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R M Clegg

Publications and source records attributed to R M Clegg.

At least 37 records · Page 2Linked to original sources
PubMed1994

The solution structure of the four-way DNA junction at low-salt conditions: a fluorescence resonance energy transfer analysis.

The four-way DNA (Holliday) junction is an important postulated intermediate in the process of genetic recombination. Earlier studies have suggested that the junction exists in two alternative conformations, depending upon the salt concentration present. At high salt concentrations the junction folds into a stacked X structure, while at low salt concentrations the data indicate an extended unstacked conformation. The stereochemical conformation of the four-way DNA junction at low salt (low alkali ion concentration and no alkaline earth ions) was established by comparing the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor molecules attached pairwise in three permutations to the 5' termini of the duplex arms. A new variation of FRET was implemented based upon a systematic variation of the fraction of donor labeled single strands. The FRET results indicate that the structure of the four-way DNA junction at low salt exists as an unstacked, extended, square arrangement of the four duplex arms. The donor titration measurements made in the presence of magnesium ions clearly show the folding of the junction into the X stacked structure. In addition, the FRET efficiency can be measured. The fluorescence anisotropy of the acceptor in the presence of Mg2+ during donor titrations was also measured; the FRET efficiency can be calculated from the anisotropy data and the results are consistent with the folded, stacked X structure.

Base Composition↗
PubMed1993

Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.

The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of double-stranded B-DNA structure; the helical geometry of double-stranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (R/R0)6] dependence of FRET efficiency characteristic for the Förster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.

Base Sequence↗
PubMed1993

The four-way DNA junction: a fluorescence resonance energy transfer study.

Fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions have been carried out in order to analyze the global structure and its dependence on the concentration of several types of ions. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The results indicate that the four-way junction isomerizes from an unstacked extended square arrangement of the four duplex arms at low ion concentration to an antiparallel stacked X-structure as the salt is added. The ion-related conformational change progresses in a continuous non-cooperative manner as the ionic strength of the solution increases.

DNA, Recombinant↗
PubMed1992

Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.

Base Sequence↗
PubMed1991

Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.

Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).

2-Aminopurine↗
PubMed1991

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

3T3 Cells↗
PubMed1990

Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities.

Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.

Absorption↗
PubMed1989

Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules.

The four-way junction between DNA helices is the central intermediate in recombination, and the manner of its interaction with resolvase enzymes can determine the genetic outcome of the process. A knowledge of its structure is a prerequisite to understanding the interaction with proteins, and there has been recent progress. Here we use fluorescence energy transfer to determine the relative distances between the ends of a small DNA junction, and hence the path of the strands. Our results are consistent with the geometry of an 'X'. The interconnected helices are juxtaposed so that the continuous strands of each helix generate an antiparallel alignment, and the two interchanged strands do not cross at the centre. The acute angle of the X structure is defined by a right-handed rotation of the helical axes about the axis perpendicular to the X plane, as viewed from the centre of the X.

Base Sequence↗
PubMed1987

Viscosity dependence of ethidium-DNA intercalation kinetics.

The kinetics of ethidium intercalation into double-stranded poly[d(G-C)] were investigated by use of repetitive pressure-jump chemical relaxation at 20 degrees C in low ionic strength (0.1 M NaCl) aqueous buffers containing either glycerol or methanol. The viscosity of the various solvents differed by more than an order of magnitude while other physical properties (e.g., dielectric constant) remained approximately constant. The single-reciprocal kinetic relaxation time (tau -1) increases linearly with DNA concentration. The observed association rate constant is lower in all organic-aqueous mixtures than in water and is inversely proportional to the viscosity. These results provide evidence for an additional step in the intercalation mechanism which is identified as an obligatory DNA conformational change preceding ethidium intercalation. From the data presented, the equilibrium constant of this local conformational change is approximately 10(-3), i.e., greatly favoring the structure incapable of intercalation. The corresponding kinetics were not directly determined; however, in order to be consistent with all of the data the forward and/or reverse rate constants of the conformational change must be larger than the rate of the intercalation reaction. Thus, it is proposed that the rate of the conformational change back to the nonintercalating B-DNA structure is greater than approximately 500 s-1, implying a rate of opening greater than approximately 0.5 s-1, in agreement with other hydrogen exchange and NMR data. The observed overall rate constant for the dissociation of ethidium is inversely proportional to the solvent density, possibly reflecting a dependence on the solvent free volume. The overall volume change of intercalation is less negative in the organic-aqueous solvent mixtures than in water.

DNA↗
PubMed1987

Diffusion-controlled association of a dye, 1-anilinonaphthalene-8-sulfonic acid, to a protein, bovine serum albumin, using a fast-flow microsecond mixer and stopped-flow.

The kinetic constants of the two fastest reactions of 1-anilinonaphthalene-8-sulfonic acid binding to bovine serum albumin are derived from the results of experiments with a microsecond fast-flow mixing technique and a stopped-flow method. The experiments are interpreted in terms of rapid bimolecular diffusion-controlled associations to two independent regions on the protein surface; this reaction mechanism contrasts with previous kinetic studies of ligand binding to bovine serum albumin which have not demonstrated the fastest kinetic processes.

Anilino Naphthalenesulfonates↗
PubMed1985

Pressure-jump study of the kinetics of ethidium bromide binding to DNA.

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1985

Binding of N-dansylgalactosamine to the lectin from Erythrina cristagalli as followed by stopped-flow and pressure-jump relaxation kinetics.

The binding kinetics of N-dansylgalactosamine to the lectin from Erythrina cristagalli have been studied using stopped-flow and pressure-jump chemical relaxation by monitoring ligand fluorescence. Both methods gave results which are consistent with a simple bimolecular association reaction. The association rate constant, k + 1 = 4.8 X 10(4) M-1 s-1, is far too low to be controlled by diffusion; the dissociation rate is 0.4-0.66 s-1, depending upon the method of determination and the experimental conditions. Identical reaction-rate parameters were obtained at pH 7.3, where soluble aggregates can be present in the lectin solution and at pH 4.7 where such aggregates are absent. The slow rates of carbohydrate binding seem to be characteristic for most lectins and lend support to the idea that they are evolutionary related and have structurally similar binding sites. Analysis of the relaxation amplitudes of the pressure-jump experiments yielded a molar reaction volume change, delta V0, upon binding of +7 ml/mol. This volume change can be caused by desolvation of the ligand upon binding.

Binding Sites↗
PubMed1985

Translational diffusion of lipids in liquid crystalline phase phosphatidylcholine multibilayers. A comparison of experiment with theory.

A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) has been undertaken in liquid crystalline phase phosphatidylcholine bilayers by using the fluorescence recovery after photobleaching technique. This work was done with the intention of comparing the experimental results with the predictions of theoretical models for diffusion in membranes. The following is shown. For NBD-PE, the dependence of the translational diffusion coefficient (Dt) upon the acyl chain length of the diffusant is not that predicted by continuum fluid hydrodynamic models for diffusion in membranes [Saffman, P.G., & Delbrueck, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113; Hughes, B. D., Pailthorpe, B. A., & White, L. R. (1981) J. Fluid Mech. 110, 349-372]. Plots of Dt vs. 1/T (Arrhenius plots) are nonlinear in dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers where the acyl chain composition of the NBD-PE is matched with that of the host bilayer lipid. This suggests that a "free volume" model may be appropriate for the description of lipid diffusion in lipid bilayers. In bilayers of phosphatidylcholines with saturated acyl chains at the same "reduced temperature", the magnitude of Dt follows the order distearoylphosphatidylcholine greater than DPPC greater than DMPC greater than DLPC. This is the inverse of what may be expected from the hydrodynamic model but is in agreement with the free volume in these bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion↗