Abstract

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg²⁺-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine–purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg²⁺ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine–purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

中文翻译：

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APE1核糖核酸酶活性的突变和动力学分析

摘要

人嘌呤/嘧啶内切核酸酶1（APE1）参与DNA修复系统。认为APE1的主要生物学功能是DNA中Mg ²⁺依赖的AP位点的水解。在结构数据，动力学研究和突变分析的基础上，建立了APE1与受损DNA相互作用的关键阶段。最近显示，APE1可以作为内切核糖核酸酶，催化某些嘧啶-嘌呤位点的mRNA水解，从而控制某些转录物的水平。另外，Mg ²⁺的存在与AP核酸内切酶活性相反，APE1的核酸内切酶活性不需要离子。这表明APE1在RNA和DNA底物上催化机理的差异，但这些差异的原因仍不清楚。在这里，进行了使用野生型APE1酶及其突变体Y171F，R177F，R181A，D210N，N212A，T268D，M270A和D308A对模型RNA底物的核糖核酸酶水解的分析。结果表明，Asn212，Asp210和Tyr171残基的突变导致AP内切核酸酶活性降低，而内切核糖核酸酶活性得以保留。同样，T268D和M270A APE1突变体对嘧啶-嘌呤序列失去特异性。R177F和R181A没有显示出酶活性的显着下降，而D308A显示了核糖核酸内切酶活性的下降。