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PubMed1989

Minor-groove binding models for acetylaminofluorene modified DNA.

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)).

2-Acetylaminofluorene↗
PubMed1990

Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides.

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.

Alkaloids↗
PubMed1989

Hairpin structures in synthetic oligodeoxynucleotides: sequence effects on the duplex-to-hairpin transition.

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.

Base Composition↗
PubMed1989

Identification and purification of the enhancer-binding factor of human immunodeficiency virus-1. Multiple proteins and binding to other enhancers.

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.

Base Sequence↗
PubMed1992

Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine.

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.

Base Sequence↗
PubMed1988

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site.

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1979

Terminal sequences of vesicular stomatitis virus RNA are both complementary and conserved.

The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).

Animals↗
PubMed1992

Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.

We have devised a protocol to obtain accurate and complete sequences of the immunoglobulin heavy- and light-chain variable-region (VH and VL) genes of single B-hybridoma cells that express defined V genes. The amplification achieved ranges from 2 x 10(13)- to 1 x 10(14)-fold. Only one potential Taq DNA polymerase error was observed in 7590 nucleotides of sequence, thus permitting the identification of naturally occurring somatic mutations. The two-step nature of the amplification protocol provides sufficient DNA for a minimum of 160 sets of sequencing reactions of both the VH and VL genes from one cell without cloning. The amplification of relatively long segments of DNA in the first step of the protocol permits second-step amplification and sequencing of regions that flank VH and VL codons. Fractionating cellular lysates prior to the first step of amplification permits the separate amplification of V genes on opposite sister chromatids and possibly on opposite strands of the same DNA duplex. Accurate sequencing of VH and VL genes of defined germ-line origin that are expressed by single B cells taken directly from the animal is thus made feasible by this approach.

Animals↗
PubMed1987

Specific A . T DNA sequence binding of RP-HPLC purified HMG-I.

HMG-I (alpha-protein) is a high mobility group protein which recognizes and binds specifically to A . T rich double stranded DNA. We have investigated, by electrophoretic shift assays and DNase I footprinting, the ability of reverse-phase high performance liquid chromatography purified HMG-I to bind to specific A . T rich duplex DNA sequences. We show here that when HMG-I is isolated and purified under denaturing conditions it retains its specific A . T DNA binding activity. These results suggest that reverse-phase high performance liquid chromatography to be the method of choice for the preparation of HMG-I.

Adenine↗
PubMed1989

Association of anthracyclines and synthetic hexanucleotides. Structural factors influencing sequence specificity.

The equilibrium and kinetic aspects of the interaction between four anthracyclines and two synthetic self-complementary hexanucleotides was investigated by fluorescence detection. Two of the studied anthracyclines are widely used antitumor drugs: doxorubicin (1, formerly adriamycin) and daunorubicin (2, formerly daunomycin). The other two, 9-deoxydoxorubicin (3) and 3'-deamino-3'-hydroxy-4'-epidoxorubicin (4), are doxorubicin analogues with modifications of the chemical groups that have been proposed as responsible for sequence specificity (Chen, K.-X., Gresh, N. and Pullman, B. (1985). J. Biomol. Struct. Dyn. 3, 445-466). One of the oligonucleotides, d(CGTACG), is identical to that used in the high resolution x-ray structure determination of the daunorubicin intercalative complex (Wang, A. H.-J., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Biochemistry 26, 1152-1163). Binding to this hexanucleotide is compared with intercalation into the d(CGCGCG) duplex, revealing sequence preferences of the four anthracyclines. Taking into account the anthracycline aggregation and the dissociation of the hexanucleotide double standard form, results can be interpreted with a model that assumes complete fluorescence quenching at intercalative sites containing the CG base pair, and a large residual fluorescence after intercalation within the TpA fragment. All four anthracyclines show preferential intercalation at sites near the ends of both hexanucleotide duplexes, partly as a result of positive cooperativity in the formation of di-intercalated species at these sites. Within the limits of experimental error, complete site specificity for the CpG fragment is found in the intercalation of 1 and 2 into d(CGTACG) duplex, whereas analogues 3 and 4 give increasing evidence of intercalation at other sites including the fluorescence-preserving TpA fragment. Site specificity is less pronounced in the association with d(CGCGCG), when cooperativity is taken into account. Kinetic data corroborate the results of equilibrium studies and are interpreted with a mechanism that includes formation of an intermediate bound species followed by drug redistribution to preferential sites. Finally, from a comparison of pertinent site binding constants, approximate free energy contributions to sequence specific DNA interaction, due to C9-OH on the aglycone and -NH3+ on daunosamine, are estimated not to exceed 2 kcal/mol.

Antibiotics, Antineoplastic↗
PubMed1985

A monoclonal antibody specific for the duplex DNA poly[d(TC)].poly[d(GA)].

Although most duplex DNAs are not immunogenic some synthetic DNAs such as poly[d(Tm5C)].poly[d(GA)] are weakly immunogenic allowing the production of monoclonal antibodies. The specificity of one of these antibodies, Jel 172, was investigated in detail by a competitive solid-phase radioimmune assay. Jel 172 bound well to poly[d(TC)].poly[d(GA)] but not to other duplex DNAs such as poly[d(TTC)].poly[d(GAA)] and poly[d(TCC)].poly[d(GGA)]. The binding to poly[d(Br5UC)].poly[d(GA)] was enhanced while that to poly[d(TC)].poly[d(IA)] was decreased compared to poly[d(TC)].poly[D(GA)]. Thus, not only is the antibody very specific for a sequence of duplex DNA but it also appears to recognize functional groups in both grooves of the helix.

Animals↗
PubMed1990

The upstream factor-binding site is not essential for activation of transcription from the adenovirus major late promoter.

An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.

Adenoviridae↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 10. Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126.

The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.

Adenosine Triphosphate↗
PubMed1986

Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally.

Human mitochondrial DNA is transcribed from two distinct, strand-specific promoters located in the displacement loop region of the genome. The transcriptional control sequences identified by deletion mapping and site-directed mutagenesis studies span short regions surrounding the initiation sites and bear no obvious sequence homology to any nuclear or procaryotic promoters. In vitro transcription analyses also revealed several minor initiation sites that are characterized by a pyrimidine-rich region followed by a purine-rich region, a feature that is shared by the two major promoters. In this paper, we report a new class of minor promoters in human mitochondrial DNA. These minor promoters were localized to the same duplex DNA sequences that direct major transcriptional events, but they had transcriptional polarity opposite to that of the major promoters. Furthermore, nucleotide changes that affected the major form of transcription similarly affected transcription in the opposite direction. For one of these minor promoters, a corresponding in vivo RNA species initiating from the same site was identified. These observations indicate that the major transcriptional promoters in human mitochondria can function bidirectionally both in vivo and in vitro.

Base Sequence↗
PubMed1986

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution.

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.

Base Sequence↗
PubMed1992

Interactions between the trp repressor and its operator sequence as studied by base analogue substitution.

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.

Bacterial Proteins↗
PubMed1986

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution.

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.

Base Sequence↗