Isolation and Characterization of Stable Protein-DNA Adducts Induced in Chromatin by Ultraviolet Light
The induction of protein-DNA adducts mediated by ultraviolet (uv) light was analyzed in two forms of chromatin isolated from cultured Chinese hamster cells: sheared, soluble chromatin, and defined, chromatin subunits (nucleosomes). Four methods of analysis were employed to quantify and qualify this photochemical reaction of stabilizing protein to DNA: (1) a membrane filter assay which retains both protein and protein-DNA complexes; (2) CsCl equilibrium density gradients in which stable complexes of protein and DNA band at densities other than their native buoyant densities; (3) gel filtration which allows separation of protein linked to DNA from the bulk, nonlinked protein; and (4) SDS-polyacrylamide gel electrophoretic analysis of the chromatin proteins. The following observations have been made: (1) the rate of linkage of protein-to-DNA is linear with uv light fluence in both forms of chromatin studied; (2) in sheared, soluble chromatin, the rate of linkage of nonhistone proteins exceeds the rate of linkage of histone proteins by two-fold (mass:mass); (3) in sheared, soluble chromatin, factors which increase condensation of chromatin (divalent metal ions, ionic strength, pH) enhance the photochemical addition reaction; (4) in both forms of chromatin, it appears as if all four core histones (H2A, H2B, H3, and H4) participate (to varying degrees) in the uv light-induced crosslinking reaction; and (5) uv light of 254-nm wavelength induces other photoproducts (protein-protein adducts and/or aggregates) besides protein-DNA adducts.
KeywordsChinese Hamster Cell Nonhistone Protein Lauroyl Sarcosinate Soluble Chromatin Sodium Lauroyl Sarcosinate
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