Reaction conditions affect the specificity of bromoacetaldehyde as a probe for DNA cruciforms and B-Z junctions.

The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.     

Torsional stress stabilizes extended base unpairing in suppressor sites flanking immunoglobulin heavy chain enhancer

DNA sequences surrounding the immunoglobulin heavy chain (IgH) enhancer contain negative regulatory elements which are important for the tissue specificity of the enhancer. We have shown that sequences located both 5' and 3' of the enhancer, corresponding to the negative regulatory elements, become stably and uniformly unpaired over an extended length when subjected to torsional stress. These DNA sequences are also included within matrix association regions. The ability of the sequences to assume a stably unpaired conformation was shown by reactivity with chloroacetaldehyde which is specific for unpaired DNA bases, as well as two-dimensional gel electrophoresis of topoisomers. The sequences located 3' of the enhancer induce base unpairing in the direction of the enhancer. This unpaired region progressively expands to include as much as 200 base pairs as the ionic concentration decreases or superhelical density increases. When an ATATAT motif within a negative regulatory element located 3' of the enhancer was mutated, the extensive base-unpairing property was abolished. This base-unpairing property of DNA may be important for negative regulation of gene expression and attachment to the nuclear matrix.     

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.     

A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.     

Kinetics and effect of salts and polyamines on T4 polynucleotide ligase.

The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.     

Meiotic double-strand breaks in yeast artificial chromosomes containing human DNA.

Meiotic recombination in the yeast Saccharomyces cerevisiae is initiated by double-strand breaks (DSB) in chromosomal DNA. These DSB, which can be mapped in the rad 50S mutant yeast strain, are caused by a topoisomerase II-like enzyme, the protein Spo11. Evidence suggests that this protein is located in the axial element of the meiotic chromosome which implies that the DSB are located in these chromosomes in the vicinity of the bases of the DNA loops. We have found that in the yeast artificial chromosomes carrying human DNA, at the level of resolution obtained by pulsed field gel electrophoresis (PFGE), the meiotic DSB in the diploid yeast are co-localized with the DNase I hypersensitive sites (HS) in a haploid strain of yeast. These HS are located close to sequences which, under stress, have the potential to form secondary structures containing unpaired nucleotides. Clusters of such sequences could be a hallmark of the bases of the chromatin loops.     

Properties of a novel oligonucleotide-releasing bidirectional DNA exonuclease from mouse myeloma

Highly purified, but not homogeneous, samples of helix-destabilizing protein 1 from mouse myeloma contain a novel oligonucleotide-releasing DNA exonuclease. This enzyme was separated from helix-destabilizing protein 1 and obtained in highly purified form. A polypeptide of Mr 41 000 is a main constituent of the purified enzyme, and this polypeptide comigrated with the exonuclease activity during the final step of the purification, Sephacryl S-200 gel filtration where the enzyme had a native Mr of 40 000. Overall purification of enzyme activity was greater than 20 000-fold. This exonuclease releases 5'-oligonucleotides in a limited processive manner in both the 5'----3' and 3'----5' directions. Activity of the enzyme is resistant to 1 mM N-ethylmaleimide, requires a divalent cation, has an alkaline pH optimum, and degrades single-stranded DNA much faster than double-stranded DNA or RNA. The predominant oligonucleotide product with uniformly labeled substrates is (pdN)2. With 3' end labeled substrates, greater than 95% of the labeled products are (pdN)4 and (pdN)5; with 5' end labeled substrates, the main labeled product is (pdA)2. The rate of product release from 3' and 5' end labeled substrates is nearly identical at 37 degrees C. A model of the action of this enzyme and a comparison with a human placenta exonuclease [Doniger, J., & Grossman, L. (1976) J. Biol. Chem. 251, 4579-4587] are discussed.     

Distortion After Monofunctional Alkylation by Mitomycin C of a Dodecamer Containing its Major Binding Site

Abstract

The structural distortions of the duplex dodecamer d(ATTAACGTTAAT)₂ monofunctionally alkylated by mitomycin C have been studied by the use of chemical probes reactivity and resonance Raman spectroscopy. This sequence contains the 5′-ACGT sequence for which mitomycin C was determined to present the best affinity (S. Kumar, R. Lipman, and M. Tomasz, Biochemistry 31, 1399 (1992)). Raman spectroscopy as well as osmium tetroxyde reactivity indicate that the distortion of the double helix structure is located around the central CG bases. Mitomycin C reacts exclusively with the 2-amino group of guanine and this binding does not disrupt the inter bases H-bonds, as indicated by chloroacetaldehyde reactivity. Although resonance Raman spectroscopy does not allow the handedness of the monoalkylated CG/GC sequence to be determined, it indicates a similarity between the base stacking and that which would be observed for alternating purine/pyrimidine sequences at high salt concentration.     

Assessment of adenyl residue reactivity within model nucleic acids by surface enhanced Raman spectroscopy

We rank the reactivity of the adenyl residues (A) of model DNA and RNA molecules with electropositive subnano size [Ag]n+ sites as a function of nucleic acid primary sequences and secondary structures and in the presence of biological amounts of Cl- and Na+ or Mg2+ ions. In these conditions A is markedly more reactive than any other nucleic acid bases. A reactivity is higher in ribo (r) than in deoxyribo (d) species [pA>pdA and (pA)n>(pdA)n]. Base pairing decreases A reactivity in corresponding duplexes but much less in r than in d. In linear single and paired dCAG or dGAC loci, base stacking inhibits A reactivity even if A is bulged or mispaired (A.A). dA tracts are highly reactive only when dilution prevents self-association and duplex structures. In d hairpins the solvent-exposed A residues are reactive in CAG and GAC triloops and even more in ATC loops. Among the eight rG1N2R3A4 loops, those bearing a single A (A4) are the least reactive. The solvent-exposed A2 is reactive, but synergistic structural transitions make the initially stacked A residues of any rGNAA loop much more reactive. Mg2+ cross-bridging single strands via phosphates may screen A reactivity. In contrast d duplexes cross-bridging enables "A flipping" much more in rA.U pairs than in dA.T. Mg2+ promotes A reactivity in unpaired strands. For hairpins Mg2+ binding stabilizes the stems, but according to A position in the loops, A reactivity may be abolished, reduced, or enhanced. It is emphasized that not only accessibility but also local flexibility, concerted docking, and cation and anion binding control A reactivity.     

A DNA endonuclease isolated from yeast nuclear extract.

We have isolated and partially purified a DNA endonuclease from nuclei of the yeast Saccharomyces cerevisiae. Although purified on the basis of its ability to degrade denatured DNA, the enzyme can also attack native DNA. Denatured oligonucleotide products of the enzyme are sensitive to venom phosphodiesterase (EC3.1.4.1.) but not to bovine spleen phosphodiesterase (EC3.1.4.18). The enzyme has an estimated molecular weight of 6.6--7.5 X 10(4), more than twice as large as the endonucleases involved in DNA repair in Escherichia coli. When analyzed on glycerol gradients, the endonuclease sedimented as a single activity against both denatured DNA and closed circular DNA duplexes. The enzyme showed a 10-fold preference for denatured over native T7 DNA substrate, and appears to produce random nicks in a supercoiled replicative form of phiX174 DNA (RFI) with no discernable preference for the unpaired bases in the supercoiled duplex. The endonuclease appears to be distinct from the yeast endonucleases previously described.     

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.     

Purification and properties of an Arthrobacter oxydans P52 carbamate hydrolase specific for the herbicide phenmedipham and nucleotide sequence of the corresponding gene.

Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.     

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.     

Differential sequence dynamics of homopolymeric and alternating AT tracts in a small plasmid DNA

The location of OsO4 bispyridine hyper- and hyporeactivity in a small deletion derivative of plasmid ColE1 (PTC12, 1727 bp) has been determined for approximately 70% of the molecule. Thymine bases in homopolymeric (dA)n.(dT)n tracts (n greater than or equal to 4) were always found to be resistant toward OsO4 modification. DNA supercoiling did not destabilize these tracts. The extent of OsO4 bispyridine reactivity of homopolymeric (dA)n.(dT)n tracts, where n = 3, was found to be dependent on the rate of base unpairing of the sequence immediately 5' and 3' to the tract. Repressed OsO4 reactivity of thymine bases in (dA)3.(dT)3 tracts was observed if immediately both 5' and 3' to the tract were stable DNA sequences composed of GC base pairs and/or a homopolymeric (dA)n.(dT)n tract (n greater than or equal to 4). Homopolymeric tracts of n = 3 not having adjacent sequences with repressed unpairing rates did not show reduced levels of OsO4 bispyridine reactivity. Alternating d(TA)n tracts (n greater than or equal to 2) were found to exhibit hyperreactivity with OsO4. The extent of this hyperreactivity was dependent on the length of the tract and superhelical torsional stress. The distribution and frequency of homopolymeric (dA)n.(dT)n (n greater than or equal to 4) tracts in Escherichia coli promoter sequences were examined, and the possible implications of these tracts on promoter function are discussed.     

Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein.

The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated. DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA. Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde). Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised. Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature. The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage. Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.     

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core.     

Characterization of O-acetyl-L-serine sulfhydrylase purified from an alkaliphilic bacterium

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55% by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25% and 12% by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21%. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the beta-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40 degrees C (when tested at pH 7.5), and at pH 6.5 (at 40 degrees C). The enzyme kept 90% its activity after incubation at 65 degrees C (at pH 7.5) for 30 min, and more than 90% after 30 min incubation at pHs 7-12 at 30 degrees C. The enzyme had a Km of 4 mM for O-acetyl-L-serine and a Vmax of 37.0 micromol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5% total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5'-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.     

A Parallel Quadruplex DNA Is Bound Tightly but Unfolded Slowly by Pif1 Helicase

DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular quadruplex. Pif1 readily unfolds a parallel quadruplex DNA substrate in a multiturnover reaction and also generates some product under single cycle conditions. The rate of ATP hydrolysis by Pif1 is reduced when bound to a parallel quadruplex compared with single-stranded DNA. ATP hydrolysis occurs at a faster rate than quadruplex unfolding, indicating that some ATP hydrolysis events are non-productive during unfolding of intramolecular parallel quadruplex DNA. However, product eventually accumulates at a slow rate.     

Base modifications in plasmid DNA caused by potassium permanganate

KMnO4 is a powerful oxidizing agent which has been used to modify DNA bases. In previous studies, mild KMnO4 treatment has been shown to preferentially modify Thy; Cyt and Gua are modified only under harsher conditions to as yet unidentified products. In the present study, denatured plasmid pCMV beta gal DNA was exposed to 0.015-1.5 mM KMnO4, pH 8.6, at 4 degrees C for 5 min, after which the DNA was hydrolyzed in formic acid, trimethylsilylated, and analyzed for modified base content by gas chromatography-mass spectrometry/selected ion monitoring. KMnO4 treatment, even at concentrations as low as 0.015 mM, caused a concentration-dependent increase in the Thy products Thy glycol and 5-hydroxy-5-methylhydantoin, the Cyt products Cyt glycol, 5,6-dihydroxycytosine, and 5-hydroxyhydantoin, the Ade product 8-hydroxyadenine, and the Gua product 8-hydroxyguanine. The Ade product 4,6-diamino-5-formamidopyrimidine and the Gua product 2,6-diamino-4-hydroxy-5-formamidopyrimidine were minimally (less than or equal to 2-fold) increased by treatment with greater than or equal to 0.8 mM KMnO4. These data demonstrate that, in addition to Thy, Cyt, Gua, and Ade bases in plasmid DNA may be modified by treatment with KMnO4, even under mild conditions. They represent the first identification of Cyt, Gua, and Ade products caused by KMnO4 treatment. Furthermore, these data suggest that previous studies which have used treatment with KMnO4 to study the mutagenicity of Thy glycol specifically or as a Thy-specific probe in DNA structure should be interpreted with caution.