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D M Crothers

Publications and source records attributed to D M Crothers.

At least 55 records · Page 3Linked to original sources
PubMed1994

Kinetics of an RNA conformational switch.

The spliced leader RNA from Leptomonas collosoma has two competing secondary structures of nearly equal free energy. Short, complementary oligonucleotides can drive the structure from one form of the other. We report stopped-flow rapid-mixing and temperature-jump measurements of the kinetics of the structural switch. At high concentrations of oligonucleotide, the rate of binding becomes limited by the rate of the structural switch, which occurs on a time scale of a fraction of a second. The low activation energy observed for the process implies a branch migration type of mechanism in which portions of the two competing helices transiently coexist.

Animals↗
PubMed1994

Detection of localized DNA flexibility.

The bending and flexibility of DNA are important in packaging, recombination and transcription. Bending decreases electrophoretic mobility in a manner depending on bend position within a fragment (circular permutation) and on the distance between bends (phasing analysis). Bending can also affect DNA ring closure (cyclization). The lack of a complete theory for the mechanism of gel retardation hampers measurement of bend magnitudes by electrophoresis, whereas cyclization is done entirely in solution and is well understood theoretically. Disagreements between bend angles estimated by the two electrophoretic assays have been ascribed to DNA flexibility. Here we test this interpretation using an internal loop as a model flexible locus. Whereas the circular permutation and helical phasing experiments are only subtly affected by the loop, DNA cyclization kinetics detects and quantifies substantial increases in torsional and bending flexibility. Furthermore, the results support a functional role for the stress of DNA bending in inducing base-pair opening.

Base Sequence↗
PubMed1994

Site selectivity of daunomycin.

We have reexamined the binding properties of the antitumor drug daunomycin using double-helical oligonucleotides 16 base pairs long that were designed to contain preferred binding sites for the drug. The preferred sites are contained in a six base pair core which is flanked on the 5' and 3' ends by tracts of adenines. The flanking sequences, which augment helix stability and reduce and effects, were chosen because daunomycin is known to bind poorly to poly(dA).poly(dT). Four major sequences were examined in the six base pair core: CGTACG, TAGCTG, TCATCC, and (TA)3 and compared with calf thymus DNA. A randomly generated 16 bp sequence containing no A tracts and a sequence containing only tracts of As and Ts were also used. Fluorometric, absorption, calorimetric, and stopped-flow techniques were used to examine the binding. The affinity of the drug for oligomers containing known binding sites was comparable to or enhanced relative to that for calf thymus bulk DNA. Association constants ranged from 1.0 x 10(8) to 3.0 x 10(7) M-1. The strongest core binding site found was CGTACG, but its affinity is only 2-fold larger than that of other core sequences. Appreciable binding to the flanking A tracts was observed. An oligonucleotide which incorporates the CGTACG sequence in a short hairpin helix binds an order of magnitude more weakly. Complex lifetimes measured by stopped flow generally increase with equilibrium stability; the kinetics confirm the existence of a set of weaker sites. The exothermic binding enthalpy for daunomycin with the CGTACG core sequence is more than twice as large as for the TATATA sequence. Binding to dA20.dT20 is endothermic, and a less exothermic component can be detected in the calorimetric binding curve of the oligomers containing flanking A tracts.

Animals↗
PubMed1994

Binding affinity and site selectivity of daunomycin analogues.

We have tested a series of daunomycin analogues for binding affinity to a group of oligonucleotides that contain binding sites specific for daunomycin and that were previously screened for relative binding affinity for daunomycin. The series of drugs differed from daunomycin in the sugar moiety, including substitution of a hydroxyl group for the charged amino group and replacement of the 2'-OH by an iodo substituent. Data were analyzed by Scatchard plots and association constants were estimated from the gamma-intercept at saturating levels of oligonucleotide. Because of the solubility problems associated with these compounds, Scatchard plots could not be extended to high levels of binding. A second method of analysis of the fluorescence data confirmed the semiquantitative association constants obtained from the intercepts of the Scatchard plots. The association constants were in the range of 10(5)-10(7) M-1. When compared with daunomycin, the compounds with hydroxyl substituted for the amino group in the sugar ring generally bound less well to the oligonucleotides, by factors of up to several hundred. Much of the binding lost upon removal of the charged amino group was restored, however, with compounds containing an iodo substituent on the sugar ring. Changing the iodo-substituted sugar from the natural L-form to the D-form diminished binding by 6-50-fold, depending on sequence. This result implies a stereospecific interaction of the natural sugar with the DNA chain. A positively cooperative curve was observed in the Scatchard plot for the D-form sugar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Major groove accessibility of RNA.

Chemical acylation experiments showed that the RNA major groove, often assumed to be too deep and narrow to permit recognition interactions, is accessible at duplex termini. Reactivity extended further into the helix in the 5' than in the 3' direction. Asymmetric and large loops between helices uncoupled them, which yielded both enhanced reactivity at terminal base pairs and weaker stabilization enthalpy compared to that in small loops or symmetric loops of the same size. Uncoupled helices have effective helix ends with accessible major grooves; such motifs are attractive contributors to protein recognition, tertiary folding, and catalysis.

Base Sequence↗
PubMed1993

The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms.

We have used a combination of physical and molecular biological techniques to examine the structure of Leptomonas collosoma spliced leader RNA. We confirm the general features of the previously proposed structure for the 3' half of the RNA, in which a single-stranded region is flanked by two stem loops. However, we find that the 5' half of the RNA, which contains the splice site, has two competing secondary structures which differ only slightly in stability and which can interconvert on a fast (<1 s) time scale. In the favored conformation, a stable hairpin helix is augmented by conserved complementarity between the splice site and the 5' end of the SL RNA. This putative helix has anomalous nuclease sensitivity and thermal stability features, suggesting that it is probably coupled by unknown tertiary interactions to other nucleotides in the 5' half-molecule. The structure offers intriguing parallels with RNA-RNA interactions in the mammalian splicing system.

Animals↗
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↗