Spin transfer from protein to DNA in X-irradiated 'dry' and hydrated chromatin: an electron spin resonance investigation of spectral components between 77 K and room temperature.

PURPOSE

To investigate the potential total and relative free-radical transfer from histones to DNA in X-irradiated chromatin, by analysing the relative contributions of individual radicals from protein and from DNA.

MATERIALS AND METHODS

Chromatin was isolated from calf thymus, freeze-dried and either used as such or after equilibration at 76% relative humidity. A mixture of histones was purchased. X-irradiation was performed at 77 K (liquid nitrogen). Data acquisition was on a Bruker ESP 380 ESR-spectrometer (X-band, 9.5 GHz) and at high magnetic fields (285 GHz, Y-band, GHMFL Grenoble). Data analysis involved computer treatment of spectra.

RESULTS

Three components were isolated from an annealing series of histones and assigned to specific radicals (X-band). Chromatin revealed the existence of radicals from both DNA, as well as from the histone compartment. The presence of the oxidized guanine base, as well as the reduced cytosine and thymine bases from DNA at 77 K was confirmed by high-field ESR. Relative radical yields were determined by superposition of individual components from DNA and histones in order to give complete reconstructions of the experimental spectra of the annealing series.

CONCLUSIONS

The relative yields of individual radicals in chromatin differ from those in histones or DNA, respectively. Their behaviour upon annealing is, on the other hand, not significantly changed. Since the total radical yield of DNA radicals is about two times higher in the chromatin complex than in pure DNA, the hypothesis of spin transfer from protein to DNA prior to radical stabilization at 77 K is substantiated.