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Results for “duplex sequencing”

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PubMed2000

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro.

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.

Attachment Sites, Microbiological↗

PubMed2001

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

Hot Temperature↗

PubMed1988

Electron microscopy of nucleic acid.

Electron microscopy (EM) of nucleic acids was first described by Kleinschmidt and Zahn 1959 (1). By complexing duplex DNA with basic proteins and spreading the complexed DNA onto the surface of an aquous phase it was possible to obtain well stretched DNA molecules that could be examined by EM. Addition of formamide to DNA spreading solution as well as to hypophase prevented single-stranded DNA and RNA from forming secondary structures thus providing conditions for analysis of these molecules. EM studies have provided information about molecular weight, molecular configuration and homogeneity/heterogeneity of the DNA preparations examined. Furthermore, intramolecular heterogeneity in base sequence could be analyzed by partial denaturation of duplex DNA. By partial denaturation mapping information of the orientation of DNA molecules was obtained. Sequence homology between two DNA molecules were studied by heteroduplex analyses where the two DNAs to be studied were denatured to full strand separation followed by reassociation. This method has been widely used to analyze DNA mutations, and to discover sequence homologies within a DNA molecule or between two DNA molecules. Heteroduplex analyses between DNA and RNA (R-loop formation) provided information about distribution of ribosomal RNA genes and gave the first information of eukaryotic post transcriptional processing of mRNA. Also the various modes for DNA replication and transcription were studied by EM. It is possible to study duplex DNA not complexed by basic proteins by EM. The DNA is then adsorbed to an activated carbon film and rotary shadowed with finely grained metals to enhance the contrast. Using this method it was possible to study binding of proteins to DNA. This method was also used in the study of chromatin structure where it provided information of the size and distribution of nucleosomes as well as packaging of DNA in chromatin fibres. The complicated nature of DNA strand exchange and repair reactions was studied by in-vitro systems where EM analysis was used in the description of the presynoptic filament and structure of the joined molecules. Furthermore, EM provided the first direct evidence for the existence of the paranemic joint, where the DNA strands are joined without Watson-Crick base-pairing.

Animals↗

PubMed1997

Altered structure of the DNA duplex recognized by yeast transcription factor Reb1p.

The Saccharomyces cerevisiae REB1 gene encodes a sequence-specific DNA binding protein that has been implicated in chromatin structure, transcription regulation and transcription termination. Previous work has shown that the DNA sequence recognized by Reb1p contains an adenosine residue that is unusually reactive toward chemical modification by dimethylsulfate and that methylation of this nucleoside increases the binding affinity of the Reb1p protein for its target. Prompted by these results, we determined the solution structure of the 13mer Reb1p DNA duplex recognition site d(GTCCGGGTAATGC).d(GCATTACCCGGAC) using 2D NMR, distance geometry and iterative 2D NOESY back-calculation structure refinement. The distance geometry-refined molecule demonstrated an unusual structure in the TAAT region of the sequence that was manifested in cross-strand base stacking, as indicated by unusually strong NOE interactions between H2 protons on three adjacent adenosine bases. This structure was compared to two published NMR studies of DNA duplexes containing the related sequence TAAC. The Reb1p DNA structure does not show the conformational mobility or the 'transient kink' at TpA steps characteristic of the related TAAT-containing sequences.

DNA↗

PubMed1979

Total synthesis of a tyrosine suppressor transfer RNA gene. XIII. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence -1 to -29 in the promoter region.

Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described. These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains. All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a [32P]phosphate group.

Base Sequence↗

PubMed1983

Vitellogenin B2 gene in Xenopus laevis: isolation, in vitro transcription and relation to other vitellogenin genes.

The isolation of the four Xenopus laevis vitellogenin genes has been completed by the purification from a DNA library of the B2 gene together with its flanking sequences. The overlapping DNA fragments analyzed cover 34 kilobases. The B2 gene which has a length of 17.5 kilobases was characterized by heteroduplex and R-loop mapping in the electron microscope and by in vitro transcription in a HeLa whole-cell extract. Its structural organization is compared with that of the closely related B1 gene. The mRNA-coding sequence of about 6 kilobases is interrupted 34 times in the B1 gene and 33 times in the B2 gene. Sequence homology between the two genes was not only found in exons. In addition, 54% of the intron sequences as well as 63% and 48.5% respectively of the 5' and 3' flanking sequences, show enough homology to form stable duplexes. These findings are compared with earlier results obtained with the two other closely related members of the vitellogenin gene family, the A1 and the A2 genes.

Animals↗

PubMed2004

Short lexitropsin that recognizes the DNA minor groove at 5'-ACTAGT-3': understanding the role of isopropyl-thiazole.

Isopropyl-thiazole ((iPr)Th) represents a new addition to the building blocks of nucleic acid minor groove-binding molecules. The DNA decamer duplex d(CGACTAGTCG)(2) is bound by a short lexitropsin of sequence formyl-PyPy(iPr)Th-Dp (where Py represents N-methyl pyrrole, (iPr)Th represents thiazole with an isopropyl group attached, and Dp represents dimethylaminopropyl). NMR data indicate ligand binding in the minor groove of DNA to the sequence 5'-ACT(5)AG(7)T-3' at a 2:1 ratio of ligand to DNA duplex. Ligand binding, assisted by the enhanced hydrophobicity of the (iPr)Th group, occurs in a head-to-tail fashion, the formyl headgroups being located toward the 5'-ends of the DNA sequence. Sequence reading is augmented through hydrogen bond formation between the exocyclic amine protons of G(7) and the (iPr)Th nitrogen, which lies on the minor groove floor. The B(I)/B(II) DNA backbone equilibrium is altered at the T(5) 3'-phosphate position to accommodate a B(II) configuration. The ligands bind in a staggered mode with respect to one another creating a six base pair DNA reading frame. The introduction of a new DNA sequence-reading element into the recognition jigsaw, combined with an extended reading frame for a small lexitropsin with enhanced hydrophobicity, holds great promise in the development of new, potentially commercially viable drug lead candidates for gene targeting.

DNA↗

PubMed2002

Nontemplated nucleotide addition by HIV-1 reverse transcriptase.

We studied the kinetics of nontemplated nucleotide addition by the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) using model substrates derived from the 3' end of HIV-1 minus-strand strong-stop DNA. The addition of a nontemplated nucleotide was highly dependent on the nature of the base (fastest addition with dATP), type of nucleoside, and pH of the reaction buffer. The salt concentration, presence or absence of nucleocapsid protein, and nature of the blunt-ended duplex (DNA/DNA versus RNA/DNA) had only limited effects. The efficiency and base specificity were strongly affected by the sequence at the 3' end of the blunt-ended duplex. In every case, nontemplated nucleotide addition was much slower than templated polymerization. The K(d) for the incoming dNTP with an RT bound to a blunt-ended duplex was at least 1000-fold higher than with a duplex with a template overhang. At concentrations normally found in vivo, ATP can compete with dNTPs for binding to the polymerase active site and reduce the efficiency of nontemplated nucleotide addition. Although a stable ternary complex RT/DNA/dNTP could be readily detected by gel retardation assays if the DNA had a template overhang, stable ternary complexes were not observed with a blunt-ended duplex substrate. At in vivo concentrations of dNTPs (5-10 microM), nontemplated nucleotide addition occurred, but it was very inefficient and the rate of nontemplated polymerization is at least 10000-fold slower than the rate of templated polymerization. We could conclude that, in vivo, the unfavorable binding of the incoming dNTP, low concentration of dNTPs, the presence of a large concentration of ATP, and the inability to form a stable ternary complex prior to the polymerization step collaborate to reduce the efficiency of nontemplated nucleotide addition.

Adenosine Triphosphate↗

PubMed2006

Binding of the universal minicircle sequence binding protein at the kinetoplast DNA replication origin.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation.

Animals↗

PubMed2005

Specific and nonspecific hybridization of oligonucleotide probes on microarrays.

Gene expression analysis by means of microarrays is based on the sequence-specific binding of RNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving sequences other than the intended target is problematic because it adds a chemical background to the signal, which is not related to the expression degree of the target gene. The article presents a molecular signature of specific and nonspecific hybridization with potential consequences for gene expression analysis. We analyzed the signal intensities of perfect match (PM) and mismatch (MM) probes of GeneChip microarrays to specify the effect of specific and nonspecific hybridization. We found that these events give rise to different relations between the PM and MM intensities as function of the middle base of the PM, namely a triplet-like (C > G approximately T > A > 0) and a duplet-like (C approximately T > 0 > G approximately A) pattern of the PM-MM log-intensity difference upon binding of specific and nonspecific RNA fragments, respectively. The systematic behavior of the intensity difference can be rationalized on the level of basepairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Nonspecific binding is characterized by the reversal of the central Watson-Crick (WC) pairing for each PM/MM probe pair, whereas specific binding refers to the combination of a WC and a self-complementary (SC) pairing in PM and MM probes, respectively. The Gibbs free energy contribution of WC pairs to duplex stability is asymmetric for purines and pyrimidines of the PM and decreases according to C > G approximately T > A. SC pairings on the average only weakly contribute to duplex stability. The intensity of complementary MM introduces a systematic source of variation which decreases the precision of expression measures based on the MM intensities.

Biophysics↗

PubMed1999

Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora.

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (zeta-eta) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.

Base Pairing↗

PubMed1991

RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange.

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.

Adenosine Triphosphate↗

PubMed1992

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.

Base Sequence↗

PubMed2003

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes.

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.

Animals↗

PubMed2004

Conformationally gated electrochemical gene detection.

The synthesis and characterization of a 26-base DNA hairpin containing both a redox-active reporter (ferrocene) and terminal thiol functionality for electrochemical gene detection is described. This electrochemical DNA sensor exploits electron-transfer dynamics that alter as a consequence of a large structural rearrangement (hairpin-to-duplex) induced by hybridization of the target DNA sequence. Melting temperature and circular dichroism studies confirm that the 26-mer DNA forms a hairpin structure in the absence of target DNA. The loop region of the DNA hairpin is shown to form a stable duplex in the presence of complementary single-stranded DNA. Atomic force microscopy and ellipsometry experiments of immobilized self-assembled DNA monolayers suggest that hybridization with complementary DNA affords a conformational change that alters the electrochemical response.

Base Sequence↗

PubMed1994

A-Z-RNA conformational changes effected by high pressure.

This paper reports evidence obtained by circular dichroism (CD) spectroscopy measurements indicating that two oligoribonucleotide duplexes with the alternating purine-pyrimidine sequences r(GC)6 or r(AU)6 change their A-RNA conformation under high pressure. Under the high-pressure conditions at which B-Z-DNA transition easily occurs, RNA acquires a conformation which only differs slightly from that of A-RNA. However, exposure of r(GC)6 or r(AU)6 to high pressure (6 kbar) in the presence of 5 M NaCl causes a conformation change of both oligoribonucleotide duplexes from their A- to their Z-RNA form. The departure of RNA or DNA duplexes from their original conformations under high pressure depends on the water structure itself and involves displacing an active (structural) water molecule outside the nucleic acid molecules. Experiments carried out until now in many laboratories have shown that B-Z or A-Z transitions of DNA or RNA, respectively, do not depend on the conditions applied, but the common mechanism for these processes seems to be dehydration. This same effect can be observed either at high salt concentrations or in the presence of an alcohol or at high pressure. Our results also support the view that the higher stability of RNA compared with DNA duplexes is due to the strong interaction of the 2'-hydroxyl groups of RNA with water molecules.

Circular Dichroism↗

PubMed1981

Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.

Base Sequence↗

PubMed1994

Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.

To better understand genetic diversity within mammalian reoviruses, we determined S2 nucleotide and deduced sigma 2 amino acid sequences of nine reovirus strains and compared these sequences with those of prototype strains of the three reovirus serotypes. The S2 gene and sigma 2 protein are highly conserved among the four type 1, one type 2, and seven type 3 strains studied. Phylogenetic analyses based on S2 nucleotide sequences of the 12 reovirus strains indicate that diversity within the S2 gene is independent of viral serotype. Additionally, we found marked topological differences between phylogenetic trees generated from S1 and S2 gene nucleotide sequences of the seven type 3 strains. These results demonstrate that reovirus S1 and S2 genes have distinct evolutionary histories, thus providing phylogenetic evidence for lateral transfer of reovirus genes in nature. When variability among the 12 sigma 2-encoding S2 nucleotide sequences was analyzed at synonymous positions, we found that approximately 60 nucleotides at the 5' terminus and 30 nucleotides at the 3' terminus were markedly conserved in comparison with other sigma 2-encoding regions of S2. Predictions of RNA secondary structures indicate that the more conserved S2 sequences participate in the formation of an extended region of duplex RNA interrupted by a pair of stem-loops. Among the 12 deduced sigma 2 amino acid sequences examined, substitutions were observed at only 11% of amino acid positions. This finding suggests that constraints on the structure or function of sigma 2, perhaps in part because of its location in the virion core, have limited sequence diversity within this protein.

Amino Acid Sequence↗