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Biomedical subjects

D M Crothers

Publications and source records attributed to D M Crothers.

At least 19 recordsLinked to original sources
PubMed1992

Stability and properties of double and triple helices: dramatic effects of RNA or DNA backbone composition.

Studies of a series of short oligonucleotide double and triple helices containing either all RNA, all DNA, or a mixture of the two show strand-dependent variation in their stability and structure. The variation in stability for both groups falls over a range of greater than 10 kilocalories per mole. In forming the triple helix, RNA is favored on both pyrimidine strands, whereas DNA is favored on the purine strand. In general, relatively unstable duplexes form particularly stable triplexes and vice versa. Structural data indicate that the strands in hybrid helices adopt a conformation that is intermediate between molecules containing all DNA and all RNA. Thus, RNA-DNA hybrids were not forced into the conformation of the RNA (A-form). The provocative stability of the triplex with an RNA third strand+DNA duplex points to novel antisense strategies and opens the possibility of an in vivo role of these structures. Overall, the data emphasize the fundamental role of sugars in determining the properties of nucleic acid complexes.

Base Sequence↗
PubMed1992

Structural modifications of camptothecin and effects on topoisomerase I inhibition.

Camptothecin (1), a potent antitumor alkaloid, is known to inhibit topoisomerase I, an enzyme that relaxes supercoiled DNA. Modifications have been made to the B, D, and E rings of this natural product. Specifically, compounds 2-10 either have an ester moiety in place of the E ring lactone, a methyl ester attached to position 14, a saturated (or nonexistent) deaza B ring, or contain a combination of these permutations. We have conducted in vitro assays against the topoisomerase I relaxation reaction which verify the necessity for a lactone in the E ring. Furthermore, steric requirements at position 14 are shown to be crucial for activity, and planarity of the A and B rings of camptothecin is also implicated in the ability of the drug to inhibit topoisomerase I. Speculation on the nature of the drug binding pocket is presented.

Animals↗
PubMed1992

RNA binding assays for Tat-derived peptides: implications for specificity.

RNA recognition by the HIV Tat protein is mediated in part by an arginine- and lysine-rich basic subdomain implicated as a signature element in proteins that bind RNA. Relative RNA binding affinities for a 14-residue peptide derived from Tat that spans the basic region are determined using a competition protocol. Binding specificity is compared with complexation by a 38-residue model for the RNA binding domain of Tat using the same approach. Binding strength for the minimal (14 residue) peptide is correlated with that for the longer peptide: both peptides recognize a short, bulged duplex. However, the shorter peptide dissociates more rapidly from the wild-type site and discriminates less well between nonspecific (double-stranded RNA) and specific sites. Relative dissociation constants for 38-residue peptide determined from direct partition and competition assays differ; the former assay consistently predicts stronger discrimination against RNAs with mutations in the stems flanking the bulge. Differences between the two assays are reconciled in terms of contributions from labile binding which is unstable to native gel electrophoresis. Kinetic stability may constitute a major specificity determinant for basic subdomain-mediated recognition of RNA.

Base Sequence↗
PubMed1992

Protein-induced bending and DNA cyclization.

We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.

Base Sequence↗
PubMed1992

Sliding and intermolecular transfer of the lac repressor: kinetic perturbation of a reaction intermediate by a distant DNA sequence.

The lac repressor associates with its operator at a rate faster than conventional diffusion allows, either because of one-dimensional diffusion of a captured repressor along the DNA (sliding) or because the tetrameric repressor can be rapidly transferred between DNA sites distant from each other in the primary sequence (direct transfer). We report measurements of relative repressor-operator association rates at physiological operator concentrations. We assay for the ability of DNA targets of equal length (approximately 200 base pairs) containing one or six operator segments to compete for repressor; as the sliding range decreases, the hexameric operator fragment should react up to six times faster than the monomeric operator fragment. We find that the advantage enjoyed by the hexameric fragment varies from little to none at low operator concentration (depending on ionic strength) to more than 3-fold at high concentration. We ascribe this behavior to sliding at low operator concentration and to an increasing contribution of bimolecular direct transfer events as concentration increases. The observations require a "semibound" intermediate state of the protein at operator sites. This species can either undergo a relatively slow (tau greater than 1 sec) unimolecular isomerization to the final complex, or the isomerization can occur in a bridged complex with another operator site, accompanied by transfer to the second operator with probability of 0.5. Bridging alters one or more rate constants in the complex.

Base Sequence↗
PubMed1991

Specificity and stringency in DNA triplex formation.

Triple-helix formation can in principle serve as a general method for sequence-specific recognition and physical separation of duplex DNA molecules. Realization of this goal depends on how much the triplex is destabilized by mismatches and other defects (specificity) and on finding conditions in which perfect complexes are stable and defect complexes are not (stringency). We have addressed the question of specificity by determining the difference in free energy between perfect and defect complexes by using UV melting curves and equilibrium competition experiments. We find that third strands that bind with either single-base bulges or single mismatches are destabilized relative to the perfect triplex by 2.5-2.9 and 3.2-4.0 kcal/mol (1 cal = 4.184 J), respectively, essentially equivalent to the corresponding values determined for duplex DNA and RNA. Also, we present a method, referred to as stringency clamping, which maintains specific binding under conditions far from normal stringency. To do this, we provide for the formation of a competing structure involving the third strand with stability between that of the perfect and imperfect complexes; the competitive interaction effectively prevents triplex formation at imperfect sites even far below their melting temperature. We illustrate the phenomenon with three different stringency clamps, two of which compete for the all-pyrimidine third strand through Watson-Crick pairing and one that competes through all-pyrimidine pairing at acidic pH. We demonstrate physical separation of two duplex DNA molecules differing by a single base pair in their target sequence for triple-helix formation.

Base Sequence↗
PubMed1991

The carbohydrate domain of calicheamicin gamma I1 determines its sequence specificity for DNA cleavage.

We have investigated the DNA cleaving properties of calicheamicinone, the synthetic core aglycone of calicheamicin gamma I1, a natural product with extremely potent antitumor activity. Our experiments have shown that the synthetic analog binds and cleaves DNA, albeit without any sequence selectivity and with less efficiency than the natural compound. We propose that a key element in the sequence recognition process is the thiobenzoate ring present in the natural compound. We have demonstrated by one-dimensional NMR that there is direct hydrogen abstraction from DNA by calicheamicinone, with enhanced binding affinity contributed by the carbohydrate domain. The reduced efficiency of hydrogen abstraction from DNA by bound calicheamicinone, compared with the natural compound, implicates the carbohydrate moiety in positioning the drug for hydrogen abstraction.

Aminoglycosides↗
PubMed1991

RNA recognition by Tat-derived peptides: interaction in the major groove?

Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.

Amino Acid Sequence↗
PubMed1991

Catabolite activator protein-induced DNA bending in transcription initiation.

We describe experiments that enable us to track the presence and direction of the DNA bend induced by Escherichia coli catabolite activator protein (CAP) through the intermediate stages of transcription initiation at the lac promoter. Transcriptional complexes examined were formed on superhelical templates to enhance specific complex formation, and detected by electrophoretic analysis after restriction digestion. We found that the bend is maintained and even increased upon formation of closed and open complexes. Our results exclude the hypothesis that the energy of the CAP-induced bend is used to promote open complex formation. We now suggest a new model, in which DNA wraps around the CAP-polymerase complex to form a writhing structure equivalent to that at the end of an interwound superhelical domain. Formation of this structure may facilitate open complex formation. We further propose that the stored bend energy may be used to help counteract strong protein-protein or protein-DNA interactions, thus assisting the process of RNA polymerase escape from the promoter.

Base Sequence↗
PubMed1991

Synthetic DNA bending sequences increase the rate of in vitro transcription initiation at the Escherichia coli lac promoter.

Appropriately phased DNA bending sequences replacing the CAP binding site upstream from the lac promoter increase by roughly tenfold the rate of specific transcription initiation from a superhelical promoter template in vitro; promoter occlusion results from polymerase binding to the upstream (dA)n.(dT)n tracts, but this phenomenon is not responsible for the observed phase-dependent transcriptional activity. The rates of open complex formation at both P1 and P2 promoters respond in a similar phase-dependent way to the synthetic curved DNA sequences.

Base Sequence↗
PubMed1991

Helical repeat and chirality effects on DNA gel electrophoretic mobility.

We determined a value of 10.34 +/- 0.04 base pairs (bp) per turn for the helical repeat of bent DNA sequences of the form A6N4-A6N5 by estimating the sequence repeat required to produce a planar curve, as judged from the maximum in the electrophoretic mobility anomaly of multimers containing different sequence repeats (10.00, 10.33, 10.50, 10.67, and 11.00 bp per turn). This result provides the basis for a method to evaluate the helical repeat of any DNA segment by comparative electrophoresis measurements. The sequence of interest is placed between two A-tract bends and the phasing is varied over an entire helical turn. Knowledge of the number of base pairs between the bends in the cis isomer, which has the lowest electrophoretic mobility, allows calculation of the average helical repeat of the inserted sequence. In the course of these experiments we observed an unexpected dependence of electrophoretic mobility on the shape of DNA molecules: in high-percentage polyacrylamide gels, those bent molecules for which we deduced a right-handed superhelical form are less retarded than their homologous left-handed isomers. To explain this finding we propose that superhelical chirality influences the choice of DNA migration pathway, leading to rotation of the DNA molecule relative to the local coordinate frame in the gel. High-percentage gels have sufficiently close contact with the right-handed DNA helical twist to differentiate the frictional consequences of right- and left-handed twisting motions.

Base Sequence↗
PubMed1991

Sequence-dependent contribution of distal binding domains to CAP protein-DNA binding affinity.

We report measurements of the relative binding affinity of CAP for DNA sequences which have been systematically mutated in the region flanking the consensus binding site. Our experiments focus on the locus one helical turn from the dyad axis where DNA bending toward the minor groove is induced upon C-AP binding. The binding free energy and extent of bending are moderately well correlated for the set of 56 sequences. Changes in binding affinity spanning a factor of about 50 could be accounted for by additive contributions of dinucleotides; with a few exceptions, the relative ranking of dinucleotide contributions to binding and bending are similar. We conclude that dinucleotides are the smallest independent unit required for quantitative interpretation of CAP-induced DNA bending and binding in the distal domains of the CAP consensus binding site. The imperfect correlation between binding strength and extent of bending implies that sequence changes affect protein binding strength not only by altering the DNA deformation energy required to form the complex, but also by affecting directly the free energy of interaction between protein and DNA.

Base Sequence↗
PubMed1990

Effects of DNA sequence and histone-histone interactions on nucleosome placement.

Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.

Base Composition↗
PubMed1990

Fragments of the HIV-1 Tat protein specifically bind TAR RNA.

Proteolytically produced carboxyl-terminal fragments of the human immunodeficiency virus type-1 (HIV-1) Tat protein that include a conserved region rich in arginine and lysine bind specifically to transactivation response RNA sequences (TAR). A chemically synthesized 14-residue peptide spanning the basic subdomain also recognizes TAR, identifying this subdomain as central for RNA interaction. TAR RNA forms a stable hairpin that includes a six-residue loop, a trinucleotide pyrimidine bulge, and extensive duplex structure. Competition and interference experiments show that the Tat-derived fragments bind to double-stranded RNA and interact specifically at the pyrimidine bulge and adjacent duplex of TAR.

Amino Acid Sequence↗
PubMed1990

Rapid high-performance liquid chromatography of nucleic acids with polystyrene-based micropellicular anion exchangers.

Nucleic acids were separated by ion-exchange chromatography on 30 x 4.6 and 100 x 4.6 mm columns packed with a micropellicular anion exchanger made of 3-microns rigid polystyrene-based non-porous microspheres with a covalently bound hydrophilic layer and DEAE functional groups at the surface. The stationary phase particles showed negligible swelling in methanol according to permeability measurements with water and methanol. Nucleic acids and their fragments including synthetic single-stranded oligonucleotides, linear, nicked and supercoiled DNAs as well as DNA restriction fragments were separated in less than 5 min, a time scale that is much smaller than that of conventional high-performance liquid chromatographic analysis for such samples. When only buffer and sodium chloride were used in the eluent for the separation of double-stranded DNA restriction fragments pGEM-3Z/Taq I, electrophoretic analysis of the effluent revealed the presence of smaller fragments in the bands of the larger ones. Upon addition of ethylenediaminetetraacetic (EDTA) salt to the eluent, however, such contamination by shorter fragments was no longer observed. In the absence of EDTA, magnesium chloride in the eluent at a concentration of 1 mM precluded the separation of the restriction fragments under otherwise identical chromatographic conditions.

Anion Exchange Resins↗