Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene
Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene
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DOI：
10.1016/j.jmb.2004.09.005
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发表时间：
2004-11-05
影响因子：
5.6
通讯作者：
Belfort, M
中科院分区：
文献类型：
--
作者：
Edgell, DR;Stanger, MJ;Belfort, M
为了最大化其宿主内含子或内含子的扩散，许多归巢核酸内切酶识别编码靶基因的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，它结合一段胸苷酸合酶 (TS) DNA，编码 TS 活性位点中的功能关键残基。使用体外选择方案，我们确定了 I-TevI 切割位点区域中对切割效率很重要的两个碱基对。这些通过比较 I-TevI 对突变体和野生型底物的切割效率得到证实。我们还表明，与其他归巢核酸内切酶不同，I-TevI 对底部链的切口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 序列中普遍保守，并且与先前鉴定的 I-BmoI 切割决定簇相同，I-BmoI 是一种相关的 GIY-YIG 核酸内切酶，可结合 TS 编码 DNA 的同源片段。另一个碱基对仅在 TS 基因的子集中保守，其中包括 I-TevI，但不包括 I-BmoI 靶序列。 I-TevI 和 I-BmoI 裂解位点要求均对应于 TS 活性位点内广泛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，这表明每种核酸内切酶都单独适应了有效切割不同的 TS 底物。 (C) 2004 Elsevier Ltd. 保留所有权利。
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. The other 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. (C) 2004 Elsevier Ltd. All rights reserved.