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D R Kearns

Publications and source records attributed to D R Kearns.

At least 19 recordsLinked to original sources
PubMed1992

The formation of A-DNA in NaDNA films is suppressed by netropsin.

Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177).

Animals↗
PubMed1990

Deuterium relaxation and internal motion in solid Li-DNA.

As part of an effort to explore the nature of the internal motion in solid polynucleotides, the spectral densities of motion J1(omega 0) and J2(2 omega 0) have been measured for oriented, partially hydrated samples of calf thymus Li-DNA deuterated in the guanine and adenine 8-positions. Both spectral densities increase with increasing hydration level, J1 is found to be 2-5 times larger than J2, and their frequency dependence appears to be omega-1 and omega-3/2, respectively. The large values of the ratio J1/J2 rule out any in-plane torsional motion as the dominant relaxation mechanism in these samples, but a drop in this ratio at high hydration levels (G13 H2O/nucleotide) may indicate increasing contributions from such torsional motion. Although a satisfactory fit to a particular motional model has yet to be achieved, our findings show that the librational motion of the C8-D bond at or below a hydration level of 10 H2O/nucleotide is approximately uniaxial, with correlation times for the motion in the range 0.2-3.0 microseconds.

DNA↗
PubMed1990

Reduced DNA flexibility in complexes with a type II DNA binding protein.

We studied internal molecular motions in Bacillus subtilis phage SPO1 DNA using the time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The torsional flexibility of this (hydroxymethyl)uracil-containing DNA is very similar to that of naturally occurring thymine-containing DNAs, as judged from fits of the time-resolved FPA decay to an elastic DNA model. Binding of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the phage SPO1, enhances the FPA, indicating a substantial decrease in the average DNA torsional flexibility in the DNA-TF1 complex. The FPA increase is correlated with a reduced ethidium binding affinity. The effects can be noticed at TF1 binding ratios less than 1 TF1 dimer/500 DNA base pairs, and the measured torsional rigidity at high TF1 binding ratios (1 TF1 dimer/15-20 DNA base pairs) is about 7 times greater than in the absence of TF1. On the basis of a discussion of various mechanisms for the observed effect we argue that it is due to protein-induced DNA bending at low binding densities although other explanations are also possible. This interpretation might have implications for understanding the biological function of TF1.

Bacteriophages↗
PubMed1990

A fluorescence study of the binding of Hoechst 33258 and DAPI to halogenated DNAs.

We have studied the time-resolved and the steady-state fluorescence of the DNA groove binders 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 with the double stranded DNAs poly(dA-dU) and poly(dI-dC) and their halogenated analogs, poly(dA-I5dU) and poly(dI-Br5dC). These studies were prompted by earlier observations that steady-state fluorescence of Hoechst 33258 is quenched on binding to halogenated DNAs (presumably due to an intermolecular heavy atom effect involving the halogen atom in the major groove), and recent studies which clearly point to a binding-site in the minor groove of DNA. Measurements of the time resolved fluorescence decay demonstrate that the fluorescence of Hoechst 33258 is quenched on binding to the halogenated DNAs, in agreement with previous observations. However, quenching studies carried out using the free halogenated bases IdUrd and BrdCyd in solution yielded bimolecular rate constants more than one order of magnitude larger than those expected for an intermolecular heavy atom effect. Moreover, the quenching of the Hoechst 33258 fluorescence was accompanied by an accelerated photochemical destruction of Hoechst 33258. We therefore conclude that the fluorescence quenching observed with halogenated DNAs is probably due to a photochemical reaction involving Hoechst 33258, rather than direct contact of Hoechst 33258 with the halogen substituents in the major groove of the DNA. The fluorescence decay measurements however, do provide clear evidence for at least two different modes of binding. Taking into account the alternating sequences used in this study and the possibility of two different conformations for bound dye, at least four different modes of binding are plausible. Our present data do not allow us to distinguish between these alternatives. The time-resolved fluorescence decays and fluorescence quantum yields of DAPI are not affected by the presence of the heavy atom substituents in the DNA major groove. Based on this observation and earlier reports that DAPI binds in one of the DNA grooves, we conclude that the high affinity sites for DAPI on DNA are located in the minor groove.

Benzimidazoles↗
PubMed1989

A type II DNA-binding protein genetically engineered for fluorescence spectroscopy: the "arm" of transcription factor 1 binds in the DNA grooves.

We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Codon↗
PubMed1989

Fluorescence studies of a single tyrosine in a type II DNA binding protein.

We studied the fluorescence properties of a single tyrosine (Tyr94) located in the C-terminal tail of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the Bacillus subtilis phage SPO1. The time-resolved fluorescence intensity of Tyr94 in free TF1 dimers decays as a single exponential, and this is consistent with a twofold symmetrical structure. The fluorescence is readily quenched by acrylamide, but it is less accessible to anionic quenchers (iodide and citrate), suggesting that the tyrosine is located on the protein surface in a negatively charged environment provided by neighboring Glu95 and Asp96 residues. TF1 dimers associate at moderate concentrations (greater than 0.02 mg/mL) as judged from concentration dependencies in the molar fluorescence intensity, the steady-state fluorescence polarization, and the bimolecular quenching constants. Nonspecific binding of TF1 to SPO1 and calf thymus (CT) DNA and various double-stranded polynucleotides quenches the Tyr94 fluorescence to varying extent. Fluorescence lifetimes of TF1 in the bound state correlate with spectral overlaps between TF1 emission and DNA absorption, demonstrating that excitation energy transfer to DNA bases contributes significantly to the observed quenching. From analysis of the observed quenching in the DNA complexes we conclude that Tyr94 is located within 10-14 A of the DNA helix axis and not in direct contact with the DNA bases. Equilibrium analyses based on fluorescence titrations show that the maximum binding density on DNA extrapolates to ca. 1 TF1 dimer/5 DNA base pairs. We find several differences in TF1 binding to SPO1 DNA, which contains hydroxymethyluracil instead of thymine, and CT DNA: (i) The tyrosine residue is less exposed to the solvent in the SPO1 DNA complex than in the CT DNA complex. (ii) D2O addition enhances the Tyr94 fluorescence when TF1 binds to SPO1 DNA but not when it binds to CT DNA. (iii) The TF1-SPO1 DNA complex is stable at higher NaC1 concentrations than is the TF1-CT DNA complex, and its formation involves the dissociation of more Na+ ions than does the TF1-CT DNA complex. On the basis of these observations and the fact that the Tyr94-containing tail of TF1 is essential for binding to SPO1 DNA, we discuss various models for the TF1-DNA complex.

Amino Acid Sequence↗
PubMed1989

Interaction of water with oriented DNA in the A- and B-form conformations.

High resolution 2H nuclear magnetic resonance (NMR) was used to investigate the interaction of D2O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D2O/nucleotide, the 2H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D2O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D2O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D2O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D2O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D2O/nucleotide Na-DNA converts entirely to the B-form and the D2O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D2O/nucleotide are caused by a mixture of A- and B-DNA in those samples.

DNA↗
PubMed1988

Static disorder and librational motions of the purine bases in films of oriented Li-DNA.

Solid-state 2H nuclear magnetic resonance line shapes have been obtained from folded films of oriented Li-DNA molecules with the purine bases selectively labeled with deuterium at the 8-position. From line shape simulations, the static base tilts as well as the anisotropic motional amplitudes were determined as a function of hydration level and temperature. It was found that the average tilt angle of the bases is close to 0 degrees and at a hydration of ten water molecules per nucleotide the distribution width of tilt angles about this average cannot be larger than 9 degrees (standard deviation). A slightly increased distribution width is observed at low hydration levels. The motional amplitudes are hydration dependent, with the tilting motion ranging from 4 degrees for the driest, up to 15 degrees for the wettest sample, and slightly larger amplitudes are observed for the twisting motion. The amplitude of the twisting motion is unaffected by a temperature decrease down to -60 degrees C, in contrast to the tilting motion that is suppressed at low temperatures.

Animals↗
PubMed1987

Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements.

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.

Calorimetry↗
PubMed1986

1H nuclear magnetic resonance study of the dynamic properties of the B and Z-forms of poly[d(A-br5C).d(G-T)].

Poly[d(A-br5C).d(G-T)], a synthetic polynucleotide with a 50% A-T base composition, undergoes a reversible, highly co-operative transition between the right-handed B and left-handed Z conformations. The latter is stabilized at both elevated temperature and ionic strength. The B and Z-forms of poly[d(A-br5C).d(G-T)] coexist in 4.6 M-NaCl at 45 degrees C. Due to slow exchange, two sets of Tim and Gim resonances are observed and can be assigned to the B and Z conformations (the chemical shifts are, respectively, Tim = 13.4, 14.1 p.p.m. (parts/million); and Gim = 11.9, 12.4 p.p.m.). Measurements of the 1H spin-lattice (R1) and spin-spin (R2) relaxation rates of the exchangeable thymine (Tim) and guanine (Gim) imino protons have been used to probe the internal dynamics of the B and Z-forms of poly[d(A-br5C).d(G-T)] and the mechanism of the B-Z transition. The proton exchange behavior in the B and Z conformations is quite different. At elevated temperature, R1 for both Tim and Gim in the B conformation is dominated by exchange with the solvent, with Tim exchanging more rapidly than Gim. This demonstrates that exchange involves the opening of single base-pairs and that neighboring A-T and G-br5C base-pairs exchange independently of each other. B-form poly[d(A-br5C).d(G-T)] is unusual in that there is an acceleration of the Tim exchange rate with increasing NaCl concentration. Conversion to the Z-form by addition of 4.5 M-NaCl dramatically reduces both the Tim and Gim exchange rates (estimated to be less than 2 s-1 at 70 degrees C). Thus, the G-br5C base-pair and, in particular, the A-T base-pair are stabilized in the Z conformation. By measuring relaxation rates at 45 to 50 degrees C where the B and Z-forms are in equilibrium, we find that the B-Z interconversion rates are less than two per second. In the B conformation at 25 degrees C, the dipolar contributions to the imino proton relaxation rates are about one-third of those expected on the basis of a rigid rod model for 65 base-pair fragments, a difference we assign to large amplitude (30 degrees high frequency (less than 100 ns) out-of-plane motions of the bases. Conversion to the Z conformation has little effect on the dipolar contributions to relaxation, i.e. on the internal motions.(ABSTRACT TRUNCATED AT 400 WORDS)

Magnetic Resonance Spectroscopy↗
PubMed1986

Effects of hydration on purine motion in solid DNA.

Deuterium quadrupole echo spectra and spin-lattice relaxation rates measured at 76.8 and 38.4 MHz as a function of relative humidity are reported for calf thymus DNA deuterated at positions A8 and G8. The amplitude of base pair motion is observed to increase slightly with increasing degree of hydration (up to approximately 20 mol of H2O/nucleotide), and the onset of motion is associated with a more than 100-fold drop in T1. This observed decrease in T1 parallels that observed previously for the phosphate backbone and appears to be characteristic of collective modes of motion. Above approximately 20 mol of H2O/nucleotide, the amplitude of the base motion increases substantially up to a point where slow components of motion lead to a complete loss of the quadrupole echo.

Base Composition↗
PubMed1986

Magnetic ordering of DNA liquid crystals.

Sonicated calf thymus DNA with an average length of approximately 100 base pairs has been found to form a cholesteric liquid crystal at a concentration of approximately 250 mg of DNA/mL of solution. Immediately after preparation, small ordered domains of a few micrometers are formed, resulting in an opaque solution. This liquid crystal can readily be oriented in the magnetic field of an NMR magnet, resulting in a clear birefringent phase. The DNA molecules align with their helix axes perpendicular to the field so that the cholesteric pitch axis was parallel with the field. A pitch length of approximately 2.5 microns for the cholesteric phase was determined both from optical measurements (optical light rotation) and from NMR measurements (solvent diffusion). The observation that DNA molecules can be magnetically oriented opens up new possibilities for studying the structure and dynamics of the aligned DNA molecules.

Crystallization↗
PubMed1986

NMR study of a synthetic DNA hairpin.

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.

Base Composition↗
PubMed1986

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].

Kinetics↗
PubMed1986

Anisotropic overall and internal motions of short DNA fragments.

Anisotropic motions of DNA fragments in the size range 6-118 base pairs are studied by the steady-state fluorescence polarization of different excitation transitions in the intercalated ethidium cation. Calculated effective tumbling and twisting times are found to be shorter than predicted for overall motions of rigid DNA, indicating that internal motions and/or dye wobbling contribute to the depolarization. The data are consistent with a model where the DNA fragments are considered to be rigid against bending but torsionally flexible, and where the dye can wobble within the intercalated site. We also discuss the possibility of correlated out-of-plane motions of the dye and the DNA bases.

DNA↗