DNA motions in the nucleosome core particle.

Abstract

We have used time-resolved triplet state anisotropy decay techniques to measure the conformational flexibility of DNA in the nucleosome. From these measurements we conclude that, in a nucleosome, the DNA helix experiences substantial internal flexibility, which occurs with a time constant near 30 nsec. We find that our data can be fit well by a modified version of the Barkley6-Zimm model for DNA motion, allowing only DNA twisting motions and the overall tumbling of the nucleosome. That fit yields a calculated torsional rigidity equal to 1.8 x 109. erg-cm, a value equal to that measured for uncomplexed DNA. We conclude from such similarity that large, fast twisting motions of the DNA helix persist, nearly unaltered, when DNA is wrapped to form a nucleosome.The nucleosome, the basic structural unit of eukaryotic chromatin,. consists of 146 base pairs (bp) of DNA helix complexed with an octamer of histones (H3, .H4, H2A, and H2B). The structure of the nucleosome has been studied extensively. As revealed by low-resolution x-ray diffraction (1) and low~angle neutron scattering (2), the nucleosome can be described as a stubby cylinder with radius of 55 A and length of 60 A. The DNA helix wraps around the octamer core to form approximately 13/4 turns of left-handed superhelix.The thermal stability and conformation of the nucleosome are known to be affected by solvent conditions and temperature (3); however, very little is known about the dynamic properties of the nucleosome.Earlier, we showed that triplet state anisotropy decay can be used to monitor nanosecond time scale twisting motions that occur in the DNA helix (4). DNA motions ofthis kind have been detected by using NMR (5, 6), fluorescence anisotropy decay (7-9), and ESR techniques (10) and are of considerable interest because such motions are a direct measure of the elasticity of DNA structure and because large-amplitude conformational flexibility is sure to affect the specific binding of proteins.Because in eukaryotes most DNA exists as a nucleosome complex in chromatin, it is of importance to understand the internal flexibility ofnucleosomal DNA. On the basis of 31P NMR measurements, Klevan et al. (11) suggested that a large motion with a time.constant near 1 nsec.occurs in the phosphate backbone ofDNA in the nucleosome. On the basis ofmore extensive 31P relaxation measurements Shindo and McGhee (12) came to similar conclusions concerning DNA motion; their best fit to the data suggested that the phosphate. backbone expresses motions in the nucleosome with time constants nearer to 30 nsec.However,. on the basis of 'H NMR linewidth measurements, Feigon and Kearns (13) concluded that DNA is in fact held rigidly in the nucleosome. Because of such controversy in the interpretation ofNMR data, we have chosen to study DNA conformational flexibility by using triplet anisotropy decay, a technique that can measure motion over a very wide, time range. *Here, we describe time-dependent triplet state anisotropy measurements ofthe nucleosome core particle, tagged ...

Site-specific carcinogen binding to DNA.

“…Such cutting can be quantitated in a simple manner by denaturing gel electrophoresis if the DNA samples are homogeneous with respect to molecular weight. The DNA used for our initial experiments is 146 + 2 bp chicken DNA, extracted from nucleosome core particles (11). Fig.…”

“…DNA fragments 146 ± 2 base pairs (bp) long were prepared from chicken erythrocyte nucleosomes (11), then modified with (± )-BPDE as described elsewhere (9), with the exception that unreacted BPDE and BP tetrol were removed by three phenol/chloroform extractions. The extent of modification was measured optically, using 6346 = 2.95 x 104 cm-' M`for the BPDE-DNA adduct and E258 = 1.3 x 104 cm-I M -I (base pairs) for DNA.…”

Photodynamic treatment of adenoviral vectors with visible light: an easy and convenient method for viral inactivation

“…5,22,23 It is generally accepted that MB strongly associates with DNA and upon illumination, induces the formation of 8-hydroxyguanine (8-OHG). [24][25][26] Singlestranded nicks were observed as result of this 8-OHG formation. 24 The data presented here strongly indicate a preference of MB for DNA, as it was the only sensitizer tested that completely impaired the functional integrity of the viral DNA after a 5-min exposure (Figure 2).…”

Corrigendum

“…1. C, Torsional spring constant calculated from equations (1) and (2); -r, exponential time constant for motion of the long helix axis estimated from a fit of the slowest part of the anisotropy decay data to a single exponential ; P, the apparent persistence length of the fragments ; E, Young's modulus, calculated from fits of data to equations (1) and (2). In these measurements, the laser pulse has a finite width and therefore must be convoluted into the initial 20 ns of anisotropy decay simulation.…”