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B S Fujimoto

Publications and source records attributed to B S Fujimoto.

26 records · Page 2Linked to original sources
PubMed1990

Dependence of the torsional rigidity of DNA on base composition.

The Escherichia coli phage 434 repressor binds as a dimer to the operator of the DNA helix. Although the centre of the operator is not in contact with protein, the repressor binding affinity can be reduced at least 50-fold by changing the sequence there: operators with A.T base pairs near their centre bind the repressor more strongly than do operators with G.C base pairs at the same positions. To explain these observations, it has been proposed that the base composition at the centre of the operator affects the affinity of the operator for repressor by altering the ease with which operator DNA can undergo the torsional deformation necessary for complex formation. In this model, the variation in binding affinity would require the torsion constant to have specific values and to change in a sequence-dependent manner. We have now measured torsion constants for DNAs with widely different base compositions. Our results indicate that the torsion constants depend only slightly on the overall composition, and firmly delimit the range of values for each. Even the upper-limit values are much too small to account for the observed changes in affinity of the 434 repressor. These results rule out simple models that rely on substantial generic differences in torsion constant between A.T-rich sequences and G.C-rich sequences, although they do not rule out the possibility of particular sequences having abnormal torsion constants.

Base Composition↗
PubMed1988

Interaction of chloroquine with linear and supercoiled DNAs. Effect on the torsional dynamics, rigidity, and twist energy parameter.

The magnitude and uniformity of the torsion elastic constant (alpha) of linear pBR322 DNA and supercoiled pBR322 DNAs with high-twist (sigma = -0.083) and normal-twist (sigma = -0.48) are measured in 0.1 M NaCl as a function of added chloroquine/base-pair ratio (chl/bp) by studying the fluorescence polarization anisotrophy (FPA) of intercalated ethidium dye. The time-resolved FPA is measured by using a picosecond dye laser for excitation and time-correlated single-photon counting detection. A general theory is developed for the binding of ligands that unwind superhelical DNAs, and the simultaneous binding of two different intercalators is treated in detail. The equilibrium constant (K) for binding chloroquine to linear pBR322 DNA and the number (r) of bound chloroquines per base pair are determined from the relative amplitude ratio of the slow (normally intercalated) and fast (free) components in the decay of the (probe) ethidium fluorescence intensity as a function of chl/bp. For chloroquine binding to supercoiled pBR322 DNAs, the intrinsic binding constant is assumed to be the same as for the linear DNA, but the twist energy parameter ET (N times the free energy to change the linking number from 0 to 1 in units of kBT) is regarded as adjustable. Using the best-fit ET, the binding ratios r are calculated for each chl/bp ratio. Twist energy parameters are also determined for ethidium binding to these supercoiled DNAs by competitive dialysis. For chloroquine binding, we obtain ET = 360 and 460 respectively for the normal-twist and high-twist supercoiled DNAs. For ethidium binding the corresponding values are ET = 280 +/- 70 and 347 +/- 50. Like other dye-binding values, these are substantially lower than those obtained by ligation methods. In the absence of chloroquine, the torsion constants of all three DNAs are virtually identical, alpha = (5.0 +/- 0.4) x 10(-12) dyn.cm. For linear pBR322 DNA, the magnitude and uniformity of alpha remain unaltered by intercalated chloroquine up to r = 0.19. This finding argues that the FPA is not significantly relaxed by diffusion of any kinks or solitons. If alpha d denotes the torsion constant between a dye and a base pair and alpha 0 that between two base pairs, then our data imply that alpha d/alpha 0 lies in the range 0.65-1.64, with a most probable value of 1.0.(ABSTRACT TRUNCATED AT 400 WORDS)

Chloroquine↗
PubMed1985

Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein.

The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNAs, the difference Dplat - D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed.

DNA Helicases↗