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Four 1.5 ns molecular dynamics (MD) simulations were performed on the d(GCTATAAAAGGG).d(CCCTTTTATAGC) double helix dodecamer bearing the Adenovirus major late promoter TATA element and three iso-composition mutants for which physical and biochemical data are available from the same laboratory. Three of these DNA sequences experimentally induce tight binding with the TATA box binding protein (TBP) and induce high transcription rates; the other DNA sequence induces much lower TBP binding and transcription. The x-ray crystal structures have previously shown that the duplex DNA in DNA-TBP complexes are highly bent. We performed and analyzed MD simulations for these four DNAs, whose experimental structures are not available, in order to address the issue of whether inherent DNA structure and flexibility play a role in establishing these observed preferences. A comparison of the experimental and simulated results demonstrated that DNA duplex sequence-dependent curvature and flexibility play a significant role in TBP recognition, binding, and transcriptional activation.
It has been suggested that a sequence in the RNA transcript that can form a stem and loop structure, followed by the sequence CAAUCAA, is the signal for rho-dependent transcription termination. We tested this hypothesis by synthesizing a DNA duplex whose sequence corresponds to a region of the lambda tR1 terminator that contains these structural features. We cloned this synthetic DNA fragment under the control of the lacUV5 promoter, and showed that it does not cause rho-dependent termination in vitro. RNA polymerase pauses during in vitro transcription across the synthetic sequence, although less efficiently than at the corresponding sequence on the lambda template. No rho-mediated termination was detected even under conditions that prolonged transcriptional pausing at the synthetic site, indicating that the synthetic sequence is defective as a transcript release site. We suggest that unlike rho-independent terminators, rho-dependent terminators require sequences in addition to those immediately before the sites of termination.
Drosophila heat shock activator protein, a rare transacting factor which is induced upon heat shock to bind specifically to the heat shock regulatory sequence in vivo, has been purified from shocked cells to more than 95 percent homogeneity by sequence-specific duplex oligonucleotide affinity chromatography. The purified protein has a relative molecular mass of 110 kilodaltons, binds to the regulatory sequence with great affinity and specificity, and strongly stimulates transcription of the Drosophila hsp70 gene. Studies with this regulatory protein should lead to an understanding of the biochemical pathway underlying the heat shock phenomenon.
A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.
Minimized potential energy calculations have been employed to locate and evaluate energetically a number of different models for DNA modified at carbon-8 of guanine by acetylaminofluorene (AAF). Three different duplex nonamer sequences were investigated. In addition to syn guanine models which have some denaturation and a Z-DNA model, we have found two new types of structures in which guanine remains syn and the AAF is placed in the minor groove of a B-DNA helix. One type features Hoogsteen base pairing between the modified guanine and protonated cytosine, with a sharply bent helix. The other (here termed the "wedge" model because the aromatic amine is wedged into the minor groove) maintains a single hydrogen bond between O6 of the modified guanine and N3 of protonated cytosine, with much less deformation of the helix, and close Van der Waals contacts between the AAF and the walls of the minor groove. Both types of structures (as well as the related forms produced by deprotonation of cytosine) are energetically important in all three sequences examined. The wedge-type model, which is most favored except in alternating G-C sequences, has been previously observed in a combined NMR and computational characterization of an aminofluorene (AF) modified guanine opposite adenine in a DNA duplex undecamer (D. Norman, P. Abuaf, B.E. Hingerty, D. Live, D. Grunberger, S. Broyde and D.J. Patel, Biochemistry 28, 7462 (1989)).
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Homopyrimidine oligonucleotides bind to homopurine-homopyrimidine sequences of duplex DNA forming a local triple helix. This binding can be demonstrated either directly by a footprinting technique, gel assays, or indirectly by inducing irreversible reactions in the target sequence, such as photocrosslinking or cleavage. Binding occurs in the major groove with the homopyrimidine oligonucleotide orientated parallel to the homopurine strand. Thymine and protonated cytosine in the oligonucleotide form Hoogsteen-type hydrogen bonds with A.T and G.C Watson-Crick base pairs, respectively. Here we report that an 11-residue homopyrimidine oligonucleotide covalently attached to an ellipticine derivative by its 3' phosphate photo-induces cleavage of the two strands of a target homopurine--homopyrimidine sequence. To our knowledge, this is the first reported case of a sequence-specific artificial photoendonuclease. In addition we show that a strong binding site for a free ellipticine derivative is induced at the junction between the triplex and duplex structures on the 5' side of the bound oligonucleotide. On irradiation, cleavage is observed on both strands of DNA. This opens new possibilities for inducing irreversible reactions on DNA at specific sites by the synergistic action of a triple helix-forming oligonucleotide and an intercalating agent.
We have synthesized and examined a number of fully and partly self-complementary palindromic oligodeoxynucleotides for their ability to assume in solution a unimolecular hairpin structure. The main results obtained by a combined optical and electrophoresis investigation show that: (i) DNA folding needs not be driven by mismatched base pairings over the dyad; fully self-complementary palindromic duplexes, comprising regular (CG)n DNA fragments, possess a considerable intrinsic propensity to make intramolecular base pairings; (ii) The duplex-hairpin interconversion is, in general, a slow process independent of the length and base composition of the palindrome; (iii) The palindromic sequences energetically least favored to form hairpin structures consist of C:G base pairs around the dyad axis and of T:A blocks in the arms of the inverted repeat; (IV) The base composition of the stem strongly influences the hairpin thermal stability. For instance, the substitution of one C:G with one A:T base pair in the stem helix of d(CG)7 diminishes the stability of the hairpin by 9 degrees C. It is found that the stability of the stem helix, in hairpins of defined sequence and with the same loop length, decreases in the order alternating-CG greater than homo-CG greater than AC(GT) greater than alternating-AT, i.e. as in polynucleotides. The thermodynamic parameters for the hairpin-coil transition are reported.
The enhancer-binding factor of human immunodeficiency virus (HIV)-1 was purified from human B cells by sequence-specific duplex oligonucleotide affinity chromatography. Gel retardation assay and footprint analysis showed that the purified factor bound specifically to the HIV-1 enhancer sequence, and protected both direct repeats in the HIV-1 enhancer. The purified factor consisted of four main polypeptides of the molecular weight of 36,000-42,000. At least three of them had enhancer-binding activity after elution from sodium dodecyl sulfate-polyacrylamide gel and renaturation. UV cross-linking analysis also showed that at least two of the polypeptides in purified fraction had a binding activity specific for the HIV-1 enhancer. The purified factor activated transcription from the HIV-1 promoter in vitro, confirming that it was indeed a transcription factor for HIV-1. The purified factor also recognized sequences in the immunoglobulin kappa gene enhancer and the major histocompatibility complex class I gene enhancer with almost the same affinity as the HIV-1 enhancer. These results suggested the existence of multiple proteins which recognize the kappa B-related sequences. This regulatory factor should help in the study of the biochemical pathway underlying HIV production from latently infected cells.
We have devised a combinatorial method, restriction endonuclease protection selection and amplification (REPSA), to identify consensus ligand binding sequences in DNA. In this technique, cleavage by a type IIS restriction endonuclease (an enzyme that cleaves DNA at a site distal from its recognition sequence) is prevented by a bound ligand while unbound DNA is cleaved. Since the selection step of REPSA is performed in solution under mild conditions, this approach is amenable to the investigation of ligand-DNA complexes that are either insufficiently stable or not readily separable by other methods. Here we report the use of REPSA to identify the consensus duplex DNA sequence recognized by a G/T-rich oligodeoxyribonucleotide under conditions favoring purine-motif triple-helix formation. Analysis of 47 sequences indicated that recognition between 13 bases on the oligonucleotide 3' end and the duplex DNA was sufficient for triplex formation and indicated the possible existence of a new base triplet, G.AT. This information should help identify appropriate target sequences for purine-motif triplex formation and demonstrates the power of REPSA for investigating ligand-DNA interactions.
The RecA protein of Escherichia coli forms a nucleoprotein filament that promotes homologous recognition and subsequent strand exchange between a single strand and duplex DNA via a three-stranded intermediate. Recognition of homology within three-stranded nucleoprotein complexes, which is probably central to genetic recombination, is not well understood as compared with the mutual recognition of complementary single strands by Watson-Crick base pairing. Using oligonucleotides, we examined the determinants of homologous recognition within RecA nucleoprotein filaments. Filaments that contained a single strand of DNA recognized homology not only in a complementary oligonucleotide but also in an identical oligonucleotide, whether their respective sugar-phosphate backbones were antiparallel or parallel, and a filament that contained duplex DNA showed the same polymorphic versatility in the recognition of homology. Recognition of self by a filament that contains a single strand reveals that RecA filaments can recognize homology via non-Watson-Crick hydrogen bonds. Recognition of multiple forms of the same sequence by duplex DNA in the filament shows that it primarily senses base-sequence homology, and suggests that recognition can be accomplished prior to the establishment of new Watson-Crick base pairs in heteroduplex products. However, unlike the initial recognition of homology, strand exchange is stereospecific, requiring the proper antiparallel orientation of complementary strands.
Triple helix forming oligonucleotides (TFOs) recognize and bind sequences in duplex DNA and have received considerable attention because of their potential for targeting specific genomic sites. TFOs can deliver DNA reactive reagents to specific sequences in purified chromosomal DNA (ref. 4) and nuclei. However, chromosome targeting in viable cells has not been demonstrated, and in vitro experiments indicate that chromatin structure is incompatible with triplex formation. We have prepared modified TFOs, linked to the DNA-crosslinking reagent psoralen, directed at a site in the Hprt gene. We show that stable Hprt-deficient clones can be recovered following introduction of the TFOs into viable cells and photoactivation of the psoralen. Analysis of 282 clones indicated that 85% contained mutations in the triplex target region. We observed mainly deletions and some insertions. These data indicate that appropriately constructed TFOs can find chromosomal targets, and suggest that the chromatin structure in the target region is more dynamic than predicted by the in vitro experiments.
59Co NMR and CD measurements show for both stereoisomers of Co(en)3(3+) a similar trend in the sequence dependence of DNA recognition, as was reported previously for Co(NH3)6(3+). In particular, specific binding is evident to DNA molecules possessing runs of two or more same-strand guanine residues. The binding of either isomer to such sequences induces structural transitions toward A-DNA characteristics. Such measurements also show significant differences between the two stereoisomers in terms of how they recognize specific duplex DNA sequences. delta-Co(en)3(3+) binds more tightly than lambda-Co(en)3(3+) to right-handed, guanine-rich DNA, whereas lambda-Co(en)3(3+) binds more tightly than delta-Co(en)3(3+) to left-handed DNA. The two stereoisomers bind in an indistinguishable manner to AT-rich DNA.
Alternating polypurine d(GA)n, sequences exhibit a considerable polymorphism. Here we report that alpha d(GA) x d(GA) sequences form an antiparallel stranded duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic properties of the alpha/beta chimeric oligodeoxynucleotide, 5'-d(GA)4(T)4 alpha d(AG)4T-3', support the formation of a hairpin structure with antiparallel strands in the stem. The optical properties of this novel antiparallel structure are different from the parallel stranded homoduplex formed by d(GA)G7. This alpha/beta hairpin has a remarkably high Tm of 44.5 degrees C in 0.4 M NaCl with a van't Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the alpha/beta hairpin to an antiparallel bimolecular duplex and by non-denaturing gel electrophoresis using duplexes containing sequence constraints. Both support the presence of alphaG-G and alphaA-A base pairing in the antiparallel 5'-d(GA)4(T)4 alpha d(AG)4T-3' intramolecular duplex. This study adds to the polymorphic nature of alternating d(GA)n sequences as well as providing novel homopurine base pairing approaches for probing polypurine polypyrimidine sequences.
A homopurine-homopyrimidine sequence of human immunodeficiency virus (HIV) proviral DNA was chosen as a target for triple-helix-forming oligonucleotides. An oligonucleotide containing three bases (thymine, cytosine, and guanine) was shown to bind to its target sequence under physiological conditions. This oligonucleotide is bound in a parallel orientation with respect to the homopurine sequence. Thymines recognize A.T base pairs to form T.A.T base triplets and guanines recognize a run of G.C base pairs to form G.G.C base triplets. A single 5-methylcytosine was shown to stabilize the triple helix when incorporated in a stretch of thymines; it recognizes a single G.C base pair in a run of A.T base pairs. These results provide some of the rules required for choosing the more appropriate oligonucleotide sequence to form a triple helix at a homopurine-homopyrimidine sequence of duplex DNA. A psoralen derivative attached to the oligonucleotide containing thymine, 5-methylcytosine, and guanine was shown to photoinduce cross-linking of the two DNA strands at the target sequence in a plasmid containing part of the HIV proviral DNA sequence. Triplex formation and cross-linking were monitored by inhibition of Dra I restriction enzyme cleavage. The present results provide a rational basis for the development of triplex-forming oligonucleotides targeted to specific sequences of the HIV provirus integrated in its host genome.
Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, reacts with DNA following metabolism to its corresponding (+)-anti-7,8-diol-9,10-epoxide [(+)-anti-B[a]PDE], giving a major adduct (+)-trans-anti-B[a]P-N2-dG. Evidence suggests that this adduct is responsible for most of the different kinds of mutations (e.g. G-->T, G-->A, etc.) induced by (+)-anti-B[a]PDE, raising the question of how can a single adduct cause many different kinds of mutations? One hypothesis is that different mutations are induced depending upon the conformation of this adduct when bypassed during DNA replication. If true, then it becomes imperative to explore different reasonable conformations for this adduct. Herein a simulated annealing protocol is employed to study the conformation of (+)-trans-anti-B[a]P-N2-dG with the B[a]P moiety in the minor groove and pointing toward the base on its 5'-side in a 5'-CGC-3' sequence context in duplex DNA. This conformation and sequence were chosen because there is a structure derived from NMR constraints for comparison. A four step procedure is followed: the adduct is docked in canonical B-DNA, after which the structure is subjected to an initial conjugate gradient minimization, followed by simulated annealing and a final conjugate gradient minimization. The quality and final energy of structures is assessed as a function of changes in six parameters, including the length of the DNA helix, the initial annealing temperature (T0), the annealing time (t), the molecular dynamics time step (tau) and two other parameters. While there is no single set of optimum parameters, reasonable low energy structures were obtained using the values t approximately 40 ps (or longer), T0 approximately 750 K and tau approximately 1.0 fs with a helix length of 7 bp. The structures that emerge all retain the basic features of the input structure, being B-DNA-like with the B[a]P moiety in the minor groove pointing toward the base on the 5'-side. However, within this broad category there are at least six subclasses of structures, of which four have lowest energy members that differ by < approximately 5 kcal/mol. The fact that a variety of distinct but related structures emerge from a single starting structure as this parameter set is varied suggests that the use of a large but manageable number of simulated annealing runs should be considered in the search for a cohort of related structures. This is especially important given that this breadth of potentially relevant structures of approximately the same energy may indeed be relevant to the hypothesis that different mutations arise from a single adduct in different conformations.
We have developed a strategy for DNA sequencing based on exonuclease III digestion followed by double strand specific endonuclease digestion and direct dideoxynucleotide sequencing reaction. This strategy eliminates the need for subcloning, oligonucleotide primers, and prior knowledge of the DNA to be sequenced. All template and primer duplexes needed for sequencing a complete insert can be prepared in one day from uncharacterized starting DNA. Sequence information can be obtained from different regions of the DNA simultaneously. The method uses double-stranded DNA to generate single-stranded template and primer, and thus produces high quality sequence results. Commercially available dideoxy-sequencing kits are well suited for this method. The strategy should be applicable for both automatic and routine laboratory DNA sequencing.