Khanam Taran, Shukla Ankita, Rai Niyati, Ramachandran Ravishankar
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, Uttar Pradesh 226031, India.
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, Uttar Pradesh 226031, India.
Biochim Biophys Acta. 2015 May;1854(5):505-16. doi: 10.1016/j.bbapap.2015.02.019. Epub 2015 Mar 5.
The Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3'-5'-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3'-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision.
结核分枝杆菌AP核酸内切酶/3'-5'外切脱氧核糖核酸酶（MtbXthA）是DNA碱基切除修复（BER）中的重要参与者。我们证明该酶具有强大的脱嘌呤/脱嘧啶（AP）核酸内切酶活性、3'-5'外切酶活性、磷酸酶活性和磷酸二酯酶活性。该酶在高离子环境中作为AP核酸内切酶发挥作用，而在低离子环境中3'-5'外切酶活性占主导。我们的分子建模和突变实验表明，E57和D251对催化作用至关重要。尽管带切口的DNA和有缺口的DNA是MtbXthA的合适底物，但缺口大小并不影响切除活性，而且，具有凹陷3'-末端的底物更受青睐。为了了解无碱基位点识别的决定因素，我们研究了以下因素的可能作用：（i）无碱基位点对面的碱基；（ii）无碱基核糖环本身；（iii）AP位点的局部扭曲；（iv）位于活性位点附近的保守残基。我们的实验表明，前三个决定因素在MtbXthA中不起作用，事实上，该酶对单链AP DNA也表现出强大的核酸内切酶活性。关于第四个决定因素，已知AP核酸内切酶的催化位点被保守的芳香族残基包围，有趣的是，直接参与无碱基位点识别的具体残基因蛋白质而异。因此，我们结合突变分析、动力学测定和基于结构的建模，确定由Y137支持的Y237介导了MtbXthA-AP-DNA复合物的形成和AP位点切割。
