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.     

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.     

Rh(DIP)3(3+): a shape-selective metal complex which targets cruciforms.

The coordination complex tris(4,7-diphenylphenanthroline)rhodium(III), Rh(DIP)3(3+), binds to and, upon photoactivation, cleaves both DNA strands near the base of a DNA cruciform. Sites of photoinduced double-stranded DNA cleavage by the rhodium complex map to regions containing cruciforms on closed circular pBR322, pColE1 and phi X174 (replicative form) DNAs. Neither cleavage nor binding by the metal complex, assayed using S1 nuclease, is found on the linear plasmid which lacks the extruded cruciform. High resolution mapping experiments reveal that Rh(DIP)3(3+) cleaves at a specific AT-rich site neighboring the stem of the minor cruciform on pBR322. The primary site of cleavage is found at position 3238 on the 3'-strand and 3250 on the 5'-strand and is remarkably specific. The pattern of cleavage, to one side only of the cruciform stem, indicates an asymmetry in the cruciform structure recognized by the complex. These results suggest that Rh(DIP)3(3+) may provide a useful reagent to probe cruciform sites. In addition, the high degree of specificity found in targeting the cruciform structure with this simple metal complex underscores the utility of shape-selection for the recognition of specific sites on a DNA strand.     

Structural interconversion of alternating purine-pyrimidine inverted repeats cloned in supercoiled plasmids.

Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a structural transition in the primary helix of both inserts. The first sequence converts into the left-handed helix, whereas the second sequence undergoes a transition into a cruciform or a Z-type structure depending on the experimental conditions employed. This has been deduced from the mapping of S1 nuclease sensitive sites, OsO4-sensitive sites, DEP modification pattern and relaxation studies. In addition, the differential effect of 5-cytosine methylation and binding of the AT-specific drug distamycin on these transitions further supports this interpretation. Thus, it is demonstrated, that the same sequence which is both inverted repeat and alternating purine-pyrimidine type may adopt either the left-handed conformation or the cruciform structure in response to the superhelical stress. Formation of the Z-type helix can be transmitted through the d(AT)n region which is 10 bp in length.     

Dynamics of cruciform extrusion in supercoiled DNA: use of a synthetic inverted repeat to study conformational populations.

An inverted repeat has been created in a plasmid by ligation of two 13 nucleotide synthetic oligonucleotides into the cloning vector pAT153. The resulting recombinant plasmid, pIRbke8, is hypersensitive to cleavage by the single-strand-specific nuclease S1, and to modification by the single-strand-selective reagent bromoacetaldehyde, when the plasmid is negatively supercoiled. The new inverted repeat is a stronger S1 site than those derived from pBR322, but, in contrast to the ColE1 and phi X174 RF inverted repeats, these repeats share a similar temperature dependence. The kinetics of EcoRI cleavage at the centre of the synthetic inverted repeat have been studied in supercoiled and linear molecules. It is found that in the supercoiled molecule this target is not refractory to EcoRI cleavage to an extent which is greater than the resolution of the experiment. We conclude that in this molecule the cruciform is in a dynamic equilibrium with the regular duplex, in which the cruciform constitutes a relatively small subpopulation of conformational species.     

A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease

T7 endonuclease preferentially cleaves purified supercoiled pBR322 and colE1 plasmids at the single-stranded regions exposed when palindromic sequences assume cruciform structures (Panayotatos, N., and Wells, R.D. (1981) Nature 289, 466-470). In vivo, however, induction of nuclease synthesis off a cloned gene caused complete degradation of the bacterial DNA but not of the plasmid vector; presumably, single-stranded regions (cruciforms?) on the genome effectively complete for the nuclease with similar sites on the plasmid (Panayotatos, N., and Fontaine, A. (1985) J. Biol. Chem. 260, 3173-3177). To overcome this competition, we introduced on the plasmid the naturally occurring colE1 palindrome which forms a more stable cruciform in vitro. In addition, we increased the target size (and the T7 endonuclease gene dosage) by raising the copy number of the plasmid 5-fold. Induction of the endonuclease encoded by this new plasmid (pLAT75) resulted not only in degradation of genomic DNA but also in intracellular nicking and linearization of the plasmid. The cleavage site in vivo was mapped at the colE1 palindrome and coincided with the site cleaved specifically in vitro by either T7 or S1 endonuclease only when this palindrome assumes the cruciform structure. These results indicate that cruciform structures exist intracellularly and demonstrate the usefulness of endonucleases as probes of DNA topology in vivo.     

Chromatin structure of the potential Z-forming sequence (dT-dG)n X (dC-dA)n. Evidence for an "alternating-B" conformation

The sequence (dT-dG)n X (dC-dA)n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG)n X (dC-dA)n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG)n X (dC-dA)n elements instead adopt an "alternating-B" conformation on the nucleosomal surface.     

Nucleosome "phasing" and cruciform structures in circular supercoiled pBR322 DNA

Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive sites are very near to AT-rich regions of pBR322 genome. A nucleosome "phasing" in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis. On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed by nucleosome formation.     

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.     

Nucleosome “phasing” and cruciform structures in circular supercoiled pBR322 DNA

Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive sites are very near to AT-rich regions of pBR322 genome. A nucleosome “phasing” in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis. On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed by nucleosome formation.     

(A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable.

Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules. These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease. Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation. These reactivities have a requirement for an alternating sequence. In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound. We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.