LRP16结合poly(ADP-ribose)关键位点的识别

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.

The Identification of Poly (ADP-ribose) Binding Site of LRP16 Protein

LRP16 is a member of macro domain protein family. It recognizes and binds poly (ADP-ribose) and is involved in the early response of DNA damage. The key amino acids to interact with poly (ADP-ribose) (PAR) remained unclear. In this study, wild type and mutants by alanine scanning of LRP16 were constructed to identify the PAR-binding sites. Prokaryotic vecter pGEX-6p-1 was used for expression. The produced mutant proteins were purified, and subjected to PAR binding assay in vitro. The dot blot results suggested that the PAR binding activity dramatically reduced when D160 and I161 of LRP16 were mutated; partial attenuation was observed when 181G ,183V and 184 D substituted with A. The findings suggested that D160 and I161 of LRP16 were the key sites for PAR binding.