FUNCTIONAL ANALYSES OF DNA PKCS AND ITS SPLICE VARIANTS

在我们对SCID马驹中DNA-PKcs缺陷的表征过程中， 我们观察到转录本区域内广泛的剪接变异， 编码磷脂酰肌醇3激酶（PI 3 K）结构域。完全类似 在人DNA-PKcs中也观察到剪接变异。变异的一个来源是 一个外显子的差异插入正好接近于 转录本编码保守的蛋白激酶基序。额外变化 是由内含子保留完成的。DNA PKcs的显著百分比 转录本在PI 3 K结构域内保留一个短内含子， 终止密码子插入在激酶中据称的催化位点之前 域保留内含子的转录本将编码截短形式的 DNA-PKcs的预测质量为~ 441 kD（而不是~ 469 kD）， 理论上缺乏蛋白激酶活性。这份成绩单表达了所有 物种研究到目前为止，是非常保守的。我们有初步的 这份抄本是翻译的证据此外，DNA-PK分析 在高水平表达这种转录本的细胞中， 激酶的调节形式。最后，这些DNA-PKcs剪接的表达， 在静息细胞中的变异明显高于在循环细胞中的变异，这表明 DNA-PKcs与细胞周期进程之间的联系。 这项研究的假设是，由于每个DNA-PKcs转录本 编码一种结构上不同的蛋白质，每一种都有不同的 功能协调发展的拟议研究的总体目标是确定 每个DNA-PKcs的功能使用生物化学和遗传方法。

During our characterization of the DNA-PKcs defect in SCID foals, we observed extensive splice variation within the region of the transcript encoding the phosphatidyl inositol 3 kinase (PI3K) domain. Completely analogous splice variation is also observed in human DNA-PKcs. One source of variation is the differential insertion of a single exon just proximal to the region of the transcript encoding the conserved protein kinase motifs. Additional variation is accomplished by intron retention. A significant percentage of DNA-PKcs transcripts retain a short intron within the PI3K domain resulting in the insertion of a stop codon prior to the purported catalytic site in the kinase domain. Transcripts retaining the intron would encode a truncated form of DNA-PKcs with a predicted mass of ~441kD (as opposed to ~469kD) which would theoretically lack protein kinase activity. This transcript is expressed in all species examined to date and is strongly conserved. We have preliminary evidence that this transcript is translated. Furthermore, analyses of DNA-PK activity in cells, which express high levels of this transcript, encodes a regulatory from of the kinase. Finally, expression of these DNA-PKcs splice variants is markedly higher in resting cells than in cycling cells suggesting a link between DNA-PKcs and cell cycle progression. The hypothesis of the proposed research is that since each DNA-PKcs transcript encodes a structurally distinct protein, each from will have distinct functions. The overall goal of the proposed research is to determine the function of each DNA-PKcs using both biochemical and genetic approaches.