STRUCTURE/FUNCTION OF A DNA ACTIVATED PROTEIN KINASE FROM HELA CELLS

来自 HELA 细胞的 DNA 激活蛋白激酶的结构/功能

• 批准号: 6240590
• 负责人: TIMOTHY HOWARD CARTER
• 金额: $ 2.97万
• 依托单位: ST. JOHN'S UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240590
• 关键词: DNA; HeLa cells; cell cycle; enzyme activity; enzyme induction /repression; enzyme mechanism; enzyme structure; gene expression; high performance liquid chromatography; immunoaffinity chromatography; immunologic assay /test; intermolecular interaction; intracellular transport; phosphorylation; protein kinase; protein purification; protein structure function; western blottings

A novel, double-stranded DNA-activated protein kinase (DNA-PK) has been extensively purified from HeLa cells. Several monoclonal antibodies have been made which specifically inhibit the enzyme's activity and recognize a single 350 kDa polypeptide on Western blots. DNA-PK will be purified to homogeneity using FPLC and immunoaffinity chromatography, and its unique biochemical properties will be studied. Interaction of the enzyme with DNA will be probed by characterizing its requirements for primary sequence and secondary structure of the activating polynucleotide. Specific reagents and assays will be developed, including additional monoclonal and polyclonal antibodies, synthetic peptide substrates, and nucleic acid probes. The latter will be molecularly cloned using cDNA expression libraries probed with monoclonal antibodies. These reagents will be used to determine the interspecies distribution and intracellular localization of DNA-PK, and also to study the regulation of DNA-PK expression at the genetic level. The biologic function(s) of DNA-PK will be elucidated by examining (l) its interactions with other cellular and viral proteins, using co-immunoprecipitation and Western blotting; and (2) its presence and activity at different stages of the cell cycle using synchronous populations of HeLa cells obtained by centrifugal elutriation and by metabolic inhibition. Because of the extraordinary activating effect of double-stranded DNA on the phosphorylation of substrate proteins by DNA-PK, the site of action of DNA-PK within the cell is likely to be chromatin or proteins that interact with chromatin. Understanding the molecular structure, mechanism and function of this heretofore unknown type of protein kinase could therefore have major significance for understanding the regulation of gene activity and/or chromatin dynamics, including the molecular processes associated with DNA replication, recombination, DNA repair, and cell division. Thus the biological function of DNA-PK and/or related protein kinases could impact a variety of human diseases, including neoplasia, developmental abnormalities, and the response to viral infection.

一种新型双链 DNA 激活蛋白激酶 (DNA-PK) 从 HeLa 细胞中广泛纯化。一些单克隆抗体具有 已制成特异性抑制酶的活性并识别 蛋白质印迹上显示单个 350 kDa 多肽。 DNA-PK 将被纯化为 使用 FPLC 和免疫亲和层析实现均质性，及其独特的 将研究生化特性。酶与 DNA 的相互作用 将通过表征其对一级序列和 激活多核苷酸的二级结构。具体试剂 并将开发检测方法，包括额外的单克隆和 多克隆抗体、合成肽底物和核酸 探针。后者将使用 cDNA 表达进行分子克隆 用单克隆抗体探测的文库。 这些试剂将被使用 确定种间分布和细胞内定位 DNA-PK的研究，并研究DNA-PK表达的调控 遗传水平。 DNA-PK 的生物学功能将通过以下方式阐明： 检查 (l) 它与其他细胞和病毒蛋白的相互作用， 使用免疫共沉淀和蛋白质印迹； (2) 它的存在 和细胞周期不同阶段的活动使用同步 通过离心淘洗和通过 代谢抑制。 由于双链DNA对人体具有非凡的激活作用 DNA-PK 作用的底物蛋白磷酸化 细胞内的 DNA-PK 可能是相互作用的染色质或蛋白质 与染色质。了解分子结构、机制和 因此，这种迄今为止未知的蛋白激酶类型的功能可以 对于理解基因活性的调控具有重要意义 和/或染色质动力学，包括相关的分子过程 DNA 复制、重组、DNA 修复和细胞分裂。因此 DNA-PK 和/或相关蛋白激酶的生物学功能可以 影响多种人类疾病，包括肿瘤、发育 异常以及对病毒感染的反应。