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DNA-PKcs在DNA双链断裂应答过程中对H2AX磷酸化起主导作用

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Keywords: DNA双链断裂,HAX磷酸化,DNA-PKcs

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Abstract:

目的研究在DNA双链断裂(doublestrandbreak,DSB)情况下,DNA-PKcs和ATM在磷酸化H2AX中所发挥的作用。方法在HeLa细胞中,以高效的DNA双链断裂诱导剂新制癌菌素(neocarzinostatin,Ncs)诱导DSB形成,Westernblot分析H2AX磷酸化的时间,剂量依赖及其去磷酸化的动态变化。DNA-PKcs抑制剂NU7026及ATM抑制剂Ku55933分别作用细胞,Westernblot检测其对H2AX磷酸化的影响。以α-satellite序列为对象,γ-H2AXCHIP检测DNA-PKcs或ATM抑制前后H2AX磷酸化情况,得出定量数据。结果γ-H2AX生成呈Ncs作用时间及剂量依赖过程。Ncs短时间作用后,γ-H2AX能够去磷酸化恢复。抑制DNA-PKcs明显影响γ-H2AX生成,而抑制ATM使得γ-H2AX生成高峰延迟。CHIP显示抑制DNA-PKcs后其DNA富集倍数仅为抑制前的27%,而抑制ATM后其富集倍数为抑制前的63%。结论DNA-PKcs可能在DNA双链断裂应答过程中对H2AX磷酸化起主导作用。

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