suggesting that such sequences can form Z-DNA without great difficulty if they are adjacent to alternating sequences. A plasmid containing three copies of a 61 base pair fragment bearing the entire Z-forming region shows equal reactivity of all three copies at any given superhelical density, implying that they compete equally and independently for the torsional strain energy which promotes the B-Z transition, and are unaffected by adjacent sequences more than 20–30 base pairs away.     

Influence of supercoiling on DNA structure: laser Raman spectroscopy of the plasmid pBR322

We report high-resolution Raman spectra obtained from the circularly closed double stranded DNA (Form I) of the plasmid pBR322 and from its corresponding linear form (Form III). Comparison of the Raman spectra of the two forms demonstrates that, at a superhelical density (σ) of ?0.069, which is of the same order as those found for most naturally occurring circularly closed DNAs, no major structural transitions occur under the influence of supercoiling. It is shown that at least 98% of all bases are fully basepaired, and that the conformation of the sugar–phosphate backbone is essentially identical to that of linear DNA. Thus, the structural influence of supercoiling, under these conditions, is confined to minor stretches of the plasmid DNA.     

Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes

Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density. The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322. Thus topoisomerase I is involved in determining the level of supercoiling in bacteria. Three of the topoisomerase I-defective strains we studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.     

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.