Background: DNA in eukaryotic cells is associated with DNA-binding proteins (histones). Posttranslational modification of histones is an important mechanism by which cells regulate processes such as transcriptional activity of DNA and repair of damaged DNA. Recently, an enzymatic mechanism has been proposed by which histones are modified by covalent attachment of the vitamin biotin, suggesting a potential role for biotin in the metabolism of DNA. Long-term objectives: (1) To identify micronutrient-dependent modifications of histones; (2) to identify enzymes that catalyze some of these modifications; and (3) to determine the physiologic significance of micronutrient-dependent modifications of histones. Specific hypotheses: (1) Specific amino acid residues in histones are biotinylated (as opposed to nonspecific biotinylation of various amino acid residues). (2) Biotinylation of histones is a reversible process; the release of free biotin from biotinytated histones is an enzyme-mediated process. (3) Biotinytation of histones is linked to DNA repair mechanisms.
Specific aims : (1) To identify biotinylation sites in human histone H4 by protein sequencing and site-directed mutagenesis. A secondary aim will test the hypothesis that biotinylation of histones is affected by other posttranslational modifications of histones. (2) To identify enzymes that mediate debiotinylation of histones by using chromatographic techniques, photobidtin, protein sequencing, and cloning. (3) To determine whether biotinytation of histones is involved in DNA repair in cells exposed to N-ethyl-N-nitrosurea and UV light. Benefits: The proposed research will likely lead to novel insights into mechanisms by which micronutrients such as biotin regulate the transcriptional activity and repair of DNA. The potential role of biotin in these processes establishes a link between cellular biotin homeostasis and DNA mutation and abnormal proliferation such as in cancer cells.
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