Global structure and mechanical properties of a 10-bp nucleosome positioning motif

Abstract

The method of DNA cyclization kinetics reveals special properties of the TATAAACGCC sequence motif found in DNA sequences that have high affinity for core histones. Replacement of 30 bp of generic DNA by three 10-bp repeats of the motif in small cyclization constructs increases cyclization rates by two orders of magnitude. We document a 13°bend in the motif and characterize the direction of curvature. The bending force constant is smaller by nearly 2-fold and there is a 35% decrease in the twist modulus, relative to generic DNA. These features are the likely source of the high affinity for bending around core histones to form nucleosomes. Our results establish a protocol for determination of the ensembleaveraged global solution structure and mechanical properties of any Ϸ10-bp DNA sequence element of interest, providing information complementary to that from NMR and crystallographic structural studies.D NA in cells is highly compacted from the unfettered size it would have if free in solution. The first stage of packaging is achieved by wrapping DNA around core histones in a welldefined conformation (1), forming a beads-on-a-string structure that is further compacted by additional proteins into folded chromatin and ultimately into metaphase chromosomes. Interphase chromatin is generally less condensed, but it contains regulatory sites such as promoters at which DNA must be distorted to enable interactions between proteins that bind distant sites on linear DNA. As a relatively stiff polymer, DNA resists the bending required for packaging, which requires free energy assistance from strong interactions with core histones and other proteins.Variations in DNA sequence can, in principle, modulate the energy cost of bending and packaging. For example, intrinsic curvature of the kind provided by A-tracts (2) should, if in the proper direction, lessen the energy required for bending. Increased local bending flexibility could reduce the energetic cost of packaging, as could an increase in twist flexibility (3), assuming that the helical phasing between distal regulatory sequence elements must be altered to form a protein complex.The stiffness of DNA (the inverse of its flexibility) is traditionally described by the persistence length P, defined as the average projection of the end-to-end vector of a very long chain on its initial direction. Classical techniques, such as light scattering (4) and rotational diffusion (5), supplemented by DNA cyclization kinetics (6), have led to estimates of around 140-180 bp for P. However, these methods are generally unable to deconvolute rigorously the contributions of curvature and flexibility to apparent persistence length (7), unless special constructs are used (8).Parameter sets exist for predicting DNA curvature based on roll and tilt values for dinucleotide (9-13) and tetranucleotide (14) steps, but the dinucleotide parameters vary considerably from set to set (15). Nor is there sufficient data to define unambiguously the sequence dependence of the experimental persistence length, ...

High-throughput approach for detection of DNA bending and flexibility based on cyclization

“…Two strategies have been developed to extract the curvature and mechanical properties from a set of J factor measurements: the total length assay and the phasing assay (13,14,27). In the first assay, the total length of the DNA construct is changed, whereas the phasing between the test sequence and the A tracts is fixed, and vice versa in the phasing assay.…”

“…The 30-bp test sequences correspond to the nucleosome positioning sequence (NPS, ref. 14), EcoRI site containing sequence, B DNA with and without nicks, and AT repeat. Nicked DNAs were made by ligation of chemically synthesized oligomers, as was the AT sequence because of insertion-deletion PCR errors (see Fig.…”

“…The maximum dead time of mixing four samples is Ϸ35 s. Different batches of ligase dilutions may differ in their activities up to 2-fold and should be scaled to the same activity level by doing tests on the same construct. For radioactive-based cyclization, the standard procedures were followed (11,14).…”

“…The mechanical configurations for the circular DNA containing the nick N are often not available from our iterative algorithm, especially for the out-of-torsional-phase constructs, probably because the involved molecules are so floppy that the mechanical configurations belonging to different topoisomers are not well separated. Therefore, its simulation was performed by the Monte Carlo method (14,26).…”

“…The traditional implementation (10,14) of cyclization involves radioactive labeling of DNA constructs and separation of the ligation mixture by PAGE to obtain data on ligation kinetics. The data are mainly interpreted by Monte Carlo simulation to determine curvature and flexibility parameters.…”