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N R Kallenbach

Publications and source records attributed to N R Kallenbach.

At least 19 recordsLinked to original sources
PubMed1992

T-T base mismatches enhance drug binding at the branch site in a four-arm DNA junction.

Base mismatches--non Watson-Crick pairing between bases--can arise in duplex DNA as a consequence of mutational events or by recombination. In a duplex, the sequence of the two bases involved, and those flanking the site of mismatch, determines the local structure and extent of destabilization of the helix. Base mismatches can arise also in recombination of nonhomologous strands, and their occurrence in Holliday recombination intermediates can influence the outcome of general or specialized recombination events. We have previously reported that the branch site in a DNA junction can interact selectively with a variety of ligands. Here we describe the thermodynamics of junctions containing T-T mismatches flanking the branch and show that these structures bind methidium and other intercalators with higher affinity than junctions lacking mismatches.

Base Composition↗
PubMed1992

Adenine affects the structure and stability of telomeric sequences.

Adenine occurs in the strand containing repeated G clusters in the telomeric DNA of a variety of organisms, including that of humans. The role of adenine has been investigated by constructing two sets of oligonucleotides each with one, two, or four copies of the telomeric sequence dTTTAGGG together with a control sequence in which T replaces the A residue, dTTTTGGG. Comparison of the stability and spectral properties of these two sequences in the presence of Na+ or K+ affords a basis for defining the role of adenine in these structures. In Na+, the A residue stabilizes the structure formed by each oligomer significantly, presumably by a base-pairing interaction with T. In K+, by contrast, there is little difference in stability. In two- and four-copy oligomers, the A sequence has a different structure from its T analog, as detected by CD spectroscopy. In the presence of either Na+ or K+, the tetraplexes of A and T interact with intercalators.

Adenine↗
PubMed1992

Conformational properties of B-Z junctions in DNA.

The structural consequences of specific base sequences in DNA can exert a strong influence on the function of DNA. It has previously been reported that the presence of multiple B-Z conformational junctions in constructed DNA oligomers results in unusually enhanced electrophoretic gel mobilities of these oligomers [Winkle, S. A., & Sheardy, R. D. (1990) Biochemistry 29, 6514-6521]. In order to investigate this phenomenon further, we designed and synthesized several DNA oligomers capable of pure Z or B-Z junction formation for polyacrylamide gel electrophoresis studies. The results indicate that both pure Z-DNA and polymorphic B-Z-DNA oligomers exhibit unusual gel migratory properties. The results of gel mobility studies in the absence and presence of cobalt hexamine indicate that a B-Z junction corresponds to a stiff bend of the helix axis, with two or more conformers accessible at the junction site. This is a different bend and mechanism than that in oligo(A) tracts.

Base Sequence↗
PubMed1992

Interaction of the dye ethidium bromide with DNA containing guanine repeats.

DNA containing one or more copies of the motifs repeated in telomere sequences has unusual conformational properties. The isolated sequence from the protozoan Oxytricha, dT4G4 has the potential to form tetramers in the presence of sodium or potassium ions. We report here that these tetramers bind ethidium tightly, with an interaction that fulfills several criteria for an intercalative mechanism in the G sequence. By contrast, the 4-fold tandem repeat of this subunit, d(T4G4)4, does not interact specifically with ethidium in the presence of Na+. This difference might have a simple structural basis: the tetramer of dT4G4 forms a stack of four G-quartets in the presence of Na+ or K+, whereas the constraint imposed by the T4 "tethers" in the repeat d(T4G4)4 allows only two layers to form in the presence of Na+. In the presence of sufficient K+, the latter can partially form a four-layer G-quartet structure, which interacts with ethidium. This idea is supported by analysis of a "relaxed" sequence, dT4G4(T7G4)3, which allows formation of four G-quartets and binds ethidium in the presence of Na+ as well as K+. Ethidium (and intercalators generally) should thus be able to retard or inhibit the action of telomerase in the presence of K+.

Animals↗
PubMed1992

Structure and stability of sodium and potassium complexes of dT4G4 and dT4G4T.

The ends of eukaryotic chromosomes contain specialized structures that include DNA with multiple tandem repeats of simple sequences containing clusters of G on one strand, together with proteins which synthesize and bind to these sequences. The unit repeat in the protozoan Oxytricha with the cluster dT4G4 can form structures containing tetrads of guanine residues, referred to G4 DNA, in the presence of metal ions such as Na+ or K+. We show here that, in the presence of Na+, dT4G4 forms a tetramer with parallel strands by means of a UV cross-linking assay. In the presence of K+, two further interactions are observed: at low temperature, higher order complexes are formed, provided the 3' end of the strand is G; a single 3'T inhibits this association in dT4G4T. At high temperature, these complexes dissociate, leading to a tetramer with a different ordered structure that melts only at very high temperatures. These results suggest that the cohesive properties of DNA containing G clusters might depend on associative interactions driven by a free 3'G terminus in the presence of K+, as well as by connecting antiparallel G hairpins as has been postulated.

Circular Dichroism↗
PubMed1992

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.

Base Composition↗
PubMed1992

Energetic contribution of solvent-exposed ion pairs to alpha-helix structure.

Understanding the role of amino acid side-chain interactions in forming secondary structure in proteins is useful for deciphering how proteins fold and for predicting folded structures of proteins from their sequence. Analysis of the secondary structure as a function of pH in two designed synthetic peptides with identical composition but different sequences, affords a quantitative estimate of the free energy contribution of a single ion pair to the stability of an isolated alpha-helix. One peptide contains repeated blocks of Glu4Lys4. The second has repeated blocks of Glu2Lys2. The former contains significant helical structure at neutral pH while the latter has none, based on ultraviolet light circular dichroism measurements and 1H nuclear magnetic resonance spectroscopy. The difference is attributed to formation of helix-stabilizing salt-bridges between Glu- and Lys+ spaced at i, i + 4 intervals in the former peptide. The free energy of formation of a single Glu(-)-Lys+ salt-bridge can be evaluated by using a statistical model of the helix-coil transition that explicitly includes salt-bridges: the result is -0.50(+/- 0.05) kcal/mol at 4 degrees C and neutral pH in 10 mM salt, in agreement with a value derived for a single salt-bridge in a helix on the surface of a globular protein.

Amino Acid Sequence↗
PubMed1991

Drug binding to a DNA BZ molecule: analysis by chemical footprinting.

The polymorphism in a DNA 16-mer (designated BZ-II) has been investigated by means of circular dichroism (CD) spectroscopy and chemical footprinting. CD spectra indicate that, in low salt, the oligomer is fully right-handed whereas, in high salt, it possesses a B-Z conformational junction: half of the duplex is right-handed while the other half is left-handed. Treatment of BZ-II with diethyl pyrocarbonate (DEPC) confirms the existence of a left-handed segment of the duplex in high salt: enhanced DEPC scission occurs at the G residues in the alternating CG sequence. The scission patterns of the upper and lower strands in BZ-II by the reactive chemical probe MPE.Fe(II), and the antitumor antibiotics dynemicin and Fe-(II).bleomycin, are different under low salt conditions. The 3'-terminal region of both upper and lower strands and the middle region of the upper strand of BZ-II are preferential cleavage sites in low salt. This result suggests that the methylated cytosines or the alternating CG domain in the molecule perturbs the DNA structure. Under high salt conditions, the reactivity of the Z-DNA segment of BZ-II for MPE.Fe(II) and Fe(II).bleomycin is dramatically enhanced, while it is reduced in the case of dynemicin. Excess propidium (PI) eliminates preferential cleavage by each of these chemical probes in high salt conditions. This is due in part to conversion of the BZ-DNA molecule into B-DNA, as is seen by a DEPC modification experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthraquinones↗
PubMed1991

Parallel and antiparallel Holliday junctions differ in structure and stability.

Two Holliday junction analogs, JA and JP, containing identical base-paired arms have been constructed from oligonucleotides. The former is constrained to adopt an antiparallel Sigal-Alberts structure, and the latter a parallel structure, by means of single strand d(T)9 tethers. We evaluate here the free energy difference between JA and JP using two different methods. One is a direct measurement of the ratio of the equilibrium constants for formation of branched structures from intact duplexes using one labeled strand and a competition assay. The second method estimates the difference in stability from the difference in thermal denaturation temperatures of JA and JP, using urea to shift the tm of the complexes. Both methods reveal a small free energy difference between the two complexes: JA is more stable than JP by -1.1(+/- 0.4) kcal (mol junction)-1, at 25 degrees C, 5 mM-Mg2+, from the first method, and by -1.6(+/- 0.3) kcal (mol junction)-1, according to the second. DNase I and the resolvase, endonuclease I from phage T7, cleave JA differently from JP in the vicinity of the branch, indicating that the structures of these two models differ at this site. Diethyl pyrocarbonate also reveals a difference in the major grooves. Comparison of the scission patterns of JA and JP by the reactive chemical probes methidium-propyl-EDTA..Fe(II), [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2,[(OP)2Cu(I)], indicates that in both cases the branch point is a site of enhanced binding for drugs, as it is in the untethered four-arm junction containing the same core sequence at the branch.

Base Composition↗
PubMed1991

Effect of sequence on the structure of three-arm DNA junctions.

We have investigated the geometry of a number of three-arm branched DNA molecules by measuring the relative electrophoretic mobilities of analogues of each junction in which one pair of arms is extended. In general, the mobilities of three species of three-arm junctions in which the duplex arms are extended pairwise differ in the presence of Mg2+. This effect is eliminated by the absence of Mg2+ or by an increase in temperature, leading us to conclude that the three-arm DNA junctions are not 3-fold symmetric, because of either preferential stacking or asymmetric kinking of the arms at the branch that occurs in the presence of Mg2+. The geometry of the junction is governed by the base sequence at the branch and 1 bp removed from the branch. The pairwise elongated analogues of junctions that contain identical base pairs at the branch or 1 bp from the branch show mobility differences; when both positions have the same sequence no mobility differences are detected even in the presence of Mg2+. Formation of a branch in three-arm DNA junctions can be seen to produce a strain or deformation that propagates about one turn of the helix from the branch, leading thymines in this region to become hyperreactive to osmium tetraoxide. Surprisingly, the effect is independent of the presence or absence of metal cations. The structure of the three-arm junction is thus quite different in character from that of four-arm junctions both in the presence and absence of high concentrations of metal cations.

Base Sequence↗
PubMed1991

Alpha-helix stabilization by natural and unnatural amino acids with alkyl side chains.

Knowledge of the role of individual side chains in forming different secondary structures such as the alpha-helix would be useful for prediction of protein structure from sequence or de novo protein design. Experimental and theoretical studies on natural and synthetic peptides and proteins indicate that individual side chains differ in their helix-forming potential. Four aliphatic side chains occur in the standard complement of amino acids: alanine and leucine are helix stabilizing, whereas isoleucine and valine are weakly destabilizing. We have synthesized a series of helical peptides containing unnatural aliphatic side chains having two to four carbons to explore some of the factors involved in alpha-helix stabilization and the basis for selection of the natural set. We find that linear side chains with two, three, or four carbons are as strongly helix stabilizing as the single methyl in alanine and that all linear side chains are stronger helix promoters than leucine. In addition, a t-butyl side chain is significantly more helix destabilizing than the sec-butyl side chain of isoleucine, the isopropyl side chain of valine, or even the unrestricted side chain of glycine. These results provide experimental evidence that restriction in conformational freedom of a side chain imposed by alpha-helix formation is a major component of the role of a side chain in stabilizing helical structure.

Amino Acids↗
PubMed1991

Resolution of branched DNA substrates by T7 endonuclease I and its inhibition.

Endonuclease I is a multipurpose enzyme implicated in the breakdown of host DNA, packaging of phage DNA, and recombination during the lytic cycle of bacteriophage T7. We investigate here some aspects of the substrate requirements for its activity in resolving branched intermediates similar to Holliday junctions (Holliday, R. (1964) Genet. Res. 5, 282-304) that arise in recombination. The enzyme is able to resolve branched substrates containing very short duplex arms: 4 base pairs suffice. It cleaves 5' to the branch, with a distinct preference for the non-crossover strands in Holliday-like model junctions. Ligands that interact strongly with the branch site can inhibit the enzyme, with KI values in the 10-50 microM range.

Base Sequence↗
PubMed1991

Conformational preference and ligand binding properties of DNA junctions are determined by sequence at the branch.

Four-arm DNA branched junctions are stable analogues of Holliday recombinational intermediates. A number of four-arm DNA junctions synthesized from oligonucleotides have now been studied. Gel mobility or chemical footprinting experiments on several immobile four-arm junctions indicate that in the presence of Mg2+, they assume a preferred conformation consisting of two helical domains, each formed by stacking a particular pair of arms on each other. We show here that a junction we designate as J1c that has the same chemical composition as one we have previously studied in detail, J1, but is formed from the four strands complementary to those of the latter, exhibits the reverse stacking preference. The pattern of self-protection of the strands of J1c exposed to Fe(II).EDTA-induced scission reveals that twofold symmetry is preserved, but the opposite pair of strands preferentially cross over. Moreover, the Fe(II).EDTA scission profiles of J1c indicate that this junction exhibits a weaker bias as to which strands cross over than is observed in J1. The preference for the dominant species in J1 is 1.3 times greater than in J1c at 4 degrees C and in the presence of 10 mM Mg2+, based on chemical reactivity data. This is confirmed by a cleavage experiment using the resolvase enzyme, endonuclease I, from bacteriophage T7. This difference could reflect either sequence-dependent differences in the equilibrium among isomers, or in the structure of these junctions. Chemical footprinting experiments using the probes MPE.Fe(II) and (OP)2Cu(I) show that the high-affinity ligand binding site in immobile junctions is determined by junction geometry.

Base Sequence↗
PubMed1991

The helix-coil transition in heterogeneous peptides with specific side-chain interactions: theory and comparison with CD spectral data.

Natural and synthetic peptides that contain detectable intramolecular alpha-helical structure in aqueous solution have been used to evaluate the helical propensities for the common amino acids. Experimental spectroscopic data must be fit to a model of the helix-coil transition in order to determine quantitative stability constants for each amino acid. We present here a statistical mechanical description of helix formation in peptides or protein fragments that takes into account multiple internal conformations, heterogeneity in the stabilizing effects of different side chains, and specific side-chain-side-chain interactions. The model enables one to calculate values of [theta]222 for a given peptide using the length dependence of the helix signal computed by a quantum mechanical treatment of the n pi * transition that dominates the 222-nm band. In addition, the helical probability at any residue in the chain is readily computed, and should prove useful as nmr spectral data become available. The free energy of specific side-chain interactions, including ion pair formation, can be evaluated. Application of the analysis to experimental data on a pair of isomeric peptides, only one of which contains ion pairs, indicates that forming a single glutamate-lysine ion pair stabilizes the alpha-helix by 0.50 kcal/mole in 10 mM sodium ion and pH 7. A survey of the CD data measured for a variety of model peptides is presented, indicating that a single set of s values and sigma constant can account for some but not all of the available results.

Amino Acid Sequence↗
PubMed1991

Site-specific interaction of the antitumor antibiotic dynemicin with branched DNA molecules.

A specific interaction of stable branched DNA molecules with the antitumor antibiotic dynemicin is reported. Dynemicin contains an anthraquinone and an enediyne unit, and belongs to the family of enediyne antitumor agents. DNA strand scission by dynemicin appears to involve interaction of the anthraquinone core with DNA and release of a phenyl diradical from the enediyne core that can abstract hydrogen atoms from the sugar phosphate backbone of DNA. The cleavage patterns of each labeled strand in two branched tetramers of four 16-mers are compared with those of the same strands in unbranched duplex controls. Differences between the profiles corresponding to scission of branched and duplex DNA molecules can be detected in most of the strands. The strongest differences define a specific site flanking the branch in each of two branched structures. At 18 degrees C, cleavage at strand positions demarcating the site of enhanced affinity in both junctions is observed to be 70-100% more efficient than at the corresponding sequence positions in the control duplex DNA molecules. The patterns of preferential cleavage at these sites are significantly altered in the presence of excess propidium diiodide, an intercalative drug.

Anthraquinones↗
PubMed1991

Determination of DNA cleavage specificity by esperamicins.

The esperamicins are members of a class of potent antitumor antibiotics that contain stained diacetylenic ring systems capable of forming DNA-cleaving diradicals upon reaction with thiols. Here we show that the diacetylenic ring core itself determines the sequence specificity for scission of duplex DNA): esperamicin A1, and three products of hydrolysis of the glycon, esperamicins C, D, and E, are found to retain a common sequence preference. The sugar residues exert a strong influence on the cleavage efficiency, presumably by interacting nonspecifically with DNA. The presence of a branch in the DNA is found locally to inhibit scission by esperamicins, and this effect is shown to be due to the core also.

Aminoglycosides↗
PubMed1990

Asymmetric structure of a three-arm DNA junction.

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗