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科学通报  2011 

Chk1C末端调控结构域对其在DNA损伤后的核定位及修复活性的影响

, PP. 1272-1281

Keywords: Chk1,核积累,结构域,DNA,损伤,修复,细胞周期

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Abstract:

DNA损伤效应激酶Chk1是一种在进化过程中高度保守的蛋白激酶,它是联系DNA完整监控器和细胞周期引擎的关键介导蛋白.通过对一系列GFP标记的Chk1调控结构域缺失突变体细胞核定位变化分析,发现Chk1C末端调控结构域中的34个氨基酸残基(334~368)参与了调控损伤诱导的Chk1核积累.同时通过构建Chk1及不同Chk1调控结构域缺失突变体的pXJ41重组质粒,并与报告质粒pEGFP-N2共转染HeLa细胞,研究不同Chk1调控结构域缺失突变体的DNA损伤修复能力,同时检测了不同Chk1调控结构域缺失突变体对细胞周期的影响,并通过Westernblot检测Chk1下游靶标Cdc25C的磷酸化情况进行了Chk1缺失突变体激酶活性分析.结果显示,C末端缺失108个氨基酸残基(368~476)的CΔ368活性高于全长Chk1,并且其过表达可诱导细胞G1/S期阻滞,延迟细胞周期进程.C末端缺失142个氨基酸残基(334~476)的CΔ334活性与全长Chk1相当,C末端缺失188个氨基酸残基(288~476)的CΔ288几乎没有酶活性,说明调控结构域包括抑制和激活元件.通过本研究对Chk1的功能有了新的更为深入的认识.

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