PubMed Health⌕ Search

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

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

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

New insight into drug-DNA interactions at individual drug binding sites probed by RNA polymerase during active transcription of the DNA.

An in vitro transcription assay has been used to probe drug-DNA interactions during active transcription of the DNA. The method relies upon the formation of a stable, synchronized population of initiated transcripts comprising a short length of RNA, achieved by the absence of one nucleotide in the initiation mixture. Subsequent equilibration of the transcription complex with drug, followed by elongation of the initiated transcripts, yields lengths of RNA determined by transcription up to each drug-occupied site. A variety of site-specific phenomena have been observed, including delayed termination of transcription 5-10 bp downstream of actinomycin binding sites; 10-20% probability of termination at most echinomycin sites; drug residence-time-dependent termination of bacteriophage polymerases; enhanced residence time compared to physicochemical measurement of drug-DNA dissociation rates. The use of two counter-directed promoters separated by 100 bp results in a sensitive bidirectional transcription footprinting procedure able to resolve adjacent drug sites separated by only 1 bp. The significance of the method of in vitro transcription is the ability to quantitate a range of parameters describing individual drug sites in situations where multiple drug-DNA interactions exist.

Base Sequence↗
PubMed1979

Selective repression of transcription by base sequence specific synthetic polymers.

We report the effect of novel synthetic polymers on deoxyribonucleic acid (DNA) directed ribonucleic acid (RNA) synthesis in vitro. Polymers contained base-selective monomers, including a GC-specific phenazine derivative and an AT-specific triphenylmethane dye. Radical chain polymerization was carried out in aqueous solution by using monomers bound to a template DNA, which was obtained from either lambda or T7 bacteriophage. Polymers were isolated and reannealed with DNA samples, including competitive mixtures of T7 and lambda DNAs. We measured transcription from DNA-polymer complexes by using Escherichia coli RNA polymerase and determined not only the reduction in total transcription levels but also the relative inhibition of lambda- or T7-specific transcription by using a hybridization assay. The results show that micromolar concentrations of individual dyes are sufficient to cause substantial inhibition of transcription when the dyes are incorporated into polymers. More significantly, a number of the polymers inhibited more strongly transcription from the DNA which had served as template for polymer synthesis than from the DNA present as competitor in the annealing process. We conclude that template synthesis of DNA-binding polymers can lead to preferential inhibition of function of the original template. The apparent relative affinity of polymer for competing DNAs can be altered by at least an order of magnitude depending on which DNA was used as the synthesis template. The results offer a new approach to improving the specificity of DNA-binding drugs.

Coloring Agents↗
PubMed1979

The 3' terminus of 16S rRNA: secondary structure and interaction with ribosomal protein S1.

We report studies of the secondary structure and S1 ribosomal protein binding properties of the colicin fragment, containing 49 residues from the 3' terminus of E. coli 16S rRNA. Temperature jump relaxation kinetic measurements reveal two helices in the structure. One of these, melting at 81 degrees C in 5 mM Mg2+, is associated with the 9-base pair hairpin helix predicted by the nucleotide sequence. The other melting transition, at 21 degrees C in 5 mM Mg2+, is assigned to a 4-base pair helix which constrains the pyrimidine tract of the colicin fragment into a bulge loop. S1 protein forms a strong 1:1 complex with the colicin fragment, with an association constant of 5 x 10(6) M-1 in 5 mM Mg2+. More protein molecules are bound, but with weaker affinity, when the S1 concentration is increased. S1 binding causes melting of the colicin fragment secondary structure, as inferred from the observed absorbance increase. The S1 binding site on the colicin fragment has been localized in the region of the bulge loop, since the melting transition corresponding to the 4-base pair helix is lost in the complex. We discuss current models for the role of S1 protein in polypeptide chain initiation in light of these and previous results.

Base Composition↗
PubMed1979

Specific chemical labeling of DNA fragments.

We describe a simple method for specific chemical labeling of DNA fragments at their 3'-termini. The procedure includes enzymatic addition of 4-thiouridine, followed by reaction in mild non-denaturing conditions with the highly reactive alpha-haloacetamido derivatives of several chemical labels. The attached reporter molecule can be removed by extended treatment with beta-mercaptoethanol. Among the potential applications of this labeling method is the study of specific protein-DNA interactions in solution.

Chemical Phenomena↗
PubMed1979

Analysis of RNA secondary structure by photochemical reversal of psoralen crosslinks.

Aminomethyltrioxsalen (AMT), a psoralen, is known to cause interstrand crosslinks in double stranded nucleic acids. We have demonstrated the photochemical reversal of this reaction, and have used this result to develop a method for identification of specific sequences which are adjacent because of RNA secondary structure formation. E. coli 5S rRNA is used as a model system. We isolated and characterized a product that is derived from the stem region of 5S RNA.

Base Composition↗