核酸外切酶
生物
核酸外切酶 III
突变体
生物化学
DNA
热球菌
同源重组
核酸内切酶
分子生物学
古细菌
基因
大肠杆菌
DNA聚合酶
作者
Guangyu Ma,Tan Lin,Peng Cao,Philippe Oger,Kunming Dong,Li Miao,Likui Zhang
标识
DOI:10.1016/j.resmic.2024.104189
摘要
Archaeal NurA protein plays a key role in producing 3′-single stranded DNA used for homologous recombination repair, together with HerA, Mre11, and Rad50. Herein, we describe biochemical characteristics and roles of key amino acid residues of the NurA protein from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tba-NurA). Tba-NurA possesses 5′–3′ exonuclease activity for degrading DNA, displaying maximum efficiency at 45oC ∼ 65oC and at pH 8.0 in the presence of Mn2+. The thermostable Tba-NurA also possesses endonuclease activity capable of nicking plasmid DNA and circular ssDNA. Mutational data demonstrate that residue D49 of Tba-NurA is essential for exonuclease activity and is involved in binding ssDNA since the D49A mutant lacked exonuclease activity and reduced ssDNA binding. The R96A and R129A mutants had no detectable dsDNA binding, suggesting that residues R96 and R129 are important for binding dsDNA. The abolished degradation activity and reduced dsDNA binding of the D120A mutant suggest that residue D120 is essential for degradation activity and dsDNA binding. Additionally, residues Y392 and H400 are important for exonuclease activity since these mutations resulted in exonuclease activity loss. To our knowledge, it is the first report on biochemical characterization and mutational analysis of the NurA protein from Thermococcus.
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