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

一种新的双链DNA激活蛋白激酶(DNA-PK)被发现 从HeLa细胞中广泛提纯。几种单抗都有 专门抑制这种酶的活性和识别 Western blotts显示一条350 kDa的多肽。DNA-PK将被提纯到 用FPLC和免疫亲和层析法测定其均一性和唯一性 将研究生物化学特性。酶与DNA的相互作用 将通过描述其对初级序列的要求和 激活多核苷酸的二级结构。特异性试剂 并将开发分析方法，包括额外的单克隆和 多克隆抗体、合成肽底物和核酸 探测器。后者将通过cdna表达进行分子克隆。 用单抗探测文库。这些试剂将用于 确定物种间的分布和细胞内的定位 DNA-PK的表达，并研究DNA-PK表达的调控。 遗传水平。DNA-PK的生物学功能(S)将通过以下方式阐明 研究(L)它与其他细胞和病毒蛋白的相互作用， 免疫共沉淀和免疫印迹；(2)它的存在 以及在细胞周期的不同阶段使用同步 离心洗脱法和离心法获得的HeLa细胞群 代谢抑制。 由于双链DNA对细胞的超常激活作用 DNA-PK对底物蛋白的磷酸化，作用部位 细胞内的DNA-PK可能是染色质或相互作用的蛋白质 染色质。了解分子结构、机理和 这种迄今未知类型的蛋白激酶的功能可以因此 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 与DNA复制、重组、DNA修复和细胞分裂有关。因此， DNA-PK和/或相关蛋白激酶的生物学功能可能 影响人类的各种疾病，包括肿瘤、发育 异常，以及对病毒感染的反应。

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.