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• Articles by P. Serwer
• Articles by R.A. Harris

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• Dynamic Bending Rigidity of a 200-bp DNA in 4mM Ionic Streng...

  Dynamic Bending Rigidity of a 200-bp DNA in 4mM Ionic Strength: A Transient Polarization Grating Study Biophysical Journal, Volume 78, Issue 3, 1 March 2000, Pages 1498-1518 Alexei N. Naimushin, Bryant S. Fujimoto and J. Michael Schurr Abstract DNA may exhibit three different kinds of bends: 1) permanent bends; 2) slowly relaxing bends due to fluctuations in a prevailing equilibrium between differently curved secondary conformations; and 3) rapidly relaxing dynamic bends within a single potential-of-mean-force basin. The dynamic bending rigidity (), or equivalently the dynamic persistence length, = governs the rapidly relaxing bends, which are responsible for the flexural dynamics of DNA on a short time scale, ≤10 s. However, all three kinds of bends contribute to the total equilibrium persistence length, , according to ≅ ++, where is the contribution of the permanent bends and is the contribution of the slowly relaxing bends. Both and are determined for the same 200-bp DNA in 4mM ionic strength by measuring its optical anisotropy, () from 0 to 10s. Time-resolved fluorescence polarization anisotropy (FPA) measurements yield () for DNA/ethidium complexes (1 dye/200 bp) from 0 to 120ns. A new transient polarization grating (TPG) experiment provides () for DNA/methylene blue complexes (1 dye/100 bp) over a much longer time span, from 20ns to 10s. Accurate data in the very tail of the decay enable a model-independent determination of the relaxation time () of the end-over-end tumbling motion, from which =500Å is estimated. The FPA data are used to obtain the best-fit pairs of and torsion elastic constant () values that fit those data equally well, and which are used to eliminate as an independent variable. When the relevant theory is fitted to the entire TPG signal (()) the end-over-end rotational diffusion coefficient is fixed at its measured value and is eliminated in favor of . Neither a true minimum in chi-squared nor a satisfactory fit could be obtained for anywhere in the range 500–5000Å, unless an adjustable amplitude of azimuthal wobble of the methylene blue was admitted. In that case, a well-defined global minimum and a reasonably good fit emerged at =2000Å and 〈ζ〉=25°. The discrimination against values <1600Å is very great. By combining the values, =500Å and =2000Å with a literature estimate, =1370Å, a value =1300Å is estimated for the contribution of slowly relaxing bends. This value is analyzed in terms of a simple model in which the DNA is divided up into domains containing bp, each of which experiences an all-or-none equilibrium between a straight and a uniformly curved conformation. With an appropriate estimate of the average bend angle per basepair of the curved conformation, a lower bound estimate, = bp, is obtained for the domain size of the coherently bent state. Previous measurements suggest that this coherent bend is not directional, or phase-locked, to the azimuthal orientation of the filament. Abstract | Full Text | PDF (289 kb)

• Partially Condensed DNA Conformations Observed by Single Mol...

  Partially Condensed DNA Conformations Observed by Single Molecule Fluorescence Microscopy Biophysical Journal, Volume 81, Issue 6, 1 December 2001, Pages 3398-3408 Philip Serwer and Shirley J. Hayes Abstract To detect partially condensed conformations of a double-stranded DNA molecule, single molecule fluorescence microscopy is performed here. The single DNA molecules are ethidium stained, 670 kilobase pair bacteriophage G genomes that are observed both during and after expulsion from capsids. Expulsion occurs in an agarose gel. Just after expulsion, the entire G DNA molecule typically has a partially condensed conformation not previously described (called a balloon). A balloon subsequently extrudes a filamentous segment of DNA. The filamentous segment becomes gently elongated via diffusion into the network that forms the agarose gel. The elongated DNA molecule usually has bright spots that undergo both appearance/disappearance and apparent motion. These spots are called dynamic spots. A dynamic spot is assumed to be the image of a zone of partially condensed DNA segments (globule). The positions of globules along an elongated DNA molecule 1) are restricted primarily to time-stable regions with comparatively high thermal motion-induced, micrometer-scale bending of the DNA molecule and 2) move within a given region on a time scale smaller than the time scale of recording. Less mobile globules are observed when either magnesium cation or ethanol is added before gel-embedding DNA molecules. These observations are explained by globules induced at equilibrium by a bending-dependent, inter-DNA segment force. Theory has previously predicted that globules are induced by electrostatic forces along an electrically charged polymer at equilibrium. The hypothesis is proposed that intracellular DNA globules assist action-at-a-distance during DNA metabolism. Abstract | Full Text | PDF (494 kb)

• Investigating Structural Changes Induced By Nucleotide Bindi...

  Investigating Structural Changes Induced By Nucleotide Binding to RecA Using Difference FTIR Biophysical Journal, Volume 82, Issue 4, 1 April 2002, Pages 2198-2210 Blaine C. Butler, Ross H. Hanchett, Helena Rafailov and Gina MacDonald Abstract Nucleotide binding to RecA results in either the high-DNA affinity form (Adenosine 5′-triphosphate (ATP)-bound) or the more inactive protein conformation associated with a lower affinity for DNA (Adenosine 5′-diphosphate (ADP)-bound). Many of the key structural differences between the RecA-ATP and RecA-ADP bound forms have yet to be elucidated. We have used caged-nucleotides and difference FTIR in efforts to obtain a comprehensive understanding of the molecular changes induced by nucleotide binding to RecA. The photochemical release of nucleotides (ADP and ATP) from biologically inactive precursors was used to initiate nucleotide binding to RecA. Here we present ATP hydrolysis assays and fluorescence studies suggesting that the caged nucleotides do not interact with RecA before photochemical release. Furthermore, we now compare difference spectra obtained in HO and DO as our first attempt at identifying the origin of the vibrations influenced by nucleotide binding. The infrared data suggest that unique -helical, structures, and side chain rearrangements are associated with the high- and low-DNA affinity forms of RecA. Difference spectra obtained over time isolate contributions arising from perturbations in the nucleotide phosphates and have provided further information about the protein structural changes involved in nucleotide binding and the allosteric regulation of RecA. Abstract | Full Text | PDF (276 kb)

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Abstract

Although its conformation has not been observed directly, double-stranded DNA in solution is usually assumed to be randomly coiled at the level of the DNA double helix. By video light microscopy of ethidium-stained DNA at equilibrium in a nonturbulent hanging drop, in the present study, the 670 kb linear bacteriophage G DNA is found to form a flexible filament that has on average 17 double helical segments across its width. This flexible filament 1) has both asymmetry and dimensions expected of a random coil and 2) has ends that move according to the statistics expected of a random walk. After unraveling the flexible filament-associated DNA double helix near the surface of a hanging drop, recompaction occurs without perceptible rotation of the DNA. Both conformational change and intermolecular tangling of the DNA are observed when G DNA undergoes nondiffusive motion in a hanging drop. The characteristics of the G DNA flexible filament are explained by the assumption that the flexible filament is a random coil of double helical segments that are unperturbed by motion of the suspending medium.