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PubMed1997

[Isolation and analysis of two cDNA-clones expressing the human lymphocyte expression library, inducing DNA-binding activity in vitro].

Two cDNA clones are sequenced which were isolated from human lymphocyte expression library using Southwestern (DNA-binding) screening with 32P-labeled Alu DNA in the presence of 100-fold excess of unbalanced poly (dI-dC). In one of the sequenced clones (vb22) an open reading frame (ORF) is detected, encoding protein with a new potential DNA-binding (zink-finger) domain, and DNA-binding activity of the protein is directly confirmed after its expression (as GST-fusion protein) in Escherichia coli. The other sequenced clone (wa12) is partially homologous to 15EST sequences present in GENBANK (April, 1996) and cloned from very different human tissues. Connection of these 15 overlapping GENBANK sequences resulted in a longer sequence covering wa12 and having ORF potentially encoding a new 10 kDa polypeptide without any apparent DNA-binding domains. This connected sequence as well as wa12 sequence having only 65 amino acids ORF are unrecognizable by computer software as the protein-coding regions, and we suppose that wa12 transcripts possess DNA-binding activity. Homopyrimidine blocks in RNA longer than 12 nucleotides are known to bind mirror duplex DNA sequences to form triplexes whose stability is comparable to that of protein-DNA complexes, and human promoters contain many such blocks.

Amino Acid Sequence↗
PubMed1994

NMR structural studies of a 15-mer DNA duplex from a ras protooncogene modified with the carcinogen 2-aminofluorene: conformational heterogeneity.

Proton NMR studies were conducted on the complementary 15-mer DNA duplex, d(5'-TACTCTTCTT[AF]GACCT).d (5'-AGGTCAAGAAGAGTA) (designated as the AF-modified duplex). The sequence represents a portion of the mouse c-Ha-ras protooncogene and was selectively modified to contain a single N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) adduct at the deoxyguanosine corresponding to the first base of codon 61. The AF-modified duplex was found to exist in multiple conformations, with one being predominant (approximately 60%). The exchangeable and nonexchangeable protons belonging to the major conformer were sufficiently well-resolved to allow the assignment of the majority of the base and sugar protons. The one-dimensional proton spectra, as well as the NOE cross-peak patterns associated with this conformer of the AF-modified duplex both in H2O and D2O spectra, were strikingly similar to those observed for the major conformer of an analogous duplex containing N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) in the same position [Cho, B.P., Beland, F. A., & Marques, M. M. (1992) Biochemistry 31, 9587-9602]. The experimental results suggest that the AF- and ABP-modified duplexes adopt essentially identical major conformations, with each arylamine moiety being positioned in the major groove of a slightly disturbed B-type DNA duplex. Nonetheless, the absence of specific NOE cross peaks in the vicinity of the modification site indicates that the local structural perturbation is more severe in the AF-modified duplex. Although insufficient data precluded a detailed characterization of the minor conformers of the AF-modified duplex, the observation of significant shielding of the AF aromatic protons suggests a more dramatic structural alteration at the adduct site, possibly involving extensive stacking with the neighboring bases. The higher content (30-40%) of the minor conformers observed for the AF-modified duplex contrasted with the low contribution (5-10%) of similar structures in the ABP-modified duplex and may be attributed to a better overlapping efficiency of the planar AF ring with the nearby bases. Since the significant local perturbation observed in the minor conformers could provide a possible mechanism for mutations, our results support the view that the structural differences in the arylamine fragments of otherwise identical adducts have a direct influence on the conformational heterogeneities, which in turn may play a significant role in arylamine carcinogenesis.

Base Sequence↗
PubMed1995

Accelerated hybridization of oligonucleotides to duplex DNA.

We report two strategies for accelerating the hybridization of oligonucleotides to DNA. We demonstrate that oligodeoxyribonucleotides and peptide nucleic acid oligomers hybridize to inverted repeats within duplex DNA by D-loop formation. Oligonucleotides and duplex template form an active complex, which can be recognized by T7 DNA polymerase to prime polymerization. Quantitation of polymerization products allowed the rate of hybridization to be estimated, and peptide nucleic acid oligomers and oligonucleotide-protein adducts anneal with association constants 500- and 12,000-fold greater, respectively, than the analogous unmodified oligonucleotides. Together, these results indicate that sequences within duplex DNA can be targeted by Watson-Crick base pairing and that chemical modifications can dramatically enhance the rate of strand association. These findings should facilitate targeting of oligomers for priming DNA polymerization, the detection of diagnostic sequences, and the disruption of gene expression. The observed acceleration of hybridization may offer a new perspective on the ability of RecA or other proteins to accelerate strand invasion.

Base Sequence↗
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↗
PubMed1999

Crystal structures of two plasmid copy control related RNA duplexes: An 18 base pair duplex at 1.20 A resolution and a 19 base pair duplex at 1.55 A resolution.

The structures of two RNA duplexes, whose sequences correspond to portions of the ColE1 plasmid copy control RNA I and RNA II, have been determined. Crystals containing the 18mers 5'-CA CCGUUGGUAGCGGUGC-3' and 5'-CACCGCUACCAACGGUGC-3' diffract to 1.20 A resolution while those containing the 19mers 5'-GCACCGUUGGUAGCGGUGC-3' and 5'-GCACCGCUACCAACGGUGC-3' diffract to 1.55 A resolution. Both duplexes are standard A form, with Watson-Crick base pairing throughout. Use of anisotropic atomic displacement factors in refinement of the 1.20 A structure dramatically improved refinement statistics, resulting in a final R(free) of 15.0% and a crystallographic R-factor of 11.6%. Perhaps surprisingly, these crystals of the 18 base pair RNA exhibit a 36-fold static disorder, resulting in a structure with a single sugar-phosphate backbone conformation and an averaged base composition at each residue. Since the sugar-phosphate backbone structure is identical in the 36 different nucleotides that are superimposed, there can be no sequence-dependent variation in the structure. The average ribose pucker amplitude is 45.8 degrees for the 18 base pair structure and 46.4 degrees for the 19 base pair structure; these values are respectively 19% and 20% larger than the average pucker amplitude reported from nucleoside crystal structures. A standard RNA water structure, based on analysis of the hydration of these crystal structures and that of the TAR RNA stem [Ippolito, J. A., and Steitz, T. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9819-9824], has been derived, which has allowed us to predict water positions in lower resolution RNA crystal structures. We report a new RNA packing motif, in which three pro-S(p) phosphate oxygens interact with an ammonium ion.

Crystallography, X-Ray↗
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↗
PubMed1994

Joints made by RecA protein in the interior of linear duplex DNA: effects of single-stranded ends, length of homology, and dynamic state.

The importance of 3' single-stranded ends in homologous recombination led us to reevaluate reactions of single strands at homologous sites in the interior of linear duplex DNA. As the length of homology increased, the yield of joints increased up to about 2 kb of homology, at which the apparent yields were the same at either 3' or 5' single-stranded ends, or away from ends, although such joints were qualitatively different. In the presence of RecA protein and ATP, joints that formed between any of these single strands and interior sequences in duplex DNA were in a dynamic state in which they constantly recycled. Consequently, their apparent yields at steady state were sensitive to conditions of reaction, such as the concentrations of DNA, salt, and ATP vs ADP, observations which rationalize conflicting reports in the literature. The dynamic state of joints in the interior of duplex DNA strengthens the conclusion [see Burnett et al. (1994)] that RecA protein dissociates joints when it cannot displace the 5' end of a strand from the recipient duplex.

Adenosine Diphosphate↗
PubMed2004

[Bridged nucleic acids (BNAs) as a basic material for genome technology].

The completion of the human genome sequencing project will greatly accelerate the development of novel and practical technologies for genome-analysis, diagnostics or therapeutics. Oligonucleotides are playing an important role in these genome technologies, because of their sequence-specific hybridization ability toward the complementary strand. Besides the sequence-specific duplex formation, oligonucleotides are able to form stable triplex structures, which is fundamental to the antigene strategy to regulate gene expression in a living cell. However, two major drawbacks are known in the triplex formation by a natural oligonucleotide: low stability of the triplex and limitations of the target DNA sequence. One promising strategy to overcome these problems is chemical modification of the oligonucleotides. We have developed various bridged nucleic acids (BNAs), and found that the oligonucleotides containing 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification form a stable parallel motif triplex with the double-stranded DNA target under physiological conditions. Some nucleobase analogues to extend the target DNA sequence were designed, synthesized and incorporated into the 2',4'-BNA structure. The obtained 2',4'-BNA derivatives containing modified nucleobases effectively recognized a pyrimidine-purine interruption. Some other examples of nucleic acid analogues to overcome the two major drawbacks in the triplex-forming oligonucleotides are also summarized.

DNA↗
PubMed2006

Defining the sequence-recognition profile of DNA-binding molecules.

Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.

Animals↗
PubMed2000

Transcriptional activation by LR1 at the Emu enhancer and switch region sites.

LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Emu immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Emu core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Emu core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions.

Animals↗
PubMed1999

Structure and recognition of sheared tandem G x A base pairs associated with human centromere DNA sequence at atomic resolution.

G x A mismatched base pairs are frequently found in nucleic acids. Human centromere DNA sequences contain unusual repeating motifs, e.g. , (GAATG)n x (CATTC)n found in the human chromosome. The purine-rich strand of this repeating pentamer sequence forms duplex and hairpin structures with unusual stability. The high stability of these structures is contributed by the "sheared" G x A base pairs which present a novel recognition surface for ligands and proteins. We have solved the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method of d(CCGAATGAGG) in which the centromere core sequence motif GAATG is embedded. Three crystal forms were refined to near-atomic resolution. The structures reveal the detailed conformation of tandem G x A base pairs whose unique hydrogen-bonding surface has interesting interactions with bases, hydrated magnesium ions, cobalt(III)hexaammine, spermine, and water molecules. The results are relevant in understanding the structure associated with human centromere sequence in particular and G x A base pairs in nucleic acids (including RNA, like ribozyme) in general.

Adenine↗
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↗
PubMed2003

Charge transport in DNA duplex/quadruplex conjugates.

DNA conjugates containing adjacent duplex and guanine quadruplex assemblies have been designed to explore charge transport into quadruplex architectures. The quadruplex assemblies have been characterized structurally using circular dichroism and by assaying for chemical protection. Using an intercalating rhodium photooxidant, noncovalently bound or tethered to the duplex end, oxidizing radicals are found to be trapped in the folded quadruplex. Damage is observed almost exclusively at the external tetrads of the quadruplex. Little damage of the center tetrad is observed, due most likely to lowered efficiency of radical trapping within the quadruplex core. This pattern of damage is distinct from that observed for repetitive G sequences within duplex DNA. The data indicate, furthermore, that in the conjugates examined, the guanine quadruplex provides a more effective trap than a 5'-GG-3' guanine doublet within duplex DNA. Within these assemblies, sufficient base-base overlap must exist at the duplex/quadruplex junction to allow for charge migration. This funneling of damage to the quadruplex, as well as the unique pattern of damage within the quadruplex, requires consideration with respect to the analysis of oxidative DNA damage within the cell.

Circular Dichroism↗
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↗
PubMed2001

Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation.

There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes with specificity. Such molecules would be useful to test hypotheses for telomere length regulation, and may have therapeutic potential. The versatility and modular nature of the zinc finger motif makes it an ideal candidate for engineering G-quadruplex-binding proteins. Phage display technology has previously been widely used to screen libraries of zinc fingers for binding to novel duplex DNA sequences. In this study, a three-finger library has been screened for clones that bind to an oligonucleotide containing the human telomeric repeat sequence folded in the G-quadruplex conformation. The selected clones show a strong amino acid consensus, suggesting analogous modes of binding. Binding was found to be both sequence dependent and structure specific. This is the first example of an engineered protein that binds to G-quadruplex DNA, and represents a new type of binding interaction for a zinc finger protein.

Amino Acid Sequence↗
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↗