PHOTOINDUCED DNA PROTEIN CROSS LINKING IN CHROMATIN

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Radiation damage to cellular DNA, induced by ultraviolet light, involves a number of component processes. One of these is the cross-linking of chromosomal proteins to DNA. This proposal is generally concerned with elucidation of this aspect of UV radiation damage in eukaryotic cells. The proposed experiments are designed to achieve an understanding of the chemical basis of UV light-induced DNA-protein cross-linking and to assess the importance of individual nucleobase-amino acid adducts, as a function of UV dose, as contributors to the total observed cross-linking of protein to DNA in eukaryotic chromatin. Specifically, the proposed research will try to answer the following questions: Which amino acids and nucleobases particiipate in the photocross-linking of histones, in particular, and chromosomal proteins, in general, to DNA via covalent adduct formation? What are the quantitative yields, as a function of UV dose of those adducts that are the most important as contributors to the photocrosss-linking? The approach to be used in answering these questions is as follows. A set of nucleobase-amino acid adducts, selected because they are deemed of potential importance in DNA-protein cross-linking, will be prepared via routes involving reaction of DNA or homopolynucleotide with amino acid or alternatively, through reaction of nucleobase or nucleoside with an appropriate amino acid derivative. These adducts will be characterized as to their chemical, chromatographic and spectroscopic properties. Using this knowledge, they will be sought in hydrosylates of UV-irradiated nucleosomes or eukaryotic chromatin. Quantitation of adducts will be achieved via use of chemically derivatized hydrosylates of cross-linked DNA-protein complexes, in conjunction with high performance liquid chromatography and chemically derivatized standards of known adduct concentration.