Vitamin-dependent modifications of histones

描述（由申请人提供）：背景：真核细胞中的DNA与DNA结合蛋白（组蛋白）相关。组蛋白的翻译后修饰是细胞调节DNA转录活性和损伤DNA修复等过程的重要机制。最近，已经提出了一种酶促机制，通过该机制，组蛋白通过共价连接维生素生物素而被修饰，这表明生物素在DNA代谢中的潜在作用。长期目标：（1）鉴定组蛋白的微量营养素依赖性修饰;（2）鉴定催化这些修饰的酶;（3）确定组蛋白的微量营养素依赖性修饰的生理学意义。具体假设：（1）组蛋白中的特定氨基酸残基被生物素化（与各种氨基酸残基的非特异性生物素化相反）。(2)组蛋白的生物素化是一个可逆的过程;游离生物素从生物素化的组蛋白中释放是一个酶介导的过程。(3)组蛋白的生物素化与DNA修复机制有关。具体目的：（1）通过蛋白质测序和定点突变技术鉴定人组蛋白H4的生物素化位点。第二个目的是检验组蛋白的生物素化受到组蛋白其他翻译后修饰的影响这一假设。(2)通过色谱技术、光比丁、蛋白质测序和克隆鉴定介导组蛋白去生物素化的酶。(3)确定组蛋白的生物素化是否参与暴露于N-乙基-N-亚硝基脲和紫外线的细胞中的DNA修复。优点：拟议的研究可能会导致对微量营养素（如生物素）调节DNA转录活性和修复机制的新见解。生物素在这些过程中的潜在作用建立了细胞生物素稳态与DNA突变和异常增殖（如癌细胞）之间的联系。

DESCRIPTION (provided by applicant): 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.