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Results for “duplex sequencing”

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PubMed1988

Initiation of adenovirus DNA replication. I. Mechanism of action of a host protein required for replication of adenovirus DNA templates devoid of the terminal protein.

The in vitro replication of adenovirus (Ad) DNA covalently attached to the 55-kDa terminal protein requires at least five proteins including the 80-kDa preterminal protein, the Ad DNA polymerase, the Ad DNA binding protein, nuclear factor I, and topoisomerase I. The replication of Ad DNA templates devoid of the terminal protein requires an additional protein, designated factor pL, which has been purified from uninfected HeLa cell nuclei (Guggenheimer, R. A., Nagata, K., Kenny, M., and Hurwitz, J. (1984) J. Biol. Chem. 259, 7815-7825). Factor pL has been found to contain an intrinsic 5'----3' exonuclease activity. When Ad DNA templates lacking the terminal protein were pretreated with factor pL, the requirement for factor pL in the replication reaction was abolished. Synthetic partially duplex oligonucleotide templates containing Ad origin sequences were constructed in order to determine the structure of the DNA molecules that are active in the absence of factor pL. These experiments indicated that factor pL degrades the 5'-end of the nontemplate (displaced) strand of the Ad origin thereby creating a single-stranded region at the 3'-end of the template strand. Such DNAs are competent for initiation of Ad DNA replication in the absence of factor pL but remain dependent on nuclear factor 1.

Adenoviridae↗

PubMed1988

[Effect of the structure of oligonucleotide substrates on interaction with methylase Eco dam].

The interaction of Eco dam methylase with various synthetic oligonucleotide substrates was investigated. The "imperfect" duplexes contained a normal GATC recognition sequence in one chain of the enzyme recognition site and had some defects in the complementary chain, i.e., the absence of one or several nucleotide residues or the presence of S-methyl thiophosphate groups at the 3'-termini. The 3'-S-methyl thiophosphate residue has the same effect on the methylation of oligonucleotide complexes as does the absence of internucleotide phosphate in the analogous complexes. The presence of both GA dinucleotides in the recognition site is necessary for a productive enzyme-substrate interaction. The experimental data suggest that Eco dam methylase does form a symmetrical enzyme-substrate complex which is similar to that formed by type II restriction enzymes.

Base Sequence↗

PubMed1996

Editing of the GLuR-B ion channel RNA in vitro by recombinant double-stranded RNA adenosine deaminase.

Double-stranded RNA (dsRNA)-specific adenosine deaminase (DRADA) has been implicated as an enzyme responsible for the editing of RNA transcripts encoding glutamate-gated ion channel subunits (GLuR) in brain. In one case, the editing alters the gene-encoded glutamine (Q) to an arginine (R) located within the channel-forming domain of the alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunit GLuR-B. The result of editing at this site, called the 'Q/R' site, is a profound alteration of the Ca2+ permeability of the GLuR channel. Using recombinantly expressed DRADA proteins, we now demonstrate in vitro that DRADA is indeed involved in editing of the GLuR-B RNA. In addition to the formation of an RNA duplex structure involving exon and intron sequences, Q/R site-selective editing by DRADA also requires a cofactor protein(s) commonly present even in non-neuronal cells. The accuracy and efficiency of this RNA editing system appear to be determined by the quantitative balance between DRADA, cofactor and substrate GLuR-B RNA.

Adenosine Deaminase↗

PubMed1996

Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking.

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, binds and cleaves duplex DNA at sites containing the sequence 5'-(T/C)CCTT. We report the identification of Tyr70 as the site of contact between the enzyme and the +4C base of its target site. This was accomplished by UV-crosslinking topoisomerase to bromocytosine-substituted DNA, followed by isolation and sequencing of peptide-DNA photoadducts. A model for the topoisomerase-DNA interface is proposed, based on the crystal structure of a 9 kDa N-terminal tryptic fragment. The protein domain fits into the DNA major groove such that Tyr70 is positioned close to the +4C base and Tyr72 is situated near the +3C base. Mutational analysis indicates that Tyr70 and Tyr72 contribute to site recognition during covalent catalysis. We propose, based on this and other studies of the vaccinia protein, that DNA backbone recognition and reaction chemistry are performed by a relatively well-conserved 20 kDa C-terminal portion of the vaccinia enzyme, whereas discrimination of the DNA sequence at the cleavage site is accomplished by a separate N-terminal domain, which is less conserved between viral and cellular proteins. Division of function among distinct structural modules may explain the different site specificities of the eukaryotic type I topoisomerases.

Amino Acid Sequence↗

PubMed1998

[p16INK4/CDKN2 gene deletions and mutations in non-small cell lung cancers].

OBJECTIVE: To investigate the genomic status of cyclindependent kinase-4 inhibitor, p16 INK4/CDKN2 gene in primary non-small cell lung cancers. METHODS: p16 gene was analysed by duplex PCR, PCR-SSCP, and sequencing in 31 primary non-small cell lung cancer (NSCLC) tissues. RESULTS: Gene deletions were found in three samples (3/31), one case had whole p16 gene deletion and the other two had deletion in exon 1 and in exon 2 respectively. Two cases of point mutations (2/30) in the gene had been found, one in exon 1 and another one in exon 2. In the 5 cases of p16 gene abnormal samples, 4 cases (two cases of deletions and two cases of mutations) were non-small cell lung cancers with lymphnode metastasis (4/20). CONCLUSION: These results indicate that inactivation of p16 gene is associated with some NSCLC (5/31), and probably be a late event in NSCLC carcinogenesis.

Adult↗

PubMed2005

A thermodynamic approach to designing structure-free combinatorial DNA word sets.

An algorithm is presented for the generation of sets of non-interacting DNA sequences, employing existing thermodynamic models for the prediction of duplex stabilities and secondary structures. A DNA 'word' structure is employed in which individual DNA 'words' of a given length (e.g. 12mer and 16mer) may be concatenated into longer sequences (e.g. four tandem words and six tandem words). This approach, where multiple word variants are used at each tandem word position, allows very large sets of non-interacting DNA strands to be assembled from combinations of the individual words. Word sets were generated and their figures of merit are compared to sets as described previously in the literature (e.g. 4, 8, 12, 15 and 16mer). The predicted hybridization behavior was experimentally verified on selected members of the sets using standard UV hyperchromism measurements of duplex melting temperatures (T(m)s). Additional experimental validation was obtained by using the sequences in formulating and solving a small example of a DNA computing problem.

Algorithms↗

PubMed1990

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs.

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.

Adenine↗

PubMed1992

Association of 2'-5' oligoribonucleotides.

Oligoribonucleotides with 2'-5' linkages have been synthesized on solid support. UV melting and CD experiments indicate complementary strands associate to give complexes with melting temperatures 30 to 40 degrees C lower than for duplexes formed by 3'-5' oligoribonucleotides with the same sequence. UV melting and imino proton NMR spectra and NOEs for (2'-5') CGGCGCCG are consistent with formation of an antiparallel duplex. The results suggest greater duplex stability was one factor favoring 3'-5' over 2'-5' linkages in evolution.

Base Sequence↗

PubMed1997

Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids.

In this study we report for the first time the binding affinity of RNase H1 for oligonucleotide duplexes. We used a previously described 17-mer antisense sequence [Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., & Freier, S. M. (1992) J. Biol. Chem. 267, 19954-19962] hybridized to a complementary oligoribonucleotide to evaluate both the binding affinity and the catalytic rate of RNase H1. The dissociation constants (Kd) of RNase H1 for the various substrates tested were determined by inhibition analysis using chemically modified noncleavable oligonucleotide heteroduplexes. Catalytic rates were determined using heteroduplex substrates containing chimeric antisense oligonucleotides composed of a five-base deoxynucleotide sequence flanked on either side by chemically modified nucleotides. We find that the enzyme preferentially binds A-form duplexes: RNase H bound A-form duplexes (RNA:RNA and DNA:RNA) approximately 60-fold tighter than B-form duplexes (DNA:DNA) and approximately 300-fold tighter than single-strand oligonucleotides. The enzyme exhibited equal affinity for both the wild type (RNA:DNA) oligonucleotide substrate and heteroduplexes containing various 2'-sugar modifications, while the cleavage rates for these chemically modified substrates were without exception slower than for the wild type substrate. The introduction of a single positively charged 2'-propoxyamine modification into the chimeric antisense oligonucleotide portion of the heteroduplex substrate resulted in both decreased binding affinity and a slower rate of catalysis by RNase H. The cleavage rates for heteroduplexes containing single-base mismatch sequences within the chimeric oligonucleotide portion varied depending on the position of the mismatch but had no effect on the binding affinity of the enzyme. These results offer further insights into the physical binding properties of the RNase H-substrate interaction as well as the design of effective antisense oligonucleotides.

Base Sequence↗

PubMed1989

A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor.

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

Base Composition↗

PubMed1995

Chemical cross-linking of MvaI and EcoRII enzymes to DNA duplexes containing monosubstituted pyrophosphate internucleotide bond.

DNA duplexes containing a monosubstituted pyrophosphate internucleotide group, instead of a phosphodiester bond, were used as cross-linking reagent for the affinity modification of the restriction endonucleases EcoRII and MvaI (R.EcoRII and R.MvaI). An active group was introduced into the enzyme's recognition site or between the recognition site and flanking sequence. The substrate properties of such DNA duplexes were determined. Cross-linking specificity was demonstrated by competition experiments with unmodified substrate, as well as by the absence of cross-linking to an active duplex lacking a recognition site. It was shown that the nucleophilicity of the buffer solution and the presence of the enzyme cofactor Mg2+ dramatically affected the cross-linking yield.

Base Sequence↗

PubMed1998

NMR structure of the extended Myb cognate sequence and modeling studies on specific DNA-Myb complexes.

The recognition sequence of the Myb protein has been recently described to be pyAACKGHH (where py = T/C, K = G/T, and H = A/C/T), modifying the earlier identification as pyAACKG [Ording, E., et al. (1994) Eur. J. Biochem. 222, 113-120]. We had earlier determined the solution structure of the minimal cognate sequence TAACGG, choosing py = T and K = G, embeded in a 12-mer DNA duplex by NMR and related computational techniques [Radha, P. K., et al. (1995) Biochemistry 34, 5913-5912]. To understand the structural significance of the above modification and the role of the variability in the recognition sequence, we have investigated here the solution structure of a different DNA segment, d-ACAACTGCAGTTGT, which contains the extended Myb cognate site, CAACTGCA. The three-dimensional structure of the 14-mer duplex has been determined from NMR data by relaxation matrix and restrained molecular dynamics calculations. The structure of the above cognate sequence in the 14-mer duplex has been compared with that of the cognate sequence, TAACGG, in the 12-mer duplex and also with that in the NMR structure of the Myb DNA binding domain (R2R3)-DNA complex determined by Ogata et al. recently [Ogata, K., et al. (1994) Cell 79, 639-648]. The comparison highlighted differences in several structural parameters for the cognate sites in the DNA segments. Modeling studies by taking out the protein from the complex and presenting it with 12-mer and 14-mer DNA structures indicated that the protein induces structural alterations to drive the cognate site to a reasonably conserved structure. The extent of similarity of the derived structures was, however, dependent on the base sequences. Base changes in the minimal cognate sequence in the 12-mer-protein complex and in the 14-mer-protein complex so as to match the sequence of Ogata et al. produced a more conserved structure of the complex. A reverse exercise, in which the Ogata DNA in the complex was mutated to match the 12-mer and 14-mer minimal cognate sequences, complemented the above observations of the subtle sequence dependence of the structure in the complex. On the other hand, base changes in the extension did not influence the DNA-protein complex structure significantly. We also observed that the structural changes in the protein were very minor when different DNA sequences or different DNA structures were presented to it. These observations would be of interest from the point of view of DNA-Myb recognition.

Crystallography, X-Ray↗

PubMed1999

Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex.

Minor adducts, derived from the covalent binding of anti-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-[BP]dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-[BP]A6-C7-T8-T9-C10-C11). (d(G12-G13-A14-A15-G16-T17-G18-A19-G20+ ++-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the [BP]dA6 lesion site without disrupting the flanking Watson-Crick dC5.dG18 and [BP]dA6.dT17 base pairs. This structure of the (+)-cis-anti-[BP]dA.dT 11-mer duplex, containing a bay region benzo[a]pyrenyl [BP]dA adduct, is compared with the corresponding structure of the (+)-trans-anti-[BPh]dA.dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo[c]phenanthrenyl [BPh]dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick [BP]dA6.dT17 base pair more pronounced in the (+)-cis-anti-[BP]dA.dT 11-mer duplex compared to its Watson-Crick [BPh]dA.dT17 base pair in the (+)-trans-anti-[BPh]dA.dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N(6) of adenine when compared to the N(2) of guanine in DNA duplexes. The [BPh]dA and [BP]dA N(6)-adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to [BP]dG N(2)-guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of [BP]dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix.

Adenine↗

PubMed1979

Distribution of inverted repeat sequences in nuclear DNA from Physarum polycephalum.

Inverted repeat sequences, capable of forming stable intra-chain foldback duplexes, are shown using electron microscopy to be located in over 90% of fragments of nuclear DNA from Physarum polycephalum. A statistical treatment of the data indicates that, on average, foldback sequence foci are spaced every 7,000 nucleotides and that they are distributed uniformly amongst the DNA chains. The majority of inverted repeat sequences give rise to the simple types of foldback structure observed in DNA from other eukaryotic species, but a significant proportion of the DNA fragments also contain novel foldback structures with a more complex appearance, referred to as 'bubbled' hairpins. The latter structures appear to be formed by the annealing of several distinct segments of homologous inverted repeat sequence, each separated by interspersed non-foldback sequences of variable sizes up to 15,000 nucleotides in length. The size, both of the foldback duplexes and of the intervening single-chain segments of DNA, are not random. Instead, they appear to form a regular, arithmetic series of lengths. These observations suggest that the different segments of Physarum DNA from which foldback structures are derived contain nucleotide sequences that share a highly ordered and unform pattern of structural organisation. These regular units of organisation in Physarum DNA in some cases extend over distances up to 50,000 nucleotides in length.

Base Sequence↗

PubMed1997

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.

Nucleic Acid Hybridization↗

PubMed2006

Nearest neighbor parameters for Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides.

Results from optical melting studies of Watson-Crick complementary heteroduplexes formed between 2'-O-methyl RNA and RNA oligonucleotides are used to determine nearest neighbor thermodynamic parameters for predicting the stabilities of such duplexes. The results are consistent with the physical model assumed by the individual nearest neighbor-hydrogen bonding model, which contains terms for helix initiation, base pair stacking and base pair composition. The sequence dependence is similar to that for Watson-Crick complementary RNA/RNA duplexes, which suggests that the sequence dependence may also be similar to that for other backbones that favor A-form RNA conformations.

Base Pairing↗

PubMed1995

Optical melting of 128 octamer DNA duplexes. Effects of base pair location and nearest neighbors on thermal stability.

The use of short oligonucleotide probes is finding increased application in DNA sequencing and genome characterization techniques, but a lack of knowledge of the hybridization properties of short duplexes hinders their use. Melting data were acquired on 128 DNA duplexes based on the length proposed in sequencing by hybridization procedures and formed from the general sequences 5'-XYZTGGAC-3',5'-GTCCAXYZ-3',5'-GCXYZGAC-3', and 5'-GTCXYZGC-3' where X, Y, and Z are either A, T, G, or C. These molecules were designed to elucidate the effects of location and nearest-neighbor stacking on the stability of base pairing in short DNA duplexes. The type of base pairs present had a major effect on stability, but was insufficient to predict stability without the inclusion of nearest-neighbor terms. Furthermore, the addition of information on position, or distance from the end, of the nearest-neighbor doublets led to statistically better fitting of the melting data. However, the positionally dependent stabilization differences are small compared with the contributions of base pairing and stacking.

Base Composition↗

PubMed1992

Triple-helix formation is compatible with an adjacent DNA-protein complex.

The effect of oligonucleotide-directed triple-helix formation on the binding of a protein to an immediately adjacent sequence has been examined. A double-stranded oligonucleotide was designed with a target site for the binding of a pyrimidine oligonucleotide located immediately adjacent to the recognition sequence for the herpes simplex virus type 1 (HSV-1) origin of replication binding protein, which is encoded by the UL9 gene of HSV-1. Since the optimal conditions for the binding of the UL9 protein and the pyrimidine oligonucleotide to the duplex DNA are markedly different, a pyrimidine oligonucleotide was designed to optimize binding affinity and specificity for the target duplex oligonucleotide. Consideration was given to length and sequence composition in an effort to maximize triple-strand formation under conditions amenable to the formation of the UL9-DNA complex. Using gel mobility shift assays, a trimolecular complex composed of duplex DNA bound to both a third oligonucleotide strand and the UL9 protein was detected, indicating that the UL9-DNA complex is compatible with the presence of a triple helix in the immediately adjacent sequences.

Animals↗