Analysis of trimethylpsoralen photoreactivity to Z-DNA provides a general in vivo assay for Z-DNA: analysis of the hypersensitivity of (GT)n B-Z junctions

We have described an exonuclease III/photoreversal procedure to map, with base pair resolution, the bases which have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand cross-links in DNA (20). This assay allows quantitation of relative rates of Me3-psoralen photobinding to bases in DNA at levels as low as one cross-link per 8,000 base pairs. This assay should be useful for a wide variety of applications of Me3-psoralen photobinding to DNA. Here, we demonstrate the applicability of the Me3-psoralen exo III assay for analysis of the conformation of the Z forming sequences (GT)12ATGT and GAATTC(TG)6TA(TG)6. We have shown previously that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation (34). More recently we have confirmed this result with the exo III assay and have shown at least a hundred fold increase in Me3-psoralen photoreactivity at the 5'AT sequence within the EcoR I sites (GAATTC) which presumably represent B-Z junctions flanking (CG)6TA(CG)6 (20). Here we demonstrate both the characteristic decrease in psoralen photobinding to 5'TAs within (GT)12ATGT and (TG)6TA(TG)6 and the hyperreactivity of B-Z junctions. These characteristic properties of Me3-psoralen photobinding provide an assay for Z-DNA that is applicable in vivo. The general applicability of this approach for assaying Z-DNA in vivo is discussed.     

Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling

Z-DNA-forming sequences, (GT)21, (GT)12ATGT, and (CG)6TA(CG)6, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. 4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)6TA(CG)6, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.     

Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells

The rate of covalent photobinding of trimethylpsoralen to DNA is greater when the DNA is wound with negative superhelical tension than when it is relaxed. In vitro the rate of photobinding is directly proportional to the negative superhelical density of the DNA. Thus measurement of the rate of photobinding provides an assay for probing in vivo unrestrained tension in the winding of the DNA double helix. This approach has been applied to measure torsional tension in DNA as it is packaged in living E. coli. Drosophila and HeLa cells. A method is described for measuring the rate of photobinding to intracellular DNA and rRNA, and for using the latter measurement as an internal control of the rate of me3-psoralen photobinding in vivo. This permits more accurate and reproducible measurement of changes in the DNA-psoralen photobinding reaction. The me3-psoralen probe interacts with intracellular bacterial DNA as expected for a purified DNA duplex wound with superhelical density sigma = -0.05 +/- 0.01. This superhelical tension is relaxed in cells when multiple single-strand breaks are introduced into the chromosomal DNA by gamma-irradiation. Similar relaxation occurs when cells are treated with the DNA gyrase inhibitor coumermycin. The results suggest that the DNA double helix is wound with torsional tension in vivo and that DNA supercoils which are equilibrated with this tension are not completely restrained in nucleosome-like structures. Torsional tension in the DNA of eucaryotic cells is not detectable in analogous measurements of the packaged DNA of HeLa and Drosophila cells. The simplest interpretation of this finding is that, within the limits of detection, all superhelical turns in the DNA are restrained in nucleosomes or nucleosome-like structures in these eucaryotic cells.     

Crystal and molecular structure of a DNA duplex containing the carcinogenic lesion O6-methylguanine

The crystal and molecular structure of the first DNA duplex containing the carcinogenic lesion O6MeG has been determined to a resolution of 1.9 A and refined to an R factor of 19%. (d[CGC-(O6Me)GCG])2 crystallizes in the left-handed Z DNA form and has crystal parameters and conformational features similar to those of the parent sequence [d(CG)3]2. The methyl groups on O6 of G4 and G10 have C5-C6-O6-O6Me torsion angles of 73 degrees and 56 degrees, respectively, and protrude onto the major groove surface. The base-pairing conformation for the methylated G.C base pairs is of the Watson-Crick type as opposed to a wobble-type conformation that had been proposed in a B DNA fragment. As in other Z DNA structures, a spine of hydration is seen in the minor groove.     

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.     

Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences

The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.     

Pliable DNA conformation of response elements bound to transcription factor p63

We show that changes in the nucleotide sequence alter the DNA conformation in the crystal structures of p63 DNA-binding domain (p63DBD) bound to its response element. The conformation of a 22-bp canonical response element containing an AT spacer between the two half-sites is unaltered compared with that containing a TA spacer, exhibiting superhelical trajectory. In contrast, a GC spacers abolishes the DNA superhelical trajectory and exhibits less bent DNA, suggesting that increased GC content accompanies increased double helix rigidity. A 19-bp DNA, representing an AT-rich response element with overlapping half-sites, maintains superhelical trajectory and reveals two interacting p63DBD dimers crossing one another at 120°. p63DBD binding assays to response elements of increasing length complement the structural studies. We propose that DNA deformation may affect promoter activity, that the ability of p63DBD to bind to superhelical DNA suggests that it is capable of binding to nucleosomes, and that overlapping response elements may provide a mechanism to distinguish between p63 and p53 promoters.     

Long (dA)n.(dT)n tracts can form intramolecular triplexes under superhelical stress.

Plasmids containing long tracts of (dA)n.(dT)n have been prepared and their conformations examined in linear and supercoiled DNA using a series of chemical and enzymic probes which are known to be sensitive to unusual DNA structures. Under superhelical stress and in the presence of magnesium the sequence T69.A69 adopts a conformation at pH 8.0 consistent with the formation of an intramolecular DNA triplex. Site specific cleavage of the supercoiled plasmid by single-strand specific nucleases occurs within the A.T insert; the 5'-end of the purine strand is sensitive to reaction with diethylpyrocarbonate while the central 5-6 bases of the pyrimidine strand are reactive to osmium tetroxide. By contrast shorter inserts of A33.T33 and A23.T23 do not appear to form unusual structures.     

The effect of zinc on the secondary structure of d(GA.TC)n DNA sequences of different length: a model for the formation *H-DNA.

Alternating d(GA.TC)n DNA sequences are known to undergo transition to *H-DNA in the presence of zinc. Here, the effect of zinc on the secondary DNA structure of d(GA.TC)n sequences of different length (n = 5, 8, 10 and 19) was determined. Short d(GA.TC)n sequences form *H-DNA with a higher difficulty than longer ones. At bacterial negative superhelical density (- sigma = 0.05), zinc still induces transition to the *H-DNA conformation at a d(GA.TC)10 sequence but shorter sequences do not form *H-DNA. Transition to *H-DNA at a d(GA.TC)8 sequence is observed under conditions which destabilize the DNA double helix such as high negative supercoiling or low ionic strength. Our results indicate that a first step in the transition to *H-DNA is the formation of a denaturation bubble at the centre of the repeated DNA sequence, suggesting that the primary role of zinc is to induce a local denaturation of the DNA double helix. Subsequently, zinc might also participate in the stabilization of the altered DNA conformation through its direct interaction with the bases. Based on these results a model for the formation of *H-DNA is proposed.     

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.     

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.     

DNA duplex with the potential to change handedness after every half a turn.

Polymorphic forms of the DNA duplex with long stretches of structural monotony are known. Several alternating purine-pyrimidine sequences have been shown to adopt left-handed Z-conformation. We report a DNA sequence d(CGCGCGATCGAT)n exhibiting alternating right-handed B and left-handed Z helical conformation after every half a turn. Further, this unusual conformation with change in handedness after every six base pairs was induced at physiological superhelical density.     

Facile interconversion of duplex structures formed by copolymers of d(CG)

Correlations between DNA sequence and reactivity have often been drawn with an implicit or explicit connection to duplex structure. An in vitro model using oligonucleotides of defined sequences has been developed to characterize a potential source of the hypersensitivity that naturally occurring regions of redundant sequence exhibit with many nucleases. S-1 nuclease was used here to diagnose the unusual hybridization of copolymeric DNA, d(CG)6, and related oligomers, through product and kinetic analysis. Fully complementary but redundant sequences reacted with this enzyme almost an order of magnitude faster than did heterogeneous fragments of DNA. Hydrolysis products of the copolymers indicated that conformations with unpaired termini were the sole substrates under these studies, and only a facile equilibrium between aligned and extended structures was required to explain the heightened reactivity of this DNA. For example, d(CG)6 was converted to d(CG)5 and d(CG)4 whereas d(CG)4C was initially processed to an octamer and then only later to a hexamer. Catalysis by S-1 exhibited no other substrate or product specificity; even the disordered bases in the loop region of a hairpin structure, d(CG)3T4(CG)3, did not provide sites of enhanced enzyme action. The rate of DNA consumption under standard conditions was proportional to the expected concentration of overhanging sequences rather than the absolute amount of DNA present. All initial attempts to saturate enzyme activity failed, and therefore, the rate of substrate formation through strand slippage was always faster than the catalytic depletion of unpaired bases. Only a low-energy transition state(s) must then separate the various hybridized species since this structural equilibration proceeded readily under conditions of 10 mM potassium phosphate, pH 7, 100 mM NaCl, and 22 degrees C.     

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.     

Further analysis of the altered secondary structure of superhelical DNA. Sensitivity to methylmercuric hydroxide a chemical probe for unpaired bases

A previous study in our laboratory of the reaction of formaldehyde with super-helical DNAs (φX replicative form and PM2) has led to a model for superhelical DNA in which there is a region or regions of altered secondary structure containing unpaired bases. Similar experiments using the nicked circular DNA gave no evidence of interruptions of base pairing. In this study we present additional data, which support the above model as well as extending our analysis of the secondary structure of superhelical DNA and the dynamics of the early denaturation process. In a series of experiments involving the binding of methyl-mercury as a chemical probe of unpaired bases, we obtained the following results. (1) Initially, both s⁰_20w and the buoyant density of the superhelical form of phage PM2 DNA increased as a function of methylmercuric hydroxide concentration, whereas the nicked form did not. (2) This initial binding is accompanied by an increase in superhelical content τ from ?41 to ?46 turns. (3) The binding analysis allows us to estimate that 3.7% of the bases contain methylmercury in this phase of the transition. This is in excellent agreement with the extent of formylation. (4) Such a preformylated molecule shows a shift in the transition to lower mercurial concentrations. These results are interpreted as follows. The initial increase in ?τ excludes the possibility that binding occurs to normal base-paired structures, since this would produce a coupled unwinding of duplex and superhelical turns. The additive effects of formylation and methylmercury binding support the concept that both chemical probes attack the same sites and induce similar structural changes. Thus the evidence clearly supports the view that superhelical DNA contains localized region(s) of interrupted base pairing. Recent studies from other laboratories using single strand-specific endonucleases are in complete agreement with this model.     

The zab domain of the human RNA editing enzyme ADAR1 recognizes Z-DNA when surrounded by B-DNA

The Zab domain of the editing enzyme ADAR1 binds tightly and specifically to Z-DNA stabilized by bromination or supercoiling. A stoichiometric amount of protein has been shown to convert a substrate of suitable sequence to the Z form, as demonstrated by a characteristic change in the CD spectrum of the DNA. Now we show that Zab can bind not only to isolated Z-forming d(CG)(n) sequences but also to d(CG)(n) embedded in B-DNA. The binding of Zab to such sequences results in a complex including Z-DNA, B-DNA, and two B-Z junctions. In this complex, the d(CG)(n) sequence, but not the flanking region, is in the Z conformation. The presence of Z-DNA was detected by cleavage with a Z-DNA specific nuclease, by undermethylation using Z-DNA sensitive SssI methylase, and by circular dichroism. It is possible that Zab binds to B-DNA with low affinity and flips any favorable sequence into Z-DNA, resulting in a high affinity complex. Alternatively, Zab may capture Z-DNA that exists transiently in solution. The binding of Zab to potential as well as established Z-DNA segments suggests that the range of biological substrates might be wider than previously thought.     

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.     

DNA conformational variations in the in vitro torsionally strained Ig ϰ light chain gene localize on consensus sequences

We have analyzed the localization and the dependence upon superhelical density of the DNA sites which modify their conformation under torsional strain in a mouse Ig Lϰ gene. The conformational variations occur on DNA sites which have been defined as protein interaction sites and consensus sequence motifs: the 5′-upstream regulatory decanucleotides, the TATA sequence, the consensus heptanucleotides of the J recombinational sequences.