PHOTOINDUCED DNA-PROTEIN CROSS-LINKING IN CHROMATIN

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Radiation damage to cellular DNA, caused by ultraviolet (UV) light, has a number of component processes. One of these is cross-linking of chromosomal proteins to DNA. The present proposal emphasizes this aspect of UV radiation damage to eukaryotic chromatin. The long term objective of our research program in this area is to elucidate the chemical nature of nucleic acid-protein photocross-linking and to assess quantitatively the importance of individual nucleobase-amino acid mixed products, as a function of UV dose, to the total cross-linking of proteins of DNA in eukaryotic chromatin. The present research proposal has specific goals of trying to answer the following questions: which nucleobases and amino acids participate in the photocross-linking of histones to DNA in chromatosomes (one of the basic structural units of eukaryotic chromatin)? What are the quantitative yields, as a function of UV dose, of those mixed products that are important as contributors to the photocross-linking? The following approach will be used to answer these questions. A set of nucleobase-amino acid (or nucleoside-amino acid) mixed products will be prepared by routes involving reaction of the nucleobase or nucleoside with amino acid or appropriate amino acid derivative. The resulting mixed products will be characterized as to their chemical, chromatographic and spectroscopic properties. Using this information, they will be sought in hydrosylates of UV-irradiated chromatosomes. Quantitation of mixed products will be done through the use of chemically derivatized hydrosylates of photocross-linked chromatosomes, in conjunction with high performance liquid chromatography and chemically derivatized standards of known mixed product concentration.