Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control
Protein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control
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DOI：
10.1073/pnas.95.13.7445
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发表时间：
1998-06-23
影响因子：
11.1
通讯作者：
Concannon, P
中科院分区：
文献类型：
--
作者：
Wright, JA;Keegan, KS;Concannon, P
在裂殖酵母中，rad 3基因产物在感知DNA结构缺陷和激活损伤反应途径中起着关键作用。基于蛋白激酶结构域的序列相似性，已经鉴定了rad 3在人类中的结构同源物（ATR）。关于ATR表达、蛋白激酶活性和细胞定位的一般信息是已知的，但其在人类细胞中的功能仍不确定。在目前的研究中，ATR蛋白通过蛋白质提取物的凝胶过滤进行了检查，发现主要作为大蛋白质复合物的一部分存在。通过定点诱变制备ATR基因的激酶失活形式，并在转染实验中用于探测该复合物的功能。将该激酶失活的ATR引入正常成纤维细胞系、来自患有共济失调-毛细血管扩张症的患者的ATM缺陷成纤维细胞系或p53突变细胞系均导致细胞活力的显著损失。表达激酶死亡ATR的克隆显示对X射线和UV的敏感性增加，并且失去检查点控制。我们的结论是ATR功能作为一个蛋白质复合物，介导的电离和紫外线辐射在人体细胞中的反应的关键部分。这些反应包括对细胞活力和细胞周期检查点控制的影响。
In fission yeast, the rad3 gene product plays a critical role in sensing DNA structure defects and activating damage response pathways, A structural homologue of rad3 in humans (ATR) has been identified based on sequence similarity in the protein kinase domain. General information regarding ATR expression, protein kinase activity, and cellular localization is known, but its function in human cells remains undetermined. In the current study, the ATR protein was examined by gel filtration of protein extracts and was found to exist predominantly as part of a large protein complex. A kinase-inactivated form of the ATR gene was prepared by site-directed mutagenesis and was used in transfection experiments to probe the function of this complex, Introduction of this kinase-dead ATR into a normal fibroblast cell line, an ATM-deficient fibroblast line derived from a patient with ataxia-telangiectasia, or a p53 mutant cell line all resulted in significant losses in cell viability. Clones expressing the kinase-dead ATR displayed increased sensitivity to x-rays and UV and a loss of checkpoint control. We conclude that ATR functions as a critical part of a protein complex that mediates responses to ionizing and UV radiation in human cells. These responses include effects on cell viability and cell cycle checkpoint control.