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D M Crothers

Publications and source records attributed to D M Crothers.

At least 109 records · Page 6Linked to original sources
PubMed1987

Effect of drug-DNA interactions upon transcription initiation at the lac promoter.

We have examined the effects of six DNA binding drugs upon initiation at the lac UV5 promoter by Escherichia coli RNA polymerase. Experiments were directed at determining the influence of added drug on open complex formation, open complex stability, initiation from the open complex, and stability of the resulting initiated complex. The narrow groove binding drugs distamycin and 4',6-diamidino-2-phenylindole dihydrochloride were more effective in inhibiting initiation through their effect on the first three of these factors than were the intercalators ethidium bromide, daunomycin, and actinomycin. The bisintercalator bis(daunomycin) inhibited open complex formation better than its parent daunomycin. With the possible exception of actinomycin, the drugs tested were not able to disrupt preformed initiated complex, in contrast to their destabilizing effect upon the open complex. Combined with other results, the data suggest that the antitumor activity of daunomycin is unlikely to result from its effect on transcription. We compare the relative effectiveness of the drugs with the known physical properties of the corresponding drug-DNA interactions. The rate of open complex formation seems to be influenced by both the on and off rates of the drug, probably due to the relative slowness of open complex formation. This is in contrast to elongation, a much quicker process, which seems to be limited by the drug off rate alone; these considerations may possibly rationalize the difference in relative effect of particular drugs upon initiation and elongation. All drugs were able actively to disrupt open complex, although to substantially different extents; some possible mechanisms for this disruption, and the insensitivity of the initiated complex, are discussed.

DNA, Bacterial↗
PubMed1987

Anthracycline antibiotics. Interaction with DNA and nucleosomes and inhibition of DNA synthesis.

We report studies of the interaction of four anthracycline antibiotics, iremycin (IM), daunomycin (DM), aclacinomycin A (AM), and violamycin B1 (VM), with naked DNA, nucleosomal core particles, and 175 base pair (bp) nucleosomes lacking histone H1. In all cases the binding strength increases in the order IM less than DM approximately AM less than VM. The binding substrates increased in affinity for the drugs in the following order: core particles less than 175-bp nucleosomes less than DNA. The apparent DNA length increment per drug bound decreases in the progression IM greater than DM greater than AM greater than VM, the same serial order as is characterized by increasing binding affinity. Dichroism amplitude measurements show that for all drugs the long-wavelength absorbance transition moment is tilted by 26-29 degrees relative to the plane perpendicular to the helix axis; this angle probably corresponds to the long axis tilt of the intercalated chromophore. Finally, it was found that the ability of the drugs to inhibit DNA synthesis by Escherichia coli DNA polymerase I increases in the same order as their binding affinity.

Aclarubicin↗
PubMed1987

Proton nuclear magnetic resonance studies on bulge-containing DNA oligonucleotides from a mutational hot-spot sequence.

A series of bulge-containing and normal double-helical synthetic oligodeoxyribonucleotides, of sequence corresponding to a frame-shift mutational hot spot in the lambda C1 gene, are compared by proton magnetic resonance spectroscopy at 500 MHz. The imino proton resonances of d(GATGGGCAG).d(CTGCCCCATC), d(GATGGGCAG).d(CTGCCCCATC), and d(GATGGGCAG).d(CTGACCCATC) are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Nonselective T1 inversion-recovery experiments are used to determine exchangeable proton lifetimes and to compare helix stability and dynamics of the three duplexes. An extra adenosine flanking the internal G.C base pairs has a strongly localized effect on helix stability, but the destabilizing effect of an extra cytidine in a C tract is delocalized over the entire G.C run. These data lead to the conclusion that the position of the bulge migrates along the run in the fast-exchange limit on the NMR time scale. Rapid migration of the bulge defect in homopolymeric sequences may help rationalize both frame-shift mutagenesis and translational frame shifting. We estimate that the unfavorable free energy of a localized bulge defect is 2.9-3.2 kcal/mol, in good agreement with earlier estimates for RNA helices.

Base Sequence↗
PubMed1987

A stressed intermediate in the formation of stably initiated RNA chains at the Escherichia coli lac UV5 promoter.

We report experiments designed to elucidate the mechanism by which RNA polymerase advances from the open complex to synthesis of a stably bound RNA chain during transcription initiation. Techniques used include deoxyribonuclease I footprinting, methylation protection, and exonuclease III digestion through upstream domains, each applied to the open, abortive and productive transcription complexes of Escherichia coli RNA polymerase with the lac promoter. The results show a slight loss of upstream open complex contacts during abortive transcription of a 6-mer and 8-mer, but a large loss of these contacts upon escape from abortive cycling into productive transcription at the 11-mer. We propose a model for early initiation in which competition between open complex polymerase-DNA contacts on one hand and initiated complex polymerase-DNA-RNA interactions on the other produces a "stressed intermediate" during formation of a short RNA-DNA duplex. The strain energy is relieved either by ejecting the short RNA, resulting in aborted initiation, or by eliminating the sigma subunit and breaking the open complex contacts, thereby escaping abortive cycling into productive transcription. Further evidence for this model is based on the observation that destabilization of interactions specific for either open complex or initiated complex has the predicted effect on the amount of abortive cycling. The model predicts a complicated relationship between overall promoter strength and DNA sequence changes that alter polymerase-DNA interactions.

DNA-Directed RNA Polymerases↗
PubMed1987

Comparison of the open complexes formed by RNA polymerase at the Escherichia coli lac UV5 promoter.

In transcription initiation at the lac UV5 promoter, Escherichia coli RNA polymerase forms two open complexes, called Ou and O1, which can be separated by electrophoresis on native polyacrylamide gels. We have compared the properties of these two open complexes, with the objective of rationalizing the functional difference previously reported between the two forms: the complex which is dominant at high temperature (Ou) is better able to escape abortive transcriptional cycling into productive mRNA elongation. Methylation protection and binding domain probing with exonuclease III were used to investigate differences in polymerase binding strength to particular DNA domains. Also, we examined the difference in the extent and temperature dependence of promoter unwinding in the two complexes, as probed by methylation of unpaired cytosines and cleavage by phage T7 endonuclease. We find that O1 has stronger promoter interactions in the DNA domain whose upstream edge is defined by an exonuclease III stop at -24. These -24 domain interactions, which presumably aid in promoter binding and nucleation of DNA unwinding, are inferred to be strong enough to hinder escape of the polymerase from the open complex contacts that are maintained during abortive initiation. The Ou complex has weaker binding to the -24 domain, partially compensated by better upstream interactions and a better ability to accommodate extensive DNA unwinding. It thus escapes abortive initiation more readily because of weaker critical open complex contacts that must be lost when stable initiation occurs from the corresponding stressed intermediates.

DNA, Bacterial↗
PubMed1986

The DNA binding domain and bending angle of E. coli CAP protein.

We use a new gel electrophoretic analysis to map the thermodynamically defined DNA binding domain of Escherichia coli CAP protein in the lac promoter. Strong binding interactions span a 28-30 bp duplex DNA region, substantially larger than that found for typical repressors. Sequence changes outside the central 28 bp of the binding site are found to affect the electrophoretically observed extent of bending. We also report a study of the DNA bending induced at a symmetrized CAP binding site, compared with the wild-type site; binding and bending are stronger at the upstream than at the downstream half of the wild-type site. Bends of the estimated 90 degrees - 180 degrees magnitude could play a vital regulatory role by producing tertiary structure in a local DNA domain, and by storing elastic energy for subsequent use in transcription or replication.

Bacterial Proteins↗
PubMed1986

Kinetics and sequence specificity of drug-DNA interactions: an in vitro transcription assay.

An assay has been developed to detect the DNA sequence specificity of drug binding sites, and the kinetics of dissociation of drug from those sites, under conditions involving active transcription of the DNA. Specific transcriptional blockage sites were detected in the presence of actinomycin D and a bisintercalator, bis(anthracycline); the rate of RNA chain growth past the drug binding sites yields the rate of dissociation of drug from these sites. Rate constants for dissociation from the whole promoter fragment measured by the detergent sequestration method were found to be significantly faster than the rate determined for dissociation from the specific transcriptional blockage site. However, the absence of significant blockage at other drug binding sites implies much more rapid drug dissociation from those sites in the transcriptional complex. We conclude that transcriptional blockage results from a DNA sequence-dependent interaction of the drug-DNA complex with RNA polymerase; the sequences that are effective for blockage appear to be GpC for actinomycin and (CpA)3 for a bis(daunomycin) compound. Transcriptional inhibition may in general show greater sequence specificity than is exhibited by simple binding.

Base Sequence↗
PubMed1986

Bending and flexibility of kinetoplast DNA.

We have evaluated the extent of bending at an anomalous locus in DNA restriction fragments from the kinetoplast body of Leishmania tarentolae using transient electric dichroism to measure the rate of rotational diffusion of DNA fragments in solution. We compare the rate of rotational diffusion of two fragments identical in sequence except for circular permutation, which places the bend near the center in one case and near one end of the molecule in the other. Hydrodynamic theory was used to conclude that the observed 20% difference in rotational relaxation times is a consequence of an overall average bending angle of 84 +/- 6 degrees between the end segments of the fragment that contains the bending locus near its center. If it is assumed that bending results from structural dislocations at the junctions between oligo(dA).oligo(dT) tracts and adjacent segments of B DNA, a bend angle of 9 +/- 0.5 degrees at each junction is required to explain the observations. The extent of bending is little affected by ionic conditions and is weakly dependent on temperature. Comparison of one of the anomalous fragments with an electrophoretically normal control fragment leads to the conclusion that they differ measurably in apparent stiffness, consistent with a significantly increased persistence length or contour length in the kinetoplast fragments.

Animals↗
PubMed1986

Higher order structure of chromatin: evidence from photochemically detected linear dichroism.

We have used photochemically detected linear dichroism to measure the separate average angular orientations of nucleosomes and linker DNA in 30-nm chromatin fibers of varying linker size (20-80 base pairs). Our results indicate that the average tilt angles vary with linker size, but not in a monotonic manner, suggesting that the constancy of geometry of the 30-nm fiber is maintained by compensatory changes of nucleosomal tilt which accommodate packing of variable lengths of linker DNA. We discuss the compatibility of our results with the various classes of models that have been proposed for the 30-nm fiber, including the continuous solenoid model and models built from the basic unit of the zig-zag ribbon. Many models can be eliminated, and all have to be modified to fit our results for chromatins with very long linkers.

Animals↗
PubMed1986

Ring closure probabilities for DNA fragments by Monte Carlo simulation.

The rate of ligation of DNA molecules into circular forms depends on the ring closure probability, commonly called the j-factor, which is a sensitive measure of the extent to which thermal fluctuations contribute to bending and twisting of DNA molecules in solution. We present a theoretical treatment of the cyclization equilibria of DNA that employs a special Monte Carlo method for generating large ensembles of model DNA chains. Using this method, the chain length dependence of the j-factor was calculated for molecules. in the size range 250 to 2000 base-pairs. The Monte Carlo results are compared with recent analytical theory and experimental data. We show that a value of 475 A for the persistence length of DNA, close to values measured by a number of other methods, is in excellent agreement with the cyclization results. Preliminary applications of the Monte Carlo method to the problem of systematically bent DNA molecules are presented. The calculated j-factor is shown to be very sensitive to the amount of bending in these fragments. This fact suggests that ligase closure measurements of systematically bent DNA molecules should be a useful method for studying sequence-directed bending in DNA.

Cyclization↗
PubMed1986

Topological distributions and the torsional rigidity of DNA. A Monte Carlo study of DNA circles.

Distributions of the linking number of circular DNA molecules, defined as the sum of twist and the writhing number, are obtained by Monte Carlo simulations of small, randomly closed DNA circles. We estimate the relative contributions of fluctuations in twist and writhe to the linking number distribution, as functions of DNA size. Published experimental data on topoisomer distributions in circular DNA molecules are interpreted to estimate the torsional rigidity of DNA in solution. We show that ignoring the writhe component of the linking number distribution, even for DNA circles as small as 250 base-pairs, leads to an underestimate for the torsional stiffness of the double helix. The value of the torsional modulus obtained from this analysis, C = 3.4 X 10(-19) erg cm, is from 10 to 40% larger than that estimated by others and more than twice as large as the values obtained from fluorescence depolarization or other time-resolved spectroscopic measurements. We also develop further the theoretical treatment of ring closure probabilities for DNA described in the previous article. It is shown that the torsional part of the ring closure probability, phi 0,1 (tau 0) is a periodic function of DNA length that contributes strongly to the ring closure probability for short chains but makes negligible contributions for chains over 1000 base-pairs in length.

Base Composition↗
PubMed1986

Condensation of chromatin: role of multivalent cations.

We have used electric dichroism to investigate the influence of multivalent cations upon the compaction of chicken erythrocyte chromatin from the unfolded, 10-nm fiber to the 30-nm solenoid and subsequent aggregation. The pattern of condensation, which consists of compaction plus aggregation, is found to be strikingly similar for a variety of cations of differing charge, including the physiologically important polyamines spermine and spermidine. With a few exceptions such as Cu2+ and Gd3+, an optimally compacted fiber with reproducible hydrodynamic properties is produced prior to the onset of aggregation. We report the concentrations of di-, tri-, and tetravalent cations required for optimal condensation; in addition, for tri- and tetravalent cations, we were able to estimate the extent of charge neutralization produced by their binding to the optimally compacted fiber. The results show that the multivalent ion concentration required for optimal compaction decreases as cationic charge increases. In addition, the effect of a mixture of dilute mono- and multivalent cations on chromatin condensation is synergistic, rather than competitive as has been found for the multivalent cation induced condensation of DNA or the B----Z conformational transition. A simple calculation indicates that the entropy of ion uptake in chromatin condensation is surprisingly constant for a range of ionic conditions; this factor may be a dominant one in determining the folding equilibrium.

Animals↗
PubMed1986

Influence of DNA-binding drugs on chromatin condensation.

We have used transient electric dichroism to study the ability of DNA-binding drugs to affect the folding of chromatin from the 10- to the 30-nm fiber, either by themselves or in conjunction with multivalent cations. Variables considered include the cationic charge of the drug, the comparative influence of intercalation and groove binding as modes of interaction, and the effect of bis-intercalation compared to mono-intercalation. In parallel with our findings with other cations, we observe that a drug must have a charge of 3+ or greater in order to condense chromatin at concentrations substantially lower than the concentration of chromatin, measured in base pairs. Drugs of low charge, whether groove binders or mono-or bis-intercalators, are unable to condense chromatin on their own. Bis-intercalators of high charge, however, are extremely efficient condensers, being able to cross-link chromatin with greater efficiency than polyamines of corresponding charge. When Mg2+ is used in combination with bis-intercalators of high charge, the order of addition of the two determines whether compaction or cross-linking is favored. Finally, the antibiotics actinomycin D, daunomycin, and distamycin, despite varied modes of binding to DNA, all inhibit the compaction of chromatin beyond a critical point in a remarkably similar manner.

Animals↗
PubMed1986

The homeo domain of a murine protein binds 5' to its own homeo box.

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that the homeo box contains the sequences required for specific protein-DNA interactions, and the 753-bp fragment contains a homeo domain binding site. These results support the hypothesis that murine homeo boxes are DNA binding domains of proteins involved in the regulation of embryonic development.

Amino Acid Sequence↗
PubMed1985

Kinetics of the daunomycin--DNA interaction.

The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the two slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by two sequential isomerization steps. The six rate constants for this mechanism were extracted from our data by relaxation analysis. The values determined for the six rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using first sodium dodecyl sulfate to dissociate bound drug and second pseudo-first-order conditions to study the fast bimolecular step, provide independent verification of three of the six rate constants. The temperature dependence of four of the six rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. We speculate that the three steps in the proposed mechanism may correspond to a rapid "outside" binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.

DNA↗
PubMed1985

Intermediates in transcription initiation from the E. coli lac UV5 promoter.

We have used nondenaturing polyacrylamide gel electrophoresis to separate intermediates in transcription initiation that result from action of E. coli RNA polymerase on the lac UV5 promoter. The resolved gel complexes are characterized by DNAase I footprinting, protein subunit content, RNA content, and transcription ability. There are two "open" complexes, whose equilibrium ratio is a function of temperature; they differ in their ability to escape abortive cycling, but not in their DNAase I footprints. We find three "initiated" complexes, containing RNA chains at least 11 nucleotides long, and lacking the sigma subunit of RNA polymerase. These experiments provide a detailed view of the early initiation steps and their thermal regulation at the E. coli lac promoter.

DNA, Bacterial↗
PubMed1984

Equilibrium studies of the cyclic AMP receptor protein-DNA interaction.

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP. The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.

Binding Sites↗