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首页> 外文期刊>Nucleic Acids Research >Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis
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Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis

机译：Tenocongensis的新型单链DNA特异性核酸内切酶EndoTT的表征

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摘要

EndoTT encoded by tte0829 of Thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA in vitro as well as binding dsDNA. In the presence of a low concentration of NaCl, EndoTT cleaved ss regions of damaged dsDNA efficiently but did not cleave DNA that was entirely ss or ds. At high concentrations of NaCl or MgCl2 or ATP, there was also specific cleavage of ssDNA. This suggested a preference for ss/ds junctions to stimulate cleavage of the DNA substrates. EndoTT has six specific sites (a-f) in the oriC region (1-70 nt) of T. tengcongensis. Substitutions of nucleotides around site c prevented cleavage by EndoTT of both sites c and d, implying that the cleavage specificity may depend on both the nucleotide sequence and the secondary structure of the ssDNA. A C-terminal sub-fragment of EndoTT (residues 107-216) had both endonucleolytic and DNA-binding activity, whereas an N-terminal sub-fragment (residues 1-110) displayed only ssDNA-binding activity. Site-directed mutations showed that G(170), R-172 and G(177) are required for the endonuclease activity of EndoTT, but not for DNA-binding, whereas D-171, R-178 and G(189) are partially required for the DNA-binding activity.
机译：Tengcongensis嗜热厌氧杆菌的tte0829编码的EndoTT在体外与单链（ss）和受损的双链（ds）DNA结合并裂解，并与dsDNA结合。在低浓度的NaCl存在下，EndoTT可以有效切割受损dsDNA的ss区，但不能切割完全为ss或ds的DNA。在高浓度的NaCl或MgCl2或ATP中，ssDNA也会发生特异性切割。这表明偏爱使用ss / ds连接来刺激DNA底物的裂解。 EndoTT在登革热菌的oriC区域（1-70 nt）具有六个特定位点（a-f）。位点c周围的核苷酸取代阻止了EndoTT对位点c和d的切割，这意味着切割特异性可能取决于ssDNA的核苷酸序列和二级结构。 EndoTT的C末端亚片段（107-216位残基）同时具有核酸内切和DNA结合活性，而N末端的亚片段（1-110位残基）仅具有ssDNA结合活性。定点突变表明EndoTT的核酸内切酶活性需要G（170），R-172和G（177），而DNA结合不是必需的，而D-171，R-178和G（189）是部分DNA结合活性所需的。

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