He Yuan, Wang Yiyi, Qin Chen, Xu Ying, Cheng Kaiying, Xu Hong, Tian Bing, Zhao Ye, Wang Liangyan, Hua Yuejin
MOE Key Laboratory of Biosystems Homeostasis and Protection, College of Life Sciences, Institute of Biophysics, Zhejiang University, Hangzhou, China.
Front Microbiol. 2020 Jun 5;11:1178. doi: 10.3389/fmicb.2020.01178. eCollection 2020.
Various endogenous and exogenous agents cause DNA damage, including apurinic/apyrimidinic (AP) sites. Due to their cytotoxic effects, AP sites are usually cleaved by AP endonuclease through the base excision repair (BER) pathway. , an extraordinary radiation-resistant bacterium, is known as an ideal model organism for elucidating DNA repair processes. Here, we have investigated a unique AP endonuclease (DrXth) from and found that it possesses AP endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-5' exonuclease but has no nucleotide incision repair (NIR) activity. We also found that Mg and Mn were the preferred divalent metals for endonuclease and exonuclease activities, respectively. In addition, DrXth were crystallized and the crystals diffracted to 1.5 Å. Structural and biochemical analyses demonstrated that residue Gly198 is the key residue involved in the substrate DNA binding and cleavage. Deletion of the gene in caused elevated sensitivity to DNA damage agents and increased spontaneous mutation frequency. Overall, our results indicate that DrXth is an important AP endonuclease involved in BER pathway and functions in conjunction with other DNA repair enzymes to maintain the genome stability.
多种内源性和外源性因素会导致DNA损伤，包括无嘌呤/无嘧啶（AP）位点。由于其细胞毒性作用，AP位点通常会被AP内切核酸酶通过碱基切除修复（BER）途径切割。耐辐射异常球菌是一种对辐射具有非凡抗性的细菌，是阐明DNA修复过程的理想模式生物。在此，我们研究了来自耐辐射异常球菌的一种独特的AP内切核酸酶（DrXth），发现它具有AP内切核酸酶、3'-磷酸二酯酶、3'-磷酸酶和3'-5'外切核酸酶活性，但没有核苷酸切口修复（NIR）活性。我们还发现Mg和Mn分别是内切核酸酶和外切核酸酶活性的首选二价金属。此外，DrXth被结晶，晶体衍射至1.5 Å。结构和生化分析表明，残基Gly198是参与底物DNA结合和切割的关键残基。在耐辐射异常球菌中缺失该基因会导致对DNA损伤剂的敏感性增加以及自发突变频率升高。总体而言，我们的结果表明DrXth是参与BER途径的重要AP内切核酸酶，并与其他DNA修复酶协同作用以维持基因组稳定性。
