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PubMed1993

Sequence dependence of the free energy of B-Z junction formation in deoxyoligonucleotides.

The NaCl-induced transition from the right-handed B form to a hybrid form containing both left and right-handed DNA, joined by a B-Z junction, was investigated. Transition curves were constructed from circular dichroism spectra collected as a function of NaCl concentration for a series of 16 bp deoxyoligonucleotides. The sequence of the series (one strand of the duplex) was: 5'CGCGCGCGAMNGACTG, where C indicates m5dC, and -MN- was varied to include the possible Py:Py stacks: -CC-, -CT-, -TC- and -TT-. Transition curves for the conversion of all deoxyoligonucleotides were found to be biphasic. Singular value decomposition was used to analyze the experimental circular dichroism spectra obtained as a function of NaCl, and showed that the transition was not a simple two-state process, but rather required at least three species to account for the experimental data. A sequential three-state model, B<-->I<-->BZ, was derived and applied to analyze experimental transition curves. Non-linear least-squares analysis was used to evaluate the salt-dependent equilibrium constants for each step in the sequential reaction model. The results indicate that the free energy change for B-Z junction formation (delta Gj) depends on the dinucleotide sequence near the junction. At a Na activity of 5, delta Gj values ranging from +1.2 to +1.7 kcal mol-1 were determined, depending on the sequence near the junction. delta Gj was found to be strongly dependent on salt concentration, with its magnitude decreasing with increasing Na activity. In addition to studies on linear duplex molecules, the B to BZ transition was also investigated in "dumbbell" forms of selected sequences. In these molecules, the ends were covalently linked by a single-stranded T4 segment. These studies show that junction formation is energetically more costly in dumbbells than in their linear counterparts. A striking correlation was found between delta Gj and two independent conformational properties of the dinucleotide steps that were introduced into the linear duplex molecules. These are the free energy of unstacking and the estimated free energy change for the conversion of the dinucleotide from the B to the A conformation. The more stable against unstacking, or the more resistant to the B to A conversion, the sequence near the junction site is, the more costly is B-Z junction formation. These correlations reveal that the DNA sequence near the junction apparently must be pliable in order to accommodate the unusual structure of the junction.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed1980

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts.

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.

Cell Division↗

PubMed1995

Novel mutagenic properties of abasic sites in Saccharomyces cerevisiae.

Abasic sites are particularly important in mutation research because they are frequently the ultimate lesion in chemical mutagenesis, and because they are believed to be a paradigm for non-pairing lesions. Although preferential insertion of dAMP ("A-rule") opposite the lesion has been observed in almost all previous studies with other organisms, we find that in budding yeast, Saccharomyces cerevisiae, the preferred nucleotide is dCMP, suggesting that yeast has a "C-rule", at least with respect to the vector constructs used. These constructs contained a single abasic site specifically located within a 28 nucleotide single-stranded region in an otherwise duplex vector. Nucleotide insertions were determined by sequence analysis of replicated vectors taken from a random set of yeast transformants. In three different sequence contexts, the frequencies of dCMP and dAMP insertion were 83% and 13%, 62% and 31%, and 85% and 8%, respectively. A similar bias in favor of cytosine insertion was found using vectors that were entirely single-stranded. However, a preference for dAMP insertion was found when Escherichia coli, rather than yeast, was transfected with samples of the same gapped duplex vector DNA. Preferential insertion of dCMP is not likely to have arisen by previously proposed mechanisms, but is also unlikely to have occurred by a primer/template misalignment mechanism, in which a nearby template guanine directs the insertion of cytosine. Predominant dCMP insertion was observed even when template guanine bases were excluded from a region extending 19 nucleotides 5', and 13 nucleotides 3', to the abasic site.

Base Sequence↗

PubMed2004

Synthesis of a 1'-aminomethylthymidine and oligodeoxyribonucleotides with 1'-acylamidomethylthymidine residues.

Reported here is a 10-step synthesis of a phosphoramidite building block of 1'-aminomethylthymidine that starts from 2-deoxyribose. The framework of the branched aminonucleoside was elaborated from a known 1-cyano-1-bromo glycosyl donor, whose reaction with the silylated nucleobase furnished the 1'-cyanide, which was reduced to the desired aminomethylnucleoside. The N-allyloxycarbonyl (Alloc)-protected nucleoside was converted to a phosphoramidite building block and incorporated into the oligonucleotides 5'-GCAT*TATTAC-3', and 5'-GCAT*TAT*TAC-3', where T* denotes 1'-acylamidomethylthymidine residues. Removal of the Alloc protecting group and acylation with the residue of pyrene-1-yl-butanoic acid were achieved on support, using microwave irradiation to ensure full conversion. The UV-melting point of the duplex of the singly and doubly modified decamers with their fully complementary target sequence is 0.1-6.9 degrees C higher than that of the unmodified control duplex, depending on the salt concentration. This suggests that the aminomethyl linker may allow for the placing of a functional "payload" in the minor groove of DNA duplexes without disrupting the helix. Oligonucleotides thus endowed with functional modifications may become useful for biomedical applications.

Base Sequence↗

PubMed2004

Locked nucleic acids and intercalating nucleic acids in the design of easily denaturing nucleic acids: thermal stability studies.

Intercalating nucleic acids (INA(R)s) with insertions of (R)-1-O-(1-pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make "quenched" complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13-mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 degrees C) in comparison with the unmodified double-stranded DNA (Tm=48.7 degrees C) and the corresponding duplexes of LNA/DNA (Tm=81.6 degrees C) and INA/DNA (Tm=66.4 degrees C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.

DNA↗

PubMed1988

Parallel stranded duplex DNA.

Three linear 21-nt oligonucleotides (C2, C3, C7) have been synthesized with different sequences of A and T residues. One pairwise combination, (C3, C7), hybridizes to form a conventional antiparallel duplex (aps-C3.C7), whereas the pair C2, C3 forms a duplex (ps-C2.C3) in which the two strands are in a parallel orientation and the A.T base-pairs in a reverse Watson-Crick configuration. The existence of the novel ps helical structure was established from the following criteria: (i) The electrophoretic mobilities of the ps and aps duplexes in native and denaturing polyacrylamide gels are similar. (ii) The ps duplex is not a substrate for T4 DNA ligase. (iii) Salt-dependent thermal transitions are observed for the two duplexes, but the melting temperatures of the ps molecules are 15 degrees C lower. (iv) The ultraviolet absorption and circular dichroism spectra of the ps duplex are indicative of a base-paired structure, but differ systematically from that of the aps helix. (v) Based on fluorescent measurements, the bis-benzimidazole drug BBI-258 shows a lower affinity for the ps compared to the aps duplex, whereas the opposite preference holds for the intercalator ethidium bromide. We conclude from the present study that parallel stranded DNA is a stable conformation which can arise by interaction between two conventional strands with appropriate sequence homology.

Circular Dichroism↗

PubMed1983

DNA sequence of the US component of the varicella-zoster virus genome.

The linear duplex DNA molecule of varicella-zoster virus is 120 000 bp in size and has the sequence arrangement UL-IRS-US-TRS, where UL and US are unique sequences and IRS and TRS are inverted repeats flanking US. The primary structure of the cloned SstI g DNA fragment containing US (5232 bp) and adjacent portions of IRS and TRS (426 bp of each) was determined, and the following model for genetic expression was derived from an analysis of the sequence. The region specifies four mRNAs encoding primary translation products with mol. wts. of 11, 44, 39 and either 74 or 70 kd. The 39-and 70-kd proteins have primary structures characteristic of membrane proteins. The mRNAs encoding the 11- and 74/70-kd proteins extend from opposite sides of US into IRS/TRS, thus sharing a common 3' terminus. These proteins do not share a common carboxy terminus because the coding region for the 11-kd protein terminates at the junction between US and IRS, whereas that for the 74/70-kd protein extends into TRS. The analysis affirms the hypothesis that the extent of inverted repeats in herpesvirus genomes is primarily a result of constraints imposed by adjacent protein coding sequences.

Amino Acid Sequence↗

PubMed2000

Interaction of the E. coli DNA G:T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence.

The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The enzyme shows a preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E. coli dcm DNA-methyltransferase (CC[A/T]GG). Thus, the preferred substrate for the vsr protein is (CT[A/T]GG), where the underlined T is opposed by a dG base. This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches. Evaluation of specificity constant (k(st)/K(D); k(st)=rate constant for single turnover, K(D)=equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e. the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT[A/T]GG and C(5Me)C[T/A]GG. Conversion of the mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates. No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence. An analysis of the fraction of active protein, by "reverse-titration" (i.e. adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate. This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a (32)P-labelled nucleic acid from the vsr protein) and burst kinetic analysis. This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity.

Base Pair Mismatch↗

PubMed1985

Displacement synthesis of globin complementary DNA: evidence for sequence amplification.

We have examined a cDNA displacement synthesis procedure in which the extent of precursor incorporation and the unusual kinetics of displacement synthesis suggest a unique replicative form of DNA and the occurrence of multiple rounds of displacement synthesis, leading to amplification of mRNA sequences. Globin double-stranded DNA containing a hairpin loop was extended by the addition of a homopolymer to the 3' end. This was followed by displacement synthesis with the Klenow fragment of DNA polymerase I that was primed by an oligonucleotide hybridized to the homopolymer. Thus, the hairpin cDNA was copied to form an open duplex with an inverted repetition of globin sequences. These molecules can then serve as templates for additional synthesis which would be primed from oligomers bound the homopolymer. Globin cDNA sequences appear to be amplified 10-fold or more by this procedure. Globin cDNA obtained by displacement synthesis was similar in size to the original template. However, displaced molecules associate to the extent that they are not readily resolved by electrophoresis or sedimentation under nondenaturing conditions. Restriction endonuclease digests of 32P-labeled displaced strands gave fragment patterns similar to rabbit globin cDNA hairpin molecules. S1 nuclease studies demonstrated that displaced complexes and replication intermediates are partially single stranded, which might account for their aggregation properties.

Animals↗

PubMed1996

Partial thermodynamic parameters for prediction stability and washing behavior of DNA duplexes immobilized on gel matrix.

Earlier we showed that reported in literature nearest-neighbor thermodynamic parameters describe poorly the thermal-induced behavior of DNA duplexes immobilized in gel. Here we present a complete set of partial thermodynamic parameters for all 10 nearest-neighbor interactions specially developed for duplexes immobilized in gel. This thermodynamic library allows to predict dissociation enthalpy and free energy of DNA duplex immobilized in gel matrix from its base sequence. The predicted values are in good agreement with the experimental ones. Dissociation enthalpy and free energy are needed for such application as (i) predicting relative stability of duplexes formed by DNA with oligonucleotides immobilized in cells of gel matrix; (ii) selecting optimal conditions for hybridization experiment; (iii) predicting washing curves and washing temperatures at irreversible temperature-stepped wash of DNA out of oligonucleotide gel matrix; (iv) selecting optimal conditions for washing gel matrix.

Acrylic Resins↗

PubMed1998

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution.

BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion. CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.

Crystallography, X-Ray↗

PubMed1997

Stabilizating effect of 5-nitroindole (universal base) on DNA duplexes immobilized on gel matrix.

Effect of attachment of 1-(2-deoxy-beta-D-ribofuranosyl)-5-nitroindole (NIDR) to the ends of target sequence of oligonucleotides immobilized on gel micromatrix on stability of duplex formed by hybridization with DNA fragment was studied. It was shown that adjunction of NIDR to 5' as well as to 3' end results in increasing stability of duplexes although in the second case the extent of stabilization effect is essentially lower. Both 5' and 3' terminal NIDR exhibited no selectivity to the opposite base while the stabilizing effect depended dramatically on the nature of the adjacent base especially in the case of 5'-end-attached universal base. The neighborhood of purine bases decreased substantially the stabilizing effect of 5' terminal NIDR. In contrast with this, the stabilizing effect of 3' terminal NIDR was reduced only slightly by adjacent pyrimidine bases.

Acrylic Resins↗

PubMed2006

DNA-methyltransferase SsoII as a bifunctional protein: features of the interaction with the promoter region of SsoII restriction-modification genes.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.

Catalytic Domain↗

PubMed1997

Sequence-independent recombination triple helices: a molecular dynamics study.

Recent experimental studies have shown that the Rec-A mediated homologous recombination reaction involves a triple helical intermediate, in which the third strand base forms hydrogen bonds with both the bases in the major groove of the Watson-Crick duplex. Such 'mixed' hydrogen bonds allow formation of sequence independent triplexes. DNA triple helices involving 'mixed' hydrogen bonds have been studied, using model building, molecular mechanics (MM) and molecular dynamics (MD). Models were built for a triplex comprising all four possible triplets viz., G.C*C, C.G*G, A.T*T and T.A*A. To check the stability of all the 'mixed' hydrogen bonds in such triplexes and the conformational preferences of such triplex structures, MD studies were carried out starting from two structures with 30 degrees and 36 degrees twist between the basepairs. It was observed that though the two triplexes converged towards a similar structure, the various hydrogen bonds between the WC duplex and the third strand showed differential stabilities. An MD simulation with restrained hydrogen bonds showed that the resulting structure was stable and remained close to the starting structure. These studies help us in defining stable hydrogen bond geometries involving the third strand and the WC duplex. It was observed that in the C.G*G triplets the N7 atom of the second strand is always involved in hydrogen bonding. In the G.C*C triplets, either N3 or O2 in the third strand cytosine can interchangeably act as a hydrogen bond acceptor.

Base Sequence↗

PubMed1992

Different interactions of spermine with a "curved" and a "normal" DNA duplex: (CA4T4G)n and (CT4A4G)n. Gel electrophoresis and circular dichroism studies.

Two nucleotide sequences, (CA4T4G)n and (CT4A4G)n, are generally considered as prototypes of a "curved" and a "normal" DNA duplex on the basis of their different behaviour with respect to physico-chemical and/or biochemical properties. We have studied their gel electrophoretic mobilities in the presence of the biologically significant tetracation spermine and have found that spermine causes a decrease of the gel electrophoretic mobility of "curved" DNA, but does not influence that of "normal" DNA. Circular dichroism spectra of the complexes between spermine and the two examined DNAs show dramatically different features. These findings suggest that different DNA superstructures are relevant in interactions not only with proteins, but also with polycations, such as spermine.

Base Sequence↗

PubMed1992

The DNA target of the trp repressor.

Unexpected features seen by high resolution X-ray crystallography at the interface of the trp repressor and the 'traditional' trp operator provoked the claim that the DNA fragment used in the crystal structure is not the true operator, and therefore that the crystal structure of the trp repressor-operator complex does not portray a specific interaction. An alternative sequence was proposed mainly on the basis of mutational studies and gel retardation analysis of short target duplexes (Staacke et al., 1990a,b). We have reexamined the sequence consensus in trpR-repressible promoters and analyzed the mutagenesis experiments of others including Staacke et al. (1990a) and found them fully consistent with the interactions of the traditional operator sequence seen in the crystal structure, and stereochemically inconsistent with the above referenced alternative model. Moreover, an in vitro trp repressor-DNA binding analysis, employing both novel DNA constructs devised to avoid previously encountered artifacts as well as full-length promoter sequences, indicates that the traditional operator used in the crystal structure is the preferred target of the trp repressor.

Bacterial Proteins↗

PubMed1999

Telomeres of polytene chromosomes in a ciliated protozoan terminate in duplex DNA loops.

The end of a telomeric DNA sequence isolated from a polytene chromosome of a hypotrichous ciliate folds back and hybridizes with downstream telomeric sequence to form a t loop that is stable in the absence of protein and DNA cross-linking. The single-stranded, telomeric DNA sequence at the end of a macronuclear molecule does not form a t loop but, instead, is complexed with a heterodimeric, telomere-binding protein. Thus, two mechanisms for capping the ends of DNA molecules are used in the same cell.

Animals↗

PubMed1992

Two-dimensional NMR studies of d(GGTTAATGCGGT).d(ACCGCATTAACC) complexed with the minor groove binding drug SN-6999.

The dodecadeoxynucleotide duplex d(GGTTAATGCGGT).d(ACCGCATTAACC) and its 1:1 complex with the minor groove binding drug SN-6999 have been prepared and studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments have been obtained for the free duplex by standard methods. The line widths of the resonances in the complex are greater than those observed for the free duplex, which complicates the assignment process. Extensive use of two-quantum spectroscopy was required to determine the scalar correlations for identifying all of the base proton and most of the 1'H-2'H-2''H spin subsystems for the complex. This permitted unambiguous sequence-specific resonance assignments for the complex, which provides the necessary background for a detailed comparison of the structure of the duplex, with and without bound drug. A series of intermolecular NOEs between drug and DNA were identified, providing sufficient structural constraints to position the drug in the minor groove of the duplex. However, the combination of NOEs observed can only be rationalized by a model wherein the drug binds in the minor groove of the DNA in both orientations relative to the long helix axis and exchanges rapidly between the two orientations. The drug binds primarily in the segment of five consecutive dA-dT base pairs d(T3T4A5A6T7).d(A18T19T20A21A22), but surprisingly strong interactions are found to extend one residue in the 3' direction along each strand to G8 and C23. The observation of intermolecular contacts to residues neighboring the AT-rich region demonstrates that the stabilization of the bis(quaternary ammonium) heterocycle family of AT-specific, minor groove binding drugs is not based exclusively on interactions with dA-dT base pairs.

Antineoplastic Agents↗