Guangling College, Yangzhou University, China; College of Environmental Science and Engineering, Marine Science & Technology Institute, Yangzhou University, China.
College of Environmental Science and Engineering, Marine Science & Technology Institute, Yangzhou University, China.
Int J Biol Macromol. 2022 Apr 15;204:617-626. doi: 10.1016/j.ijbiomac.2022.02.033. Epub 2022 Feb 10.
RecJ is ubiquitous in bacteria and Archaea, and play an important role in DNA replication and repair. Currently, our understanding on biochemical function of archaeal RecJ is incomplete due to the limited reports. The genome of the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes one putative RecJ protein (Tga-RecJ). Herein, we report biochemical characteristics and catalytic mechanism of Tga-RecJ. Tga-RecJ can degrade ssDNA in the 5'-3' direction at high temperature as observed in Thermococcus kodakarensis RecJ and Pyrococcus furiosus RecJ, the two closest homologs of the enzyme. In contrasted to P. furiosus RecJ, Tga-RecJ lacks 3'-5' ssRNA exonuclease activity. Furthermore, maximum activity of Tga-RecJ is observed at 50 °C ~ 70 °C and pH 7.0-9.0 with Mn, and the enzyme is the most thermostable among the reported RecJ proteins. Additionally, the rates for hydrolyzing ssDNA by Tga-RecJ were estimated by kinetic analyses at 50 °C ~ 70 °C, thus revealing its activation energy (10.5 ± 0.6 kcal/mol), which is the first report on energy barrier for ssDNA degradation by RecJ. Mutational studies showed that the mutations of residues D36, D83, D105, H106, H107 and D166 in Tga-RecJ to alanine almost completely abolish its activity, thereby suggesting that these residues are essential for catalysis.
RecJ 在细菌和古菌中普遍存在，在 DNA 复制和修复中发挥重要作用。由于报道有限，目前我们对古菌 RecJ 的生化功能的了解还不完全。嗜热和耐辐射的古菌 Thermococcus gammatolerans 的基因组编码一个推定的 RecJ 蛋白（Tga-RecJ）。在此，我们报告了 Tga-RecJ 的生化特性和催化机制。Tga-RecJ 可以在高温下以 5'-3'方向降解 ssDNA，就像 Thermococcus kodakarensis RecJ 和 Pyrococcus furiosus RecJ 这两种酶的最接近同源物一样。与 P. furiosus RecJ 不同，Tga-RecJ 缺乏 3'-5' ssRNA 外切核酸酶活性。此外，Tga-RecJ 的最大活性在 50°C70°C 和 pH7.0-9.0 下观察到 Mn，并且该酶是报道的 RecJ 蛋白中最耐热的。此外，通过动力学分析在 50°C70°C 下估计了 Tga-RecJ 水解 ssDNA 的速率，从而揭示了其活化能（10.5±0.6kcal/mol），这是 RecJ 降解 ssDNA 的能量障碍的首次报道。突变研究表明，Tga-RecJ 中残基 D36、D83、D105、H106、H107 和 D166 突变为丙氨酸几乎完全消除了其活性，因此表明这些残基对催化至关重要。
