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首页> 外文期刊>Journal of Molecular Biology >Coincidence of Cleavage Sites of Intron Endonuclease I-TevI and Critical Sequences of the Host Thymidylate Synthase Gene.
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Coincidence of Cleavage Sites of Intron Endonuclease I-TevI and Critical Sequences of the Host Thymidylate Synthase Gene.

机译：内含子核酸内切酶I-TevI​​切割位点与宿主胸苷酸合酶基因的关键序列的重合。

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摘要

To maximize spread of their host intron or intein, many homing endonucleases recognize nucleotides that code for important and conserved amino acid residues of the target gene. Here, we examine the cleavage requirements for I-TevI, which binds a stretch of thymidylate synthase (TS) DNA that codes for functionally critical residues in the TS active site. Using an in vitro selection scheme, we identified two base-pairs in the I-TevI cleavage site region as important for cleavage efficiency. These were confirmed by comparison of I-TevI cleavage efficiencies on mutant and on wild-type substrates. We also showed that nicking of the bottom strand by I-TevI is not affected by mutation of residues surrounding the bottom-strand cleavage site, unlike other homing endonucleases. One of these two base-pairs is universally conserved in all TS sequences, and is identical with a previously identified cleavage determinant of I-BmoI, a related GIY-YIG endonuclease that binds a homologous stretch of TS-encoding DNA. Theother base-pair is conserved only in a subset of TS genes that includes the I-TevI, but not the I-BmoI, target sequence. Both the I-TevI and I-BmoI cleavage site requirements correspond to functionally critical residues involved in an extensive hydrogen bond network within the TS active site. Remarkably, these cleavage requirements correlate with TS phylogeny in bacteria, suggesting that each endonuclease has individually adapted to efficiently cleave distinct TS substrates.
机译：为了最大化其宿主内含子或内含子的扩散，许多归巢内切核酸酶识别编码靶基因重要且保守的氨基酸残基的核苷酸。在这里，我们检查了I-TevI​​的切割要求，该切割结合了一段胸苷酸合酶（TS）DNA，该DNA编码TS活性位点中的功能关键性残基。使用体外选择方案，我们确定了I-TevI​​切割位点区域中的两个碱基对，对切割效率很重要。通过比较在突变体和野生型底物上的I-TevI​​切割效率，证实了这些。我们还表明，与其他归巢核酸内切酶不同，I-TevI​​对底部链的刻蚀不受底部链切割位点周围残基突变的影响。这两个碱基对之一在所有TS序列中都普遍保守，并且与先前鉴定的I-BmoI的切割决定簇相同，I-BmoI是一种相关的GIY-YIG核酸内切酶，结合了TS编码DNA的同源片段。另一个碱基对仅在包含I-TevI​​但不包含I-BmoI目标序列的TS基因子集中保守。 I-TevI​​和I-BmoI切割位点的要求都对应于TS活性位点内广泛的氢键网络中涉及的功能关键残基。值得注意的是，这些切割要求与细菌中的TS系统发育相关，表明每种核酸内切酶均已单独适应有效地切割不同的TS底物。

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