Picosecond laser cross: linking histones to DNA in chromatin: implication in studying histone/DNA interactions

摘要

Abstract: A picosecond UV laser radiation was used to cross-link proteins to DNA in nuclei, whole cells and different chromatin preparations. All histones as well as high-mobility group 1 protein were identified immunochemically in the covalently linked protein-DNA complexes. Irradiation of the nucleohistone resulted in cross-linking 20% of bound histones to DNA as a result of two-quantum photoreaction with a maximum quantum yield 3.10$+$MIN@4$/ for double stranded DNA. When nuclei, total hromatin, H1-depleted chromatin and core particles were irradiated and then trypsinized or treated with clostripain to cleave respectively the N-, C- and N- terminal histone tails, no histones have been found covalently linked to DNA. However whilst the yield of cross-links was similar in total and H1-depleted chromatin in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. This finding we consider as a direct evidence for interaction of nonstructured N-tails of core histones with linker DNA. Cross-linking in core particles depends on the ionic strength. All histones were identified in the complex formed up to 0.4 M NaCl, no cross-linking was observed when irradiation was carried out at salt concentration higher than 0.4 M. The cross-linking ability was preserved both upon physiological acetylation of histones known to be restricted to the N- terminal tails and with chemically acetylated chromatin. This finding is direct evidence that postsyntetic histone acetylation does not release the N-terminal tails from interaction with DNA.!