Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation
Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation
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DOI：
10.1093/nar/gkn1007
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发表时间：
2009-02-01
影响因子：
14.9
通讯作者：
Stoddard, Barry L.
中科院分区：
文献类型：
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作者：
Takeuchi, Ryo;Certo, Michael;Stoddard, Barry L.
LAGLIDADG 归巢核酸内切酶 (LHE) I-Anil 采用了极其有效的二级 RNA 剪接活性，这对其宿主有利，同时与低效的 DNA 切割保持平衡。一项选择实验鉴定了酶中的点突变，这些点突变可以协同作用来提高核酸内切酶的活性。氨基酸取代增加了靶亲和力，改变了热裂解曲线并显着增加了转染细胞中的靶向重组。 RNA剪接活性不受这些突变的影响。 DNA 切割活性的改善主要集中在酶的两个活性位点之一，对应于假设充当通用碱基的赖氨酸残基的重排。筛选中分离的大多数构建体含有一个或多个突变，这些突变将氨基酸同一性恢复为在I-Anil的一个或多个密切同源物中发现的残基。这意味着先前降低 I-Anil 核酸内切酶活性的突变被识别并逆转，有时与其他“人工”突变相结合，以优化其体内活性。
The LAGLIDADG homing endonuclease (LHE) I-Anil has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-Anil. This implies that mutations that have previously reduced the endonuclease activity of I-Anil are identified and reversed, sometimes in combination with additional 'artificial' mutations, to optimize its in vivo activity.