spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes.

Accessibility to DNA wrapped in nucleosomes is essential for nuclear processes such as DNA transcription. Large conformational changes in nucleosome structure are required to facilitate protein binding to target sites within nucleosomal DNA. Transient unwrapping of DNA from nucleosome ends can provide an intrinsic exposure of wrapped DNA, allowing proteins to bind DNA that would otherwise be occluded in the nucleosome. The molecular details underlying these mechanisms remain to be resolved. Here we show how DNA unwrapping occurs progressively from both nucleosome ends. We performed single-pair fluorescence resonance energy transfer (spFRET) spectroscopy with alternating laser excitation (ALEX) on nucleosomes either in free solution or confined in a gel after PAGE separation. We combined ALEX-spFRET with a correlation analysis on selected bursts of fluorescence, to resolve a variety of unwrapped nucleosome conformations. The experiments reveal that nucleosomes are unwrapped with an equilibrium constant of approximately 0.2-0.6 at nucleosome ends and approximately 0.1 at a location 27 basepairs inside the nucleosome, but still remain stably associated. Our findings, obtained using a powerful combination of single-molecule fluorescence techniques and gel electrophoresis, emphasize the delicate interplay between DNA accessibility and condensation in chromatin.

Indication of a cosmological variation of the proton-electron mass ratio based on laboratory measurement and reanalysis of H2 spectra.

Based on highly accurate laboratory measurements of Lyman bands of H2 and an updated representation of the structure of the ground X 1sigma(g)+ and excited B 1sigma(u)+ and C 1pi(u) states, a new set of sensitivity coefficients K(i) is derived for all lines in the H2 spectrum, representing the dependence of their transition wavelengths on a possible variation of the proton-electron mass ratio mu = m(p)/m(e). Included are local perturbation effects between B and C levels and adiabatic corrections. The new wavelengths and K(i) factors are used to compare with a recent set of highly accurate H2 spectral lines observed in the Q 0347-383 and Q 0405-443 quasars, yielding a fractional change in the mass ratio of deltamu/mu = (2.4 +/- 0.6) x 10(-5) for a weighted fit and deltamu/mu = (2.0 +/- 0.6) x 10(-5) for an unweighted fit. This result indicates, at a 3.5sigma confidence level, that mu could have decreased in the past 12 Gyr.