Direct interaction of p21 with p50, the small subunit of human DNA polymerase delta
Direct interaction of p21 with p50, the small subunit of human DNA polymerase delta
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DOI：
10.4161/cc.5.4.2425
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发表时间：
2006-02-15
期刊：
影响因子：
4.3
通讯作者：
Lee, Marietta Y. W. T.
中科院分区：
文献类型：
--
作者：
Li, Hao;Xie, Bin;Lee, Marietta Y. W. T.
利用酵母双杂交筛选技术，以人DNA聚合酶delta （pol delta）的p50亚基为诱饵，发现p21与pol delta的p50亚基相互作用。用酶联免疫吸附试验和纯化蛋白的下拉实验证实p21和p50之间存在直接相互作用。p50和p21之间的相互作用位点通过GST缺失突变体的下拉实验绘制。127 ~ 193个残基构成p21在p50上结合的主要相互作用区，而p50与p21的c端26个残基结合。高度纯化的小牛胸腺pol δ与组蛋白激酶活性相关，添加纯化的重组p21以剂量依赖的方式抑制激酶活性。p50在体内可被磷酸化，在体外可被CDK2/cyclinA磷酸化。在体内通过MCF7细胞共免疫沉淀获得了p21与p50关联的证据。研究还表明，在阿霉素处理的MCF7细胞中，p21与p50以及pol delta复合物的其他组分的关联增加。我们的研究结果表明，p50可能在某些DNA损伤（如阿霉素治疗）中靶向或锚定p21到pol - delta复合物上。
Using a yeast two-hybrid screening technique and the p50 subunit of human DNA polymerase delta (pol delta) as a bait, p21 was found to interact with the p50 subunit of pol delta. A direct interaction between p21 and p50 was confirmed by using ELISA and pull-down assays with purified proteins. The interaction sites between p50 and p21 were mapped by pull down assays with GST deletion mutants. Residues 127-193 constitute the primary interaction region on p50 to which p21 binds, while p50 binds to the C-terminal 26 residues of p21. A histone kinase activity was associated with the highly purified calf thymus pol delta and addition of purified recombinant p21 inhibited the kinase activity in a dose dependent manner. p50 is phosphorylated in vivo and can be phosphorylated by CDK2/cyclinA in vitro. In vivo evidence of p21 association with p50 was obtained by coimmunoprecipitation using MCF7 cells. It was also shown that the association of p21 with p50 and other components of the pol delta complex increased in MCF7 cells treated with adriamycin. Our results suggested that p50 might target or anchor p21 to pol delta complex upon certain DNA damage such as adriamycin treatment.