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LRP16作为macro domain家族成员,可识别、结合poly(ADP-ribose),参与DNA损伤修复的早期反应.但究竟LRP16通过其何种氨基酸位点识别、结合poly(ADP-ribose)(PAR)尚不十分清楚.本研究首先通过对LRP16蛋白的结构分析,查找LRP16结合PAR的候选氨基酸位点,然后利用丙氨酸扫描技术构建系列LRP16(点)突变体,并且进行原核蛋白表达与纯化,将获得的蛋白质进行斑点杂交实验,检测LRP16突变体蛋白与PAR的结合活性.测序结果显示,LRP16点突变基因序列成功插入到原核表达载体pGEX-6p-1中;斑点杂交实验显示,LRP16中第160位D和第161位I突变成A后,其与PAR结合能力明显减弱,而第181位G、183位V、184位D同时突变为A,LRP16与PAR的结合活性有部分减弱.结果表明,LRP16中第160位和第161位的氨基酸是其与PAR结合的关键位点.
As a member of the macro-domain family, LRP16 recognizes and binds to poly (ADP-ribose) and participates in the early response to DNA damage repair.However, LRP16 does not recognize ADP-ribose (PAR) It is very clear.In this study, firstly, the structure of LRP16 protein was searched to find the candidate amino acid sites of LRP16 binding PAR, and then a series of LRP16 (point) mutants were constructed by using alanine scanning technology and the expression and purification of prokaryotic protein would be obtained Of proteins were tested by dot blot hybridization to detect the binding activity of LRP16 mutant proteins to PAR.The sequencing results showed that the sequence of LRP16 point mutation was successfully inserted into prokaryotic expression vector pGEX-6p-1. Dot blot hybridization showed that LRP16 protein D and 161 I mutated to A, its ability to bind to PAR was significantly weakened, while the first 181 G, 183 V, 184 D simultaneously mutated to A, LRP16 binding activity with PAR partially weakened. The results show that , Amino acids 160 and 161 of LRP16 are the key sites for PAR binding.