DNA拓扑异构酶Ⅱβ结合蛋白1(TopBP1)磷酸化功能位点推测及功能分析

Prediction and functional analysis of DNA topoisomerase Ⅱ β-binding protein 1(TopBP1) phosphorylation sites

为阐明DNA拓扑异构酶Ⅱβ结合蛋白1(TopBP1)参与DNA损伤修复应答的分子机制,本研究通过生物信息学分析,发现多个潜在的TopBP1磷酸化位点T860,S887,T1104及T1167,利用分子生物学手段从人cDNA文库中扩增并获得TopBP1克隆质粒,将上述磷酸化位点突变为丙氨酸,观察突变质粒转染细胞对DNA损伤修复的应答反应.结果显示,在粒子射线照射或化疗药物处理细胞后,第1104丙氨酸突变(T1104A)的TopBP1蛋白导致pRPA32-S33的磷酸化水平大幅降低,同时细胞周期检验点失活,严重阻滞了DNA应激反应,证实T1104是TopBP1参与DNA损伤修复的关键活性位点.

To elucidate the molecular mechanism of DNA topoisomerase Ⅱ binding protein 1(TopBP1) involved in DNA damage repair response, this study found several potential phosphorylation sites(T860, S887, T1104 and T1167) of TopBP1 through bioinformatics analysis. The TopBP1 was amplified from the human cDNA library and cloned into plasmid via molecular biology, and the above phosphorylation site was mutant to alanine to observe the response of the cells transfected with the mutant plasmid to DNA damage repair. Results showed that TopBP1 protein with the 1104 th alanine mutation(T1104 A) significantly reduced the phosphorylation level of pRPA32-S33 and inactivated the cell cycle checkpoint after the irradiation or chemotherapy drug treatment, which severely blocked DNA stress response. This study confirms that T1104 is the key activity site of TopBP1 involved in DNA damage repair.