H-DNA Structure Research Articles

NMR studies of pH-dependent conformational polymorphism of alternating (C-T)n sequences.

Alternating (C-T)n sequences are involved in the H-DNA structure associated with (GA)n.(CT)n sequences. Low pH values facilitate H-DNA formation. We have undertaken a detailed analysis of the structural consequences of the (C-T)n sequence as a function of pH. The structures of three DNA oligonucleotides, d(CT)4, d(TC)4 and d(TC)15, have been studied by NMR. We found that their conformations are polymorphic and pH dependent. There are at least three major conformational species: an antiparallel-stranded (APS) duplex with entirely C:T base pairs at pH 7, an antiparallel-stranded (APS) duplex with entirely C+:T base pairs at pH 3, and a possible parallel-stranded (PS) duplex with C+:C and T:T base pairs near pH 5. In the intermediate pH range, the APS duplex may have varying numbers of C+:T and C:T base pairs, and there may be a fast exchange going on between APS duplex species involving these two kinds of base pairs. However, the transition between the APS and PS duplexes is slow. Structural refinement of the two octamers, d(TC)4 and d(CT)4, at pH = 6.9 and pH = 3 using 2D-NOE data suggests that the molecules are likely in the duplex form at 5 degrees C. We lack evidence that the structure at pH 3 is a PS structure with T nucleotides residing in the exterior of the helix. Titration of the longer oligonucleotide, d(TC)15, showed a prominent pKa of approximately 6, approaching the value of 7.0 obtained from the titration of poly-(dC).

Inhibition of gene transcription by purine rich triplex forming oligodeoxyribonucleotides.

Several oligodeoxynucleotides (ODNs) were designed in order to interact with the purine rich element of the IRE (Interferon Responsive Element) of the 6-16 gene by triplex formation. An ODN of 21 bases, the sequence being identical to that of the purine strand of the IRE (48% G), but in reverse orientation, was able to interact with the IRE (KD: 20 nM). The binding was Mg2+ dependent. The two purine strands of the triplex were oriented antiparallel as confirmed by DNAase I and copper-phenanthroline footprinting experiments. An ODN in which A were replaced by T, also interacted with the same target, but with a lower affinity. Exonuclease III action indicated that the two IRE repeats of the 6-16 promoter interacted with each other through Hoogsteen base pairing, the third strand being parallel to the paired Watson-Crick strand. This led to a potential H-DNA structure which could be destabilized by adding ODNs able to form a triplex structure. 6-16 IRE driven-reporter gene constructs lost their interferon stimulability when co-transfected with triplex forming ODNs. The range of effective ODN concentrations was compatible with the affinity determined when measuring their direct interactions with the DNA.

Evolutionary consequences of nonrandom damage and repair of chromatin domains.

Some evolutionary consequences of different rates and trends in DNA damage and repair are explained. Different types of DNA damaging agents cause nonrandom lesions along the DNA. The type of DNA sequence motifs to be preferentially attacked depends upon the chemical or physical nature of the assaulting agent and the DNA base composition. Higher-order chromatin structure, the nonrandom nucleosome positioning along the DNA, the absence of nucleosomes from the promoter regions of active genes, curved DNA, the presence of sequence-specific binding proteins, and the torsional strain on the DNA induced by an increased transcriptional activity all are expected to affect rates of damage of individual genes. Furthermore, potential Z-DNA, H-DNA, slippage, and cruciform structures in the regulatory region of some genes or in other genomic loci induced by torsional strain on the DNA are more prone to modification by genotoxic agents. A specific actively transcribed gene may be preferentially damaged over nontranscribed genes only in specific cell types that maintain this gene in active chromatin fractions because of (1) its decondensed chromatin structure, (2) torsional strain in its DNA, (3) absence of nucleosomes from its regulatory region, and (4) altered nucleosome structure in its coding sequence due to the presence of modified histones and HMG proteins. The situation in this regard of germ cell lineages is, of course, the only one to intervene in evolution. Most lesions in DNA such as those caused by UV or DNA alkylating agents tend to diminish the GC content of genomes. Thus, DNA sequences not bound by selective constraints, such as pseudogenes, will show an increase in their AT content during evolution as evidenced by experimental observations. On the other hand, transcriptionally active parts may be repaired at rates higher than inactive parts of the genome, and proliferating cells may display higher repair activities than quiescent cells. This might arise from a tight coupling of the repair process with both transcription and replication, all these processes taking place on the nuclear matrix. Repair activities differ greatly among species, and there is a good correlation between life span and repair among mammals. It is predicted that genes that are transcriptionally active in germ-cell lineages have a lower mutation rate than bulk DNA, a circumstance that is expected to be reflected in evolution. Exception to this rule might be genes containing potential Z-DNA, H-DNA, or cruciform structures in their coding or regulatory regions that appear to be refractory to repair.(ABSTRACT TRUNCATED AT 400 WORDS)

The identification of a tandem H-DNA structure in the c-MYC nuclease sensitive promoter element

Previous studies have shown that the c-myc nuclease sensitive element (NSE) is capable of forming H-DNA in vitro. The NSE sequence exhibits strong purine/pyrimidine strand asymmetry. To study the NSE further, we have isolated the element from other c-myc sequences and have shown that the NSE alone is sufficient for the formation of H-DNA in supercoiled plasmids. We also show that the NSE forms a complex structure containing both H-y3 and H-y5 H-DNA. We term this structure tandem H-DNA.

H‐DNA formation by the coding repeat elements of neisserial opa genes

The coding repeat region of opa genes from Neisseria gonorrhoeae and Neisseria meningitidis determines the expression state of their respective genes through high-frequency addition of deletion of pentanucleotide coding repeat units (CRs; CTTCT). In vitro analyses of cloned opa gene CR regions using single-strand specific nucleases, oligonucleotide protection experiments, and modifications of non-B-DNA residues indicate that the regions form structures resembling H-DNA under acidic conditions in the presence of negative supercoiling. The purine/pyrimidine strand bias and H-palindromic nature of the repeat region are consistent with sequence requirements for H-DNA formation. Sequences flanking the repeat elements are required to form the H-DNA structure in vitro as judged by the pattern of exposed non-B-DNA residues in CR sequences synthesized as oligonucleotides to form beta-galactosidase::CR translational fusions. The fusions phase vary by addition and deletion of CR elements and the rate of phase variation increases upon induction of the fusion genes. The opa gene CR region is the first reported bacterial H-DNA structure and is unique in that it lies within the coding sequence for the gene.

Suppression of Cyclobutane and <6-4> Dipyrimidines Formation in Triple-Stranded H-DNA

We have determined the effect of H-DNA formation on the distributions of two ultraviolet (UV) light induced photoproducts--cyclobutane dipyrimidines and mean value of 6-4 dipyrimidines. A region of DNA containing the sequence (dT-dC)18.(dA-dG)18 was treated under conditions that specifically yield the triple-stranded H-y3 or H-y5 DNA structure and then irradiated with UV. The positions of cyclobutane dipyrimidines and mean value of 6-4 dipyrimidines were determined by T4 endonuclease V cleavage and by hot piperidine cleavage, respectively. Formation of H-DNA structures greatly decreased the photoproduct yield in the (dT-dC)18.(dA-dG)18 region but not elsewhere in the DNA. Suppression of photoproduct formation is greater in half of the repeat, reflecting whether the DNA is in the H-y3 or H-y5 conformation. Within the repeat, the suppression was less in the middle and toward the ends. Models for the suppression of photoproduct formation in H-DNA and the possible utility of our findings are discussed.

Complete nucleotide sequence of the gene for human C1 inhibitor with an unusually high density of Alu elements

The complete (17159 bp) nucleotide sequence of the gene for the human C1 inhibitor has been determined. The transcription initiation site was examined by primer extension using human liver mRNA, and the messenger 5'-end sequence was determined on clones obtained by the anchored polymerase chain reaction. The gene of this serpin molecule is split by seven introns, with junctions of phases zero and one. An outstanding feature of the intron sequences is the occurrence of 17 AluI repeats of all four ancestral subgroups, indicating that the gene has been invaded during consecutive waves of Alu amplification, including a recent one. These Alu repeats form the sites of deletion and insertion in several known lesions in the C1-inhibitor gene. There is no obvious promoter site of the TATA-box type at the 5' end of the gene, but instead it contains a region of potential H-DNA structure similar to that found upstream of the human c-myc gene.

DNA protein interactions at the interferon-responsive promoter elements: potential for an H-DNA conformation

The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two B1 and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the B1 complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OL1 were recovered in B2. For each probe, the possibility of the part of the sequence involved in B1 complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secondary structure, the formal existence of a H-DNA structure has not been demonstrated. Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations.

Spectroscopic and calorimetric investigation on the DNA triplex formed by d(CTCTTCTTTCTTTTCTTTCTTCTC) and d(GAGAAGAAAGA) at acidic pH

The equimolar mixture of d(CTCTTCTTTCTTTTCTTTCTTCTC) (dY24) and d(GAGAAGAAAGA) (dR11) [designated (dY24).(dR11)], forms at pH = 5 a DNA triplex, which mimicks the H-DNA structure. The DNA triplex was identified by the following criteria: (i) dY24 and dR11 co-migrate in a poly-acrylamide gel, with a mobility and a retardation coefficient comparable to those observed for an 11-triad DNA triplex, previously characterized in our laboratories (1); (ii) the intercalator ethidium bromide shows a poor affinity for (dR11).(dY24) at pH = 5, and a high affinity at pH = 8; (iii) the (dR11).(dY24) mixture is not a substrate for DNase I at pH = 5; (iv) the CD spectrum of (dR11).(dY24), at pH = 5, is consistent with those previously reported for triple-stranded DNA. The (dR11).(dY24) mixture exhibits a thermally induced co-operative transition, which appears to be monophasic, reversible and concentration dependent. This transition is attributed to the disruption of the DNA triplex into single strands. The enthalpy change of the triplex-coil transition was measured by DSC (delta Hcal = 129 +/- 6 kcal/mol) and, assuming a two-state model, by analysis of UV-denaturation curves (average of two methods delta HUV = 137 +/- 13 kcal/mol). Subtracting from delta Hcal of triplex formation the contributions due to the Watson-Crick helix and to the protonation of the C-residues, we found that each pyrimidine binding into the major groove of the duplex, through a Hoogsteen base pair, is accompanied by an average delta H = -5.8 +/- 0.6 kcal/mol. The effect on the stability of the (dR11).(dY24) triplex due to the substitution of a T:A:T triad with a T:T:T one was also investigated.

Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements.

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.