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B W Kalisch

Publications and source records attributed to B W Kalisch.

9 recordsLinked to original sources
PubMed1990

Length-dependent formation of parallel-stranded DNA in alternating AT segments.

Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.

Base Sequence↗
PubMed1990

Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.

DNA↗
PubMed1988

Relative stability of parallel- and antiparallel-stranded duplex DNA.

We have recently shown that DNA containing homopolymeric A-T base pairs can form a parallel-stranded intramolecular duplex [van de Sande et al. (1988) Science (Washington, D.C.) 241, 551-557]. In the present paper, we demonstrate that parallel-stranded DNA can also be formed in unconstrained bimolecular DNA of appropriate sequence homology. Three deoxyoligonucleotides, a 21-mer [dCCCATATATATTTTTTTTCCC], a ps-15-mer [dTATATATAAAAAAAA], and an aps-15-mer [dAAAAAAAATATATAT], have been synthesized. Annealing of 21-mer and aps-15-mer results in the formation of a conventional antiparallel duplex (aps); however, the combination of 21-mer and ps-15-mer forms a duplex in which the two strands are in a parallel orientation (ps). The parallel-stranded structure was established from the following criteria: (i) The parallel-stranded structure shows a 1:1 stoichiometry of the constituent strands. (ii) Gel electrophoretic mobility of the ps and aps duplexes are similar under native conditions. (iii) Spectroscopic properties of the ps duplex are characteristics for a base-paired structure but are different from the aps structure. (iv) Both duplexes undergo a thermally induced helix to coil transition; however, the melting temperature for the ps duplex is 22 degrees C lower. (v) The minor groove binding drug Hoechst 33258 shows a reduced affinity for the ps compared to the aps duplex. (vi) The parallel-stranded duplex is not a substrate for DNA Escherichia coli polymerase I (Klenow fragment) or AMV reverse transcriptase. Parallel-stranded DNA can exist under normal solution conditions, but competition experiments show it to be thermodynamically less favorable than the conventional antiparallel form.

Base Composition↗
PubMed1988

Parallel stranded DNA.

A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.

DNA↗
PubMed1986

Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments.

A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers). These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site. The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer. They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end. This was evidenced by mobility shifts of the splinker-ligated restriction fragments. The splinker-ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus. The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase. As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence.

Base Composition↗
PubMed1985

Synthesis and characterization of poly[d(G-z5C)]. B-Z transition and inhibition of DNA methylase.

Deoxy-5-azacytidine 5'-triphosphate was synthesized and used as a substrate for the enzymatic synthesis of the polynucleotide poly[d(G-z5C)]. Whereas the triphosphate decomposes in solution, the azacytosine analogue incorporated into DNA is stable under conditions preserving the double-helical structure. Poly[d(G-z5C)] undergoes the transition to the left-handed Z conformation at salt (NaCl and MgCl2) concentrations approximately 30% higher than those required for unsubstituted poly[d(G-C)]. However, the incorporation of azacytidine potentiates the formation at room temperature of the Z helix stabilized by the transition metal Mn2+; in the case of poly[d(G-C)], a heating step is required. The spectral properties of the two polymers in the B and Z forms are similar. Both left-handed forms are recognized by anti-Z DNA immunoglobulins, indicating that the DNAs bear common antigenic features. Poly[d(G-z5C)] is not a substrate for the DNA cytosine 5-methyltransferase from human placenta. It is a potent inhibitor of the enzyme when tested in a competitive binding assay. These results are compatible with a very strong, possibly covalent, mode of interaction between methyltransferases and DNA containing 5-azacytosine.

Azacitidine↗
PubMed1979

The effect of antibiotics on the T4 polynucleotide ligase catalyzed template dependent polymerization of oligodeoxythymidylates.

The poly(dA) dependent T4 polynucleotide ligase catalyzed polymerization of oligodeoxythymidylates is dependent upon duplex stability. The antibiotics ethidium bromide, netropsin and Hoechst 33258 stabilize the duplex poly(dA) . P(dT)n (n = 6-10) to thermal denaturation. Ethidium bromide to DNA ratio of 1.25 and netropsin or Hoechst 33258 to DNA ratio of 0.1 the Tm of d(pT) 10 . poly (dA) was increased by 10 degrees and 25 degrees C respectively. The T4 polynucleotide ligase activity was not inhibited under these conditions and temperature optimum of joining of d(pT) 10 . poly(dA) was increased 5 degrees to 10 degrees by the binding of the antibiotics. Duplexes containing shorter oligodeoxythymidylates required lower concentrations of the antibiotics netropsin or Hoechst 33258 to show no inhibition of T4 polynucleotide ligase. The temperature optima of joining the duplexes d(pT)6 . POLY(DA) and d(pT) 8 . poly(dA) were increased by 5 degrees C upon binding of the antibiotics. Polyacrylamide gel analysis of the T4 polynucleotide ligase catalyzed joining of the oligodeoxythymidylates showed that the presence of antibiotics affected the product distribution of the polymerized oligomers.

Anti-Bacterial Agents↗