Background

Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5′ side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA.

Methods

Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition.

Results

Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3′-5′-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre–steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied.

Conclusions

It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg²⁺ coordination. Finally, the substitution of Met270 also destabilized the enzyme–substrate complex but did not affect the catalytic reaction.

Significance

It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.

中文翻译：

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活性位点氨基酸残基在人嘌呤/嘧啶内切核酸酶APE1切割DNA和RNA底物中的作用。

背景

人嘌呤/嘧啶内切核酸酶APE1是DNA碱基切除修复途径的参与者之一。APE1通过水解病变5'侧的磷酸二酯键来处理AP位点和许多其他类型的DNA损伤。APE1还充当内切核糖核酸酶，即可以切割未损坏的RNA。

方法

使用稳态前动力学分析，我们检查了某些催化重要氨基酸在APE1酶促途径中的作用，并描述了它们参与目标核苷酸识别的机制。

结果

比较分析含有腺苷无碱基位点，5，6-二氢尿苷或α-端异构体的受损DNA的切割效率，以及未突变DNA的3'-5'-核酸外切酶降解和突变APE1酶水解RNA底物的核酸进行含有活性位点氨基酸残基（D210N，N212A，T268D，M270A或D308A）取代的修饰。研究了在无碱基位点的识别和裂解过程中酶和DNA底物分子构象变化的详细稳态前动力学。

结论

揭示了取代T268D显着干扰了初始DNA结合，而Asn212对于DNA弯曲阶段和催化是至关重要的。取代基D210N增加了结合功效并阻断了催化反应，但是D308A由于破坏了Mg ²⁺配位而降低了结合功效。最后，Met270的取代也使酶-底物复合物不稳定，但不影响催化反应。

意义

发现活性位点氨基酸残基的测试取代影响了络合物形成过程以及催化反应的不同阶段。