Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.
Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.
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DOI：
10.1021/acs.chemrestox.2c00172
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发表时间：
2023-02-20
影响因子：
4.1
通讯作者：
Sowers, Lawrence C.
中科院分区：
文献类型：
--
作者：
Baljinnyam, Tuvshintugs;Conrad, James W.;Sowers, Mark L.;Chang-Gu, Bruce;Herring, Jason L.;Hackfeld, Linda C.;Zhang, Kangling;Sowers, Lawrence C.
最近，我们将人胸腺嘧啶DNA糖基酶的29个氨基酸序列与热自养分枝杆菌DNA错配糖基酶(MIG)的催化域连接起来，构建了杂合胸腺嘧啶DNA糖基酶(HyTDG)，从而提高了糖基酶的整体活性。先前的研究表明，MIG催化部位的酪氨酸到赖氨酸(Y126K)突变可以将糖基酶活性转化为裂解酶活性。我们对hyTDG进行了相应的突变，产生了一种hyTDG裂解酶(Y163K)。在这里，我们报告了杂交突变体具有强大的裂解酶活性，在较宽的温度范围内具有活性，并且在多种缓冲条件下都是活性的。HyTDG裂解酶切割一个类似于核酸内切酶III(Endo III)的基本位点。在β-巯基乙醇(β-ME)存在下，碱性不饱和醛形成β-ME加合物。与hyTDG糖基酶一样，hyTDG-裂解酶保持其切割相对G的偏好，并且hyTDG-裂解酶和hyTDG糖基酶可以协同作用来切割T：G错配。这里描述的hyTDG裂解酶在研究DNA损伤和修复方面应该是一个有价值的工具。未来的研究将利用这些酶来使用下一代测序来量化细胞、组织和基因组DNA中的T：G错配对。
Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking a 29-amino acid sequence from the human thymine DNA glycosylase with the catalytic domain of DNA mismatch glycosylase (MIG) from M. thermoautotrophicum, increasing the overall activity of the glycosylase. Previously, it was shown that a tyrosine to lysine (Y126K) mutation in the catalytic site of MIG could convert the glycosylase activity to a lyase activity. We made the corresponding mutation to our hyTDG to create a hyTDG-lyase (Y163K). Here, we report that the hybrid mutant has robust lyase activity, has activity over a broad temperature range, and is active under multiple buffer conditions. The hyTDG-lyase cleaves an abasic site similar to endonuclease III (Endo III). In the presence of β-mercaptoethanol (β-ME), the abasic site unsaturated aldehyde forms a β-ME adduct. The hyTDG-lyase maintains its preference for cleaving opposite G, as with the hyTDG glycosylase, and the hyTDG-lyase and hyTDG glycosylase can function in tandem to cleave T:G mismatches. The hyTDG-lyase described here should be a valuable tool in studies examining DNA damage and repair. Future studies will utilize these enzymes to quantify T:G mispairs in cells, tissues, and genomic DNA using next-generation sequencing.
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影响因子：
4.1
作者：
Ghodke, Pratibha P.;Matse, Johannes H.;Guengerich, F. Peter
通讯作者：
Guengerich, F. Peter
DOI：
10.1016/j.jbc.2022.101638
发表时间：
2022-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hsu CW;Sowers ML;Baljinnyam T;Herring JL;Hackfeld LC;Tang H;Zhang K;Sowers LC
通讯作者：
Sowers LC
影响因子：
2.9
作者：
Cui, Zhengfang;Theruvathu, Jacob A.;Sowers, Lawrence C.
通讯作者：
Sowers, Lawrence C.
影响因子：
5.6
作者：
Hans F;Senarisoy M;Bhaskar Naidu C;Timmins J
通讯作者：
Timmins J
影响因子：
4.8
作者：
Fitzgerald, Megan E.;Drohat, Alexander C.
通讯作者：
Drohat, Alexander C.