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Crosslinking of histone H1 with non-histone proteins in interphase nuclei under UV-irradiationтезисы доклада
- Авторы: Prusov A.N., Smirnova T.A., Kolomijtseva G.Ya
- Сборник: BOOK OF ABSTRACTS Third International Conference on Radiation and Applications in Various Fields of Research, RAD
- Тезисы
- Год издания: 2015
- Место издания: RAD Association, Niš, Serbia Budva, Montenegro, Будва, Черногория
- Первая страница: 119
- Аннотация: UV irradiation at λ = 254 nm (a mercury quartz lamps low pressure BUF-15) of nuclei with condensed chromatin (in the presence of 5 mM Mg ++) resulted in a dose-dependent decrease of histone H1 yield by extraction with HClO4 reaching its plateau of around 50% of the total content of H1 in the nucleus. Mild acid hydrolysis of DNA during extraction has virtually no effect on the output of H1. Immunoblotting of proteins from nuclei pellets after acid extraction with antibodies to histone H1 shows staining of bands in 45-80 kD region. We can assume that, along with the known effects of UV exposure with λ = 254 nm of chromatin (base modification, DNA-protein crosslinks, DNA strand breaks) in the nuclei appears a new type of adducts - H1-protein crosslinks. Apparently, in our conditions of the irradiation output of H1- DNA crosslinks is unessential. H1-linked proteins are not found in the pellets of irradiated nuclei with decondensed chromatin and do not appear at the refolding of chromatin by the transfer of nuclei into medium with 5 mM Mg ++. Apparently, in these manipulations the histone H1 spatial proximity to other proteins is lost or their steric interposition is broken. Sensitivity to sulfhydryl agents in the medium during irradiation suggests a radical mechanism for the formation of H1-protein crosslinks. The formation of H1-protein crosslinks upon UV irradiation of nuclei in the presence of the antibiotic distamycin (DM), a competitor of H1 for matrix DNA depended on its concentration. DM at saturating concentrations (DM / DNA = 0.1) prevents the formation of crosslinks. At the same time, a low concentration of DM (DM / DNA = 0.01), which interacts mainly with the AT-rich sites in DNA, does not reduce the proportion of crosslinked histone H1.Perhaps H1 located on the AT-rich nucleosome linkers does not cross-link with non-histone proteins. Further information was obtained by comparing the quantitative data of polyglutamic acid (PG) extraction of H1 from UV-irradiated nuclei in the absence and presence of low concentrations of DM. At a ratio PG/DNA = 6 the H1 extraction was 30% in the control experiments, but after UV irradiation PG extract only 3% from total H1 and histone H1 output in pellet decreases to about 50% (according to data of SDS-electrophoresis). DM in low concentration increases PG-extraction of H1 from control and irradiated nuclei on 25% but only from H1 remaining in the pellet without affecting 50% crosslinked H1. This fact confirms our suggestion that histone H1 localized on AT-rich nucleosome linkers, is apparently not involved in the H1-protein crosslinks. Perhaps this fraction of H1 is not extracted by PG (without DM) since it is in the "locked" state within a condensed chromatin and is not available for macromolecular polyanion. Since it is known that non-histone proteins are located on the surface of the chromatin, it can be assumed that in crosslinkings take part mainly the molecules of histone H1 located on GC-rich linkers.
- Добавил в систему: Прусов Андрей Николаевич