Biomedical subjects

D M Crothers

Publications and source records attributed to D M Crothers.

At least 91 records · Page 5Linked to original sources

PubMed1989

Modulation of mitomycin cross-linking by DNA bending in the Escherichia coli CAP protein-DNA complex.

We have examined the comparative reactivity of mitomycin cross-linking sites in DNA molecules either free in solution or complexed with Escherichia coli CAP protein. Sites in the region to which the protein is bound show strongly variable cross-linking by the drug. The reactivity of a CpG site located where the minor groove is narrowed by bending toward the protein was decreased by about 4-fold, compared to free DNA. The reactivity of a site placed so that the minor groove is widened by the bend was reduced by about 25%, and the reactivity of a (CpG)3 sequence facing primarily away from the protein was reduced 25-fold by CAP binding. These results support the view that local DNA structure plays a critical role in determining the efficiency of cross-linking.

Base Sequence↗

PubMed1989

Cooperativity in A-tract structure and bending properties of composite TnAn blocks.

The existence of intrinsically curved DNA molecules incorporating short runs of adenines is undisputed, but none of the current models can explain the entire experimental data set. Recently, Burkhoff and Tullius [Burkhoff, A. M., & Tullius, T. D. (1988) Nature 331, 455-457] offered an explanation for Hagerman's observations on A4T4N2 vs T4A4N2 polymers [Hagerman, P. J. (1986) Nature 321, 449-450], which showed that A4T4N2 multimers migrate anomalously slowly on polyacrylamide gels and T4A4N2 multimers migrate normally. In A4T4N2 multimers Burkhoff and Tullius observe a hydroxy-radical cutting pattern associated with bent DNA and a B-like cutting pattern in T4A4N2. They attribute this difference in cutting pattern to a clash in the TA step of T4A4N2 and suggest that TA4N5 might already adopt an unbent B-DNA conformation [Tullius T. D., & Burkhoff, A. M. (1988) in Structure and Expression. Vol. 3: DNA Bending and Curvature (Olson, W. K., Sarma, M. H., Sarma, R. H., & Sundaralingam, M., Eds.) pp 77-85, Adenine Press, Guilderland, NY]. We show that the conformation adopted by TnAn blocks is similar to that of AnTn blocks. Two A-tract structures of opposite polarity coexist in both blocks. Moreover, we demonstrate a cooperative buildup of a T-tract structure adjacent to an A-tract structure that cannot be predicted by any of the current models. We conclude that AA steps do not assume the same conformation in long tracts of A's as in isolated AA steps. Therefore, the assumption of nearest-neighbor models, that global curvature is an additive phenomenon of local effects, is invalid.

Adenine↗

PubMed1989

Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex.

Characterization of ternary complexes containing an Escherichia coli lac promoter DNA fragment, CAP protein and RNA polymerase, separated on non-denaturing polyacrylamide gels and footprinted in the gel slice, reveals a striking stabilization of CAP against dissociation in the open complex, compared to the CAP-DNA complex lacking RNA polymerase. The stabilization is lost when half a helical turn of DNA is inserted between CAP and polymerase sites, but is partially restored with an 11 base-pair insert; stimulation of transcription parallels the stabilization effect. This behavior suggests a direct protein-protein interaction. Comparison of initiation kinetics for wild-type and a mutant in which the P2 promoter has been inactivated shows that CAP both strengthens binding in the closed complex and accelerates isomerization to the open complex; the latter effect accounts for the bulk of the observed transcriptional activation.

Bacterial Proteins↗

PubMed1989

Conformation of a bulge-containing oligomer from a hot-spot sequence by NMR and energy minimization.

Two-dimensional nmr data on a bulge-containing oligodeoxyribonucleotide, 5'dGATGGGCAG.dCTGACCCATC, and a regular oligomer of similar sequence, 5'dGATGGCAG.dCTGCCATC, are presented. The nonexchangeable protons are assigned from sequential nuclear Overhauser effect spectroscopy (NOESY) connectivities. The two-dimensional NOE (NOESY) and correlated (COSY) spectra of the bulge-containing oligomer are compared to those of the perfect 8-mer. Experimental proton-proton distances are determined from NOESY spectra acquired with mixing times of 100, 150, and 200 ms, using comparable distances in the B-DNA region of the molecule as a calibration. With this approach, measured distances do not depend systematically on mixing time. Energy minimization techniques are used to calculate a three-dimensional structure for the bulge-containing oligomer in agreement with the nmr data. The helix is of the B family, with the extra adenine stacked into the helix, and the helix axis is bent by 20 degrees.

Base Sequence↗

PubMed1989

Kinin receptors on epithelial cells and smooth muscle of the trachea.

Kallidin and bradykinin produce a biphasic response, contraction followed by relaxation on the isolated guinea-pig tracheal muscle. Two population of receptors appear to regulate the action of kinins on the trachea. Receptors on the epithelium seem to release chemical mediators that control the relaxation response. The question whether similar or different receptors modulate contraction of the smooth muscle may be answered by the use of appropriate antagonists. The occurrence of kinins in bronchial lavage of patients with asthma raises the question of the source of tissue kallikrein involved in the formation of kallidin. We have identified immunoreactive tissue kallikrein in the submucous glands of the trachea.

Acetylcholine↗

PubMed1989

Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP.

We describe an experimental approach to the measurement of protein subunit exchange in which biotinylated subunits mediate attachment of 35S-labeled subunits to a streptavidin column as a result of the exchange process. Application of the method to Escherichia coli catabolite activator protein (CAP) revealed that in the absence of cAMP, the dimerization equilibrium constant is 3 x 10(10) M-1, with a dimer lifetime of 300 min. Exchange of CAP subunits is accelerated at least 1000-fold by the presence of nonspecific DNA, under low ionic strength conditions. Catalysis of exchange also occurs at physiological ionic conditions. In contrast, physiological concentrations of cAMP stabilize CAP with respect to subunit exchange in either the presence or the absence of DNA. We discuss the functional implications of monomerization of gene-regulatory proteins resulting from kinetic and thermodynamic lability of their dimers.

Chromatography, Affinity↗

PubMed1989

Artificial nucleosome positioning sequences.

We have used the emerging rules for the sequence dependence of DNA bendability to design and test a series of DNA molecules that incorporate strongly into nucleosomes. Competitive reconstitution experiments showed the superiority in histone octamer binding of DNA molecules in which segments consisting exclusively of A and T or G and C, separated by 2 base pairs (bp), are repeated with a 10-bp period. These repeated (A/T)3NN(G/C)3NN motifs are superior in nucleosome formation to natural positioning sequences and to other repeated motifs such as AANNNTTNNN and GGNNNCCNNN. Studies of different lengths of repetitive anisotropically flexible DNA showed that a segment of approximately 40 bp embedded in a 160-bp fragment is sufficient to generate nucleosome binding equivalent to that of natural nucleosome positioning sequences from 5S RNA genes. Bending requirements along the surface of the nucleosome seem to be quite constant, with no large jumps in binding free energy attributable to protein-induced kinks. The most favorable sequences incorporate into nucleosomes more strongly by 100-fold than bulk nucleosomal DNA, but differential bending free energies are small when normalized to the number of bends: a free energy difference of only about 100 cal/mol per bend (1 cal = 4.184 J) distinguishes the best bending sequences and bulk DNA. We infer that the distortion energy of DNA bending in the nucleosome is only weakly dependent on DNA sequence.

Animals↗

PubMed1989

Structural basis for DNA bending.

We report proton NMR studies on DNA oligonucleotides that contain A tracts of lengths known to produce various degrees of bending. Spectra of duplexes in the series 5'-(GGCAnCGG).(CCGTnGCC) (n = 3, 4, 5, 7, 9) reveal substantial structural changes within the An.Tn tract as its length is increased. Chemical-shift comparisons show that A tracts with fewer than about seven members do not contain regions of uniform [or poly(dA).poly(dT)-like] structure. Long An tracts (n greater than or equal to 7) appear to consist of an internal segment of homopolymeric conformation flanked by regions of transitional structure that occupy about four A.T pairs on the 5' side and two A.T pairs perhaps the directly adjacent G.C pair on the 3' side. In shorter duplexes (n less than 7), these two transitional regions overlap and an apparent mutual incompatibility causes length-dependent changes that are most pronounced near the 3' end. Throughout the series, there is a striking monotonic relationship between the location of an A.T pair in the A tract and the relative position of its ThyH3 resonance. The direction of the chemical-shift dispersion is opposite to that expected from consideration of ring-current effects alone; this discrepancy suggests a gradual decrease in ThyH3...N1Ade hydrogen-bond length as one moves from the 5' to the 3' end of the A tract and from short to long A tracts. Nuclear Overhauser effect measurements reveal that the interproton distances AdeH2...H1'Ade and AdeH2...H1'Thy vary along each A tract, except in the central regions of the longer ones where they are fairly constant and in good agreement with the poly(dA).poly(dT) structure proposed by Lipanov, A.A. & Chuprina, V. P. [(1987) Nucleic Acids Res. 15, 5833-5844]. This model features a substantial negative base-pair tilt, which has been suggested previously as the source of A-tract bending. In contrast, the nuclear Overhauser effect distances are inconsistent with at least one known crystallographic A-tract structure [DiGabriele, A. D., Sanderson, M. R. & Steitz, T. A. (1989) Proc. Natl. Acad. Sci. USA 86, 1816-1820], which lacks appreciable base-pair tilt.

DNA↗

PubMed1988

Binding of 9-aminoacridine to bulged-base DNA oligomers from a frame-shift hot spot.

Complexes of 9-aminoacridine and two derivatives with oligomers based on the sequence of a hot spot for frame-shift mutations, 5'dGATGGGGCAG, are investigated by proton NMR and equilibrium dialysis. Competition dialysis experiments show that the drug binds bulge-containing oligomers more strongly than regular duplexes of similar sequence and length, with one apparent strong site. A duplex containing an extra cytidine in a run of C's has the highest affinity for 9-aminoacridine among the sequences tested. An oligomer containing five consecutive G.C pairs shows cooperative drug binding, indicating that G tracts of this length may have an altered helical structure. Complexes of a regular 8-mer and a 9-mer containing a bulged guanosine are examined in detail by two-dimensional NMR techniques. 9-Aminoacridine preferentially binds at TpG sites in the 8-mer but binds primarily at the bulged guanosine in the G-bulge 9-mer. Drug-DNA NOE's in the 8-mer complex are compared with the crystal structure of 9-aminoacridine and 5-iodo-CpG [Sakore et al. (1979) J. Mol. Biol. 135, 763-785]. The NMR data suggest that the drug intercalates across the base pairs of both strands with the amino group projecting into the minor groove.

Aminacrine↗

PubMed1988

Phased psoralen cross-links do not bend the DNA double helix.

Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies base on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5 degrees [Pearlman, D. A., Holbrook, S. R., Pirkle, D. H., & Kim, S.-H. (1985) Science (Washington, D.C.) 227, 1304-1308]. On the other hand, Sinden and Hagerman [Sinden, R. R., & Hagerman, P. J. (1984) Biochemistry 23, 6299-6303] find that, in solution, psoralen cross-linked DNA is not bent. Here we use gel electrophoresis to test the validity of the current models. We have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28 degrees unwinding of the helix by HMT [Wiesehahn, G., & Hearst, J. E. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2703-2707], one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. We find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. Our observations place an upper limit of 10 degrees on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.

Cross-Linking Reagents↗

PubMed1988

DNA sequence determinants of CAP-induced bending and protein binding affinity.

The sites of DNA bending induced by binding catabolite activator protein are identified and shown to coincide with positions where DNA grooves face the protein. The bendability of DNA with different sequences at these bend centres parallels the bending preference of the sequences in nucleosomal DNA. Anisotropic DNA bendability significantly affects the structure and strength of regulatory protein-DNA complexes.

Bacterial Proteins↗

PubMed1988

Structural model for an oligonucleotide containing a bulged guanosine by NMR and energy minimization.

We present three-dimensional structural models for a DNA oligomer containing a bulged guanosine based on proton NMR data and energy minimization computations. The nonexchangeable proton resonances of the duplex 5'd(GATGGGCAG).d(CTGCGCCATC) are assigned by nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy connectivities, and the NMR spectrum is compared with that of a regular 8-mer of similar sequence, 5'd(GATGGCAG).d(CTGCCATC). Experimental proton-proton distances are obtained from NOESY spectra acquired with mixing times of 100, 150, and 200 ms. A refined three-dimensional structure for the bulge-containing duplex is calculated from regular B DNA starting coordinates by using the AMBER molecular mechanics program [Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C., Alagona, G., Profeta, S., & Weiner, P. (1984) J. Am. Chem. Soc. 106, 765-784]. We compare structures obtained by building the helix in three and four base pair increments with structures obtained by direct minimization of the entire nine base sequence, with and without experimental distance constraints. The general features of all the calculated structures are very similar. The helix is of the B family, with the extra guanine stacked into the helix, and the helix axis is bent by 18-23 degrees, in agreement with gel mobility data for bulge-containing sequences [Rice, J. A. (1987) Ph.D. Thesis, Yale University].

Base Sequence↗

PubMed1988

Preferential location of bulged guanosine internal to a G.C tract by 1H NMR.

A series of double-helical oligodeoxyribonucleotides of sequence corresponding to a frame-shift mutational hot spot in the lambda CI gene, 5'-dGATGGGGCAG, are compared by proton magnetic resonance spectroscopy at 500 MHz of the exchangeable protons. Duplexes containing an extra guanine in a run of two, three, and four G.C base pairs are compared to regular helices of the same sequence and to another sequence containing an isolated bulged G, 5'-dGATGGGCAG.dCTGCGCCATC. The imino proton resonances are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Resonances assigned to the G tract in bulge-containing duplexes are shifted anomalously upfield and are very broad. Imino proton lifetimes are determined by T1 inversion-recovery experiments. The exchange rates of G-tract imino protons in bulged duplexes are rapid compared to those in regular helices and are discussed in terms of the apparent rate of solvent exchange for the isolated G bulge. Delocalization of a bulged guanosine in homopolymeric sequences can explain the observed changes in chemical shift and relaxation times across the entire G.C run, and the chemical shifts can be fit by a simple model of fast exchange between base-paired and unpaired states for the imino protons. This allows us to calculate the relative occupancies of each bulge site. In these sequences, we find the extra base prefers positions internal to the G tract over those at the edge.

Bacteriophage lambda↗

PubMed1988

The major adduct of the antitumor drug cis-diamminedichloroplatinum(II) with DNA bends the duplex by approximately equal to 40 degrees toward the major groove.

We used gel electrophoresis methods to show that reaction of DNA with cis-diamminedichloroplatinum(II) results in a substantial (approximately equal to 40 degrees) bend in the double helix at the intrastrand crosslink between the N7 atoms of adjacent guanosine nucleosides, which bond to the platinum(II) complex with loss of two chloride ions. Multimers of a 22-base-pair (bp) oligonucleotide platinated at a single site show strong anomalies in their electrophoretic mobilities as a consequence of coherent addition of in-phase platinum-induced bends. Increase of the sequence repeat of the platinated site to 27 bp yields multimers of nearly normal mobility because the bends are out of phase. The direction of Pt-induced bends relative to adenosine-tract bends was determined from the electrophoretic mobility of multimers in which repeated platination sites are interdigitated at various phasings relative to repeated adenosine tracts. Optimum bending occurs when 1.5 helical turns separate the cis-[Pt(NH3)2[d(pGpG)]](N7,N7) adducts from the center of the adenosine tracts. This phasing produces coherent addition of adenosine tract bends toward the minor groove at their centers and Pt-induced bends toward the major groove, where the platinum is located.

Base Composition↗

PubMed1988

Calibration of DNA curvature and a unified description of sequence-directed bending.

Chemically synthesized duplex oligodeoxynucleotides having different average numbers of adenine tracts (A6) per helix turn were ligated into multimers and analyzed by electrophoresis on polyacrylamide gels. The magnitude of the anomaly in gel mobility is found to be a quadratic function of the curvature of the DNA molecule. Parameters that describe intrinsic DNA bending, expressed as the tilt and roll components of the helix-axis deflection at the junctions between the adenine tract and adjacent B-DNA, were adjusted to fit the measured relative curvature of regularly repeated DNA bending sequences known from other studies and synthesized for this study. The model developed here retains the predominance of bending in the direction of tilt at the junctions but incorporates an appreciable roll component at the 5' end of an adenine tract, opening the minor groove there. This feature is consistent with chemical "footprinting" experiments on molecules containing adenine tracts. The overall direction of bending is effectively toward the minor groove, viewed from the center of an A5 or A6 tract. A possible underlying structure, which can also be described by a wedge bending model, is that derived from fiber diffraction studies of poly(dA).poly(dT). However, alternative models for the adenine tract, such as propeller twisted DNA, cannot be eliminated, although they do not lead to the correct direction of bending. The results permit calculation of the helix-axis trajectory of natural DNA molecules containing adenine-tract bends.

Adenine↗

PubMed1987

Lac repressor is a transient gene-activating protein.

We show, using a combination of methods, that contrary to the usual view, lac repressor increases, by more than 100-fold, the initial binding of RNA polymerase to E. coli lac UV5 promoter DNA. Kinetic studies revealed that the repressor acts to block the isomerization step in transcription initiation. When IPTG, a gratuitous inducer, is added, formation of open complex and productive transcription proceed. Because of the large increases in the binding constant, at low polymerase concentrations the presence of lac repressor (and then inducer) actually increases the rate of the first round of productive transcription, thus allowing the system to respond rapidly to the release of repression. This dual role of stabilization of a pretranscriptional complex coupled with blockage of transcription initiation may be a more general model for genetic regulation than that provided by the concept of simple repression.

DNA, Bacterial↗

PubMed1987

Kinetics of the stages of transcription initiation at the Escherichia coli lac UV5 promoter.

The kinetics of initiation by Escherichia coli RNA polymerase on the lac L8UV5 promoter was studied by a gel retardation method that separates protein-DNA complexes from free DNA. The binding constant of the closed complex, the forward and reverse rate constants of isomerization from closed to open complex, and the forward rate constant from the open to initiated complex were measured. Both the forward and reverse isomerization rates were found to be temperature dependent, and the activation energies for these steps were determined. The rates of open complex formation and dissociation were not affected by the addition of ribonucleotide triphosphates; however, the extent of dissociation was greatly reduced if the triphosphates added allowed a short, unstable RNA product to form. The dissociation rate was not affected by heparin, a polyanion competitor that sequesters the polymerase. The rate of initiated complex formation appeared to be dependent on whether the initiating moiety was a mononucleotide triphosphate or dinucleoside monophosphate and on the sequence of the dinucleoside. These results are compared to those found on both the lac L8UV5 and other bacterial and phage promoters by less direct measurements. We use the values obtained for the individual rate constants to investigate the predicted steady-state kinetics of initiation-limited transcription, with the conclusion that the rate-limiting step is formation of the open complex in the limit of low polymerase concentration. However, when RNA polymerase is saturating, the rate is determined by the transition from open complex into the stably initiated ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA-Directed RNA Polymerases↗

PubMed1987

Chemical determinants of DNA bending at adenine-thymine tracts.

DNA fragments having homopolymeric adenine-thymine tracts phased with the helix screw are known to be bent. According to our working model, adenine-thymine tracts adopt a polymorphic structure (H-DNA), and juxtaposition of H-DNA with B-DNA results in bending at the junction between the two structures. We incorporated different base analogues in addition to the four ordinary bases into oligonucleotides; ligated multimers of oligonucleotide duplexes were run on polyacrylamide gels. By comparison of gel mobility data for different sequences, we identified factors both necessary and irrelevant for bending, corresponding to the formation of H-DNA. The 5-methyl group on pyrimidines is not essential, and the 2-amino group on purines interferes with the formation of H-DNA, either because it provides a third H bond between the bases or because it alters water structure in the minor groove. The strong base stacking of A may be an important contributing factor to stabilization of the anomalous DNA structure responsible for bending.

Adenine↗