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D M Crothers

Publications and source records attributed to D M Crothers.

At least 127 records · Page 7Linked to original sources
PubMed1984

Kinetics and mechanism in the reaction of gene regulatory proteins with DNA.

We have measured the kinetic properties of the Escherichia coli cAMP receptor protein (CAP) and lac repressor interacting with lac promoter restriction fragments. Under our reaction conditions (10 mM-Tris X HCl (pH 8.0 at 21 degrees C), 1 mM-EDTA, 10 microM-cAMP, 50 micrograms bovine serum albumin/ml, 5% glycerol), the association of CAP is at least a two-step process, with an initial, unstable complex formed with rate constant kappa a = 5(+/- 2.5) X 10(7) M-1 s-1. Subsequent formation of a stable complex occurs with an apparent bimolecular rate constant kappa a = 6.7 X 10(6) M-1 s-1. At low total DNA concentration, the dissociation rate constant for the specific CAP-DNA complex is 1.2 X 10(-4) s-1. The ratio of formation and dissociation rate constants yields an estimate of the equilibrium constant, Keq = 5 X 10(10) M-1, in good agreement with static results. We observed that the dissociation rate constant of both CAP-DNA and repressor-DNA complexes is increased by adding non-specific "catalytic" DNA to the reaction mixture. CAP dissociation by the concentration-dependent pathway is second-order in added non-specific DNA, consistent with either the simultaneous or the sequential participation of two DNA molecules in the reaction mechanism. The results imply a role for distal DNA in assembly-disassembly of specific CAP-DNA complexes, and are consistent with a model in which the subunits in the CAP dimer separate in the assembly-disassembly process. The dissociation of lac repressor-operator complexes was found to be DNA concentration-dependent as well, although in contrast to CAP, the reaction is first-order in catalytic DNA. Added excess operator-rich DNA gave more rapid dissociation than equivalent concentrations of non-specific DNA, indicating that the sequence content of the competing DNA influences the rate of repressor dissociation. The simplest interpretation of these observations is that lac repressor can be transferred directly from one DNA molecule to another. A comparison of the translocation rates calculated for direct transfer with those predicted by the one-dimensional sliding model indicates that direct transfer may play a role in the binding site search of lac repressor.

Bacterial Proteins↗
PubMed1983

A characterization of the low temperature structural transition of Escherichia coli 5 S RNA by partial enzymatic digestion.

The low temperature structural transition (low leads to high) of 5 S RNA from Escherichia coli is investigated by partial digestion with ribonuclease T1. In addition to a general masking of guanines from the nuclease, differential changes of accessibility are observed when Mg2+ and salt concentrations are increased to bring about the low leads to high transition. Residue G13 becomes more exposed in the high form while residues G54, G56, G61, G72, and G83-86 become less exposed. The observed cutting rate at other sites is unchanged. A possible conformational change is discussed which could explain the observed changes in RNase T1 digestion patterns as well as the physical chemical observations.

Escherichia coli↗
PubMed1983

CAP binding to B and Z forms of DNA.

We have examined the interaction between the cyclic AMP receptor protein (CAP) and a small DNA fragment containing its specific recognition sequence by circular dichroism spectroscopy. The binding of CAP to this fragment induces a B to "C-like" change in the CD spectrum, which is different from that observed for non-specific binding. A one-to-one (CAP dimer to DNA) binding stoichiometry was deduced from spectroscopic titration data, as was a non-specific binding site size of 17 bp/dimer. In addition, we have compared the non-specific binding affinity of CAP for the B and Z forms of synthetic DNA copolymers. A slight preference for the B form was found. These results do not support the recent specific suggestion that CAP binds to a left-handed form of DNA (1), but indicate more generally that an optically detectable conformational change takes place in DNA on binding CAP.

Base Composition↗
PubMed1983

Binding of daunomycin to calf thymus nucleosomes.

We report equilibrium, hydrodynamic, and electric dichroism studies of the complex of daunomycin with H1-depleted 175 base pair nucleosomes, along with some comparative data for ethidium. In contrast to ethidium, daunomycin binding to nucleosomes is strongly reduced relative to the affinity for free DNA. The salt concentration dependence of the binding constant indicates that approximately one Na+ ion is released from both nucleosomes and free DNA upon daunomycin binding. The early melting transition of nucleosomes is preferentially stabilized by low levels of both drugs, but more markedly by ethidium. Ethidium also stabilizes the second nucleosome melting transition, but daunomycin barely does so. Dichroism and rotational relaxation time measurements indicate that daunomycin unfolds nucleosomes in a manner analogous to the influence of ethidium, although about twice as much daunomycin as ethidium is required complete the unfolding process. The data support an unfold structure in which the nucleosome elongates along the DNA superhelical axis. Levels of daunomycin greater than about 0.15 per DNA base pair promote nucleosome aggregation. To relate our results to the activity of daunomycin as an antitumor agent, we propose that the drug, because of its special intercalation geometry, strongly prefers free DNA regions over the bent helices found in nucleosomes and chromatin. The result of this preference should be an increased local concentration of the drug in the genetically active regions of nuclear DNA in which nucleosomal structure is less prevalent. Presumably the abundance of such regions in tumor cells makes them especially sensitive to daunomycin.

Animals↗
PubMed1983

CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects.

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.

Carrier Proteins↗
PubMed1983

A computer graphics study of sequence-directed bending in DNA.

We present theoretical results to account for the unusual physical properties of a 423 bp DNA restriction fragment isolated from the kinetoplast of the trypanosomatid Leishmania tarentolae. This fragment has an anomalously low electrophoretic mobility in polyacrylamide gels and a rotational relaxation time smaller than that of normally-behaved control fragments of the same molecular weight. Our earlier work (Proc. Natl. Acad. Sci. USA 79, 7664, 1982) has attributed these anomalies to the highly periodic distribution of the dinucleotide ApA in the DNA sequence. As originally proposed by Trifonov and Sussman (Proc. Natl. Acad. Sci. USA 77, 3816, 1980) local features of the DNA structure such as a small bend at ApA, if repeated with the periodicity of the helix, will cause systematic bending of the molecule. Computer graphics representations of DNA chain trajectories are presented for different structural models. It is shown that the structural model of Calladine (J. Mol. Biol. 161, 343, 1982) which is based on crystallographic data, is unsuccessful in predicting the systematic bending of DNA in solution.

Animals↗
PubMed1982

Orientation of nucleosomes in the thirty-nanometer chromatin fiber.

We have measured the linear dichroism of Mg2+-stabilized and dimethylsuberimidate cross-linked 30-nm chromatin fibers, using electric fields to produce orientation. The limiting dichroism of Mg2+-stabilized fibers at low monovalent ion concentration is -0.09, with indistinguishable results for avian erythrocyte and calf thymus chromatin. Correction of the apparent dichroism signal for an absorbance change that becomes significant at high electric field yields the dichroism of -0.09 and reveals that the fibers reach saturation of their orientation by about 15 kV/cm. Calf thymus chromatin fibers cross-linked at 100 mM NaCl concentration have a dichroism of +0.05, with a dependence of orientation on field similar to that observed for Mg2+-stabilized fibers. Comparative sedimentation studies of Mg2+-stabilized fibers and fibers in 100 mM cross-linking buffer revealed a 25% sedimentation coefficient increase accompanying the dichroism increase from -0.09 to +0.05. We interpret the results to mean that the nucleosomal disk diameters form an angle of about 30 degrees to the chromatin fiber axis in Mg2+-stabilized fibers at low monovalent ion concentration. When 100 mM NaCl is added, the fiber becomes more compact, and the disk diameter angles increase by 8 degrees, to about 38 degrees. Cross-linking itself may also contribute slightly to fiber compaction. The results are consistent with a large longitudinal compressibility of the 30-nm fiber, a feature required for the bendability necessary when the fiber is further coiled to form structures such as chromosomes. Our results indicate that compression is accommodated by small changes in the angular orientation of the nucleosomal disks.

Animals↗
PubMed1982

Self-association of daunomycin.

Daunomycin, a potent anthracycline antibiotic, self-associates in aqueous solution at concentrations greater than 10 microM. We report here visible absorbance, sedimentation equilibrium, and proton nuclear magnetic resonance (NMR) experiments that characterize this self-association. In contrast to earlier reports that the process is a simple dimerization, we find that an indefinite association model best fits our data, with an intrinsic association constant, Ki, equal to 1500 M-1. From the temperature dependence of the observed NMR spectra, an enthalpy of approximately -8.0 kcal/mol is calculated. The NMR data show that the aromatic protons of the anthracycline portion of the drug are most affected by aggregation, probably due to stacking of the anthracycline rings. Knowledge of the applicable model for the self-association process, and the equilibrium constant that describes the process, enables us to assess quantitatively the possible effects of drug aggregation on the interpretation of drug-DNA binding data. For the ionic conditions most commonly used in such studies, the amount of aggregated daunomycin will be slight and may safely be ignored.

Chemical Phenomena↗
PubMed1982

Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: equilibrium binding studies on interaction of daunomycin with deoxyribonucleic acid.

We have used equilibrium dialysis and fluorescence and absorbance titration to study the interaction of daunomycin with DNA. Our data at 200 mM Na+ are best fit by the neighbor exclusion model, with K = 7.0 x 10(5) M-1 and an exclusion parameter of three to four base pairs. The binding is dependent on ionic strength, with d log K/d log [Na+] = -0.84, from which we may estimate quantitatively ion release and the binding free energy corrected for the free energy of counterion release. From the temperature dependence of the binding constant, we find the binding to be exothermic, with a van't Hoff enthalpy of -12.8 kcal/mol. Competition dialysis experiments show that G+C base pairs are slightly preferred as binding sites for the drug and suggest that daunomycin binds preferentially to G+C pairs at low r. Cesium chloride density gradient sedimentation experiments provide an experimental demonstration of this preference. Daunomycin increases the Tm for DNA melting by some 30 degrees C as binding approaches saturation, with biphasic melting at low drug/base pair ratios. The data from these equilibrium studies are consistent with intercalative binding of daunomycin and provide a solid foundation for further structural and kinetic studies.

Base Composition↗
PubMed1982

Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: geometry of intercalation of iremycin and daunomycin.

The structure of iremycin [10-(alpha-L-rhodosaminyl)-gamma-rhodomycinone] hydrochloride has been confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. We studied the interaction of iremycin and the related compound daunomycin with DNA by transient electric dichroism and by sedimentation analysis of supercoiled closed duplex DNA. The apparent length increase of sonicated calf thymus DNA (150 +/- 20 base pairs) in 2.5 mM sodium cacodylate buffer (pH 7) at 12 degrees C was determined to be 0.40 +/- 0.02 nm/bound drug). The Cu(II) complex of iremycin with a metal/drug ratio of 0.7 induces a length increase of DNA of 0.44 +/- 0.02 nm/added drug. The alignment of the iremycin chromophore with respect to the DNA helix axis was determined from the electric dichroism of the complex. The tilt (long axis) and twist (short axis) of the chromophore are both 28 +/- 4 degrees, whereas for daunomycin the long axis is perpendicular to the helix axis and the short axis is twisted by about 25 degrees. Intercalation of iremycin between DNA base pairs is supported by unwinding of the supercoiled closed duplex form of pBR 322 plasmid DNA from Escherichia coli. In 2.5 mM sodium cacodylate buffer at pH 7 and at 25 degrees C, the unwinding induced by iremycin is 15.0 +/- 1.5 degrees/bound drug. Under identical conditions daunomycin shows on unwinding angle of 15.4 +/- 1.5 degrees. The superhelical density of pBR 322 DNA (sigma 0) was determined to be -0.087 +/- 0.002 at standard conditions (0.2 M NaCl, 37 degrees C).

Anthracyclines↗
PubMed1982

Unfolding of 175-base-pair nucleosomes.

Calf thymus nucleosomes containing 175 base pairs of DNA unfold in two steps as the salt concentration is lowered, as detected by electric dichroism measurements. The transition midpoints are at 2.9 and 1.1 mM ionic strength at 7 degrees C with at most a small dependence on temperature. We identify the product of the 2.9 mM transition as an expanded disklike structure similar to the product of the 1.0--1.3 mM unfolding transition of 146-base-pair nucleosomes. The product of the 1.1 mM transition in 175-base-pair nucleosomes is elongated into a more asymmetric particle.

Animals↗
PubMed1982

Bent helical structure in kinetoplast DNA.

We have investigated the unusual physical properties of a restriction fragment of Leishmania tarentolae kinetoplast DNA. A gel-purified fragment comprising slightly more than half of a minicircle was determined by Maxam-Gilbert sequence determination to be 490 base pairs (bp) in length. This fragment has dramatically anomalous electrophoretic behavior; it has an apparent size of 450 bp on a 1% agarose gel but migrates as 1,380 bp on a 12% polyacrylamide gel. However, in gel filtration on Sephacryl S-500, the fragment elutes with an apparent size of 375 bp. Finally, it behaves anomalously in electric dichroism experiments. Field-free rotational relaxation times from transient electric dichroism studies are highly sensitive to effective molecular dimensions. The rotational relaxation time of the kinetoplast fragment is smaller than that of a 309-bp control fragment from pBR322. Because rigorous control experiments rule out the possibility that this fragment is modified, these anomalous properties must be dictated by the sequence itself. Fragment behavior indicates that it has an unusually compact configuration; we propose that this molecule contains a region of systematically bent B-DNA. This model accounts for the fragment's difficulty in snaking through the pores of a polyacrylamide gel, its ease in diffusing into Sephacryl beads, and its smaller rotational relaxation time. Bending of this molecule may be caused by periodicities in the DNA sequence.

Journal Article↗