Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif
Cofactor analogue-induced chemical reactivation of endonuclease activity in a DNA cleavage/methylation deficient TspGWI N473A variant in the NPPY motif
复制标题
NPPY 基序中 DNA 切割/甲基化缺陷 TspGWI N473A 变体中辅因子类似物诱导的核酸内切酶活性化学再激活
DOI：
--
复制
发表时间：
2014
影响因子：
2.8
通讯作者：
P. Skowron
中科院分区：
文献类型：
--
作者：
A. Zylicz;Joanna Jeżewska;P. Skowron
TspGWI是一种新的栖热菌限制性内切核酸酶-甲基转移酶（REases-MTases）家族的原型酶，它经历了由sinefungin（SIN）引起的特异性转变的新现象。在这里，我们研究了突变TspGWI N473 A，含有一个单一的氨基酸（aa）取代的NPPY基序的MTase。尽管氨基酸取代位于MTase多肽片段内，但DNA切割和修饰几乎完全消除，表明REase和MTase相互缠绕。值得注意的是，TspGWI N473 A REase功能可以通过添加SIN完全重建。我们假设SIN特异性地结合酶并恢复DNA切割能力蛋白质的三级结构。这表明别构效应物在栖热菌属物种酶的DNA切割中的重要作用。这是第一例由S-腺苷甲硫氨酸（SAM）辅因子类似物抑制REase突变。此外，TspGWI N473 A克隆强烈影响E.大肠杆菌分裂控制，充当“自私基因”。该突变体缺乏竞争MTase活性，因此可能适用于DNA操作。在这里，我们提出了一个新的战略REase活性/特异性改变的一个单一的aa取代，活性基序位置的生物信息学分析的基础上，结合（a）aa序列工程（B）改变蛋白质的酶性质，和（c）使用辅因子类似物切割重建和刺激的案例研究。
We reported previously that TspGWI, a prototype enzyme of a new Thermus sp. family of restriction endonucleases-methyltransferases (REases-MTases), undergoes the novel phenomenon of sinefungin (SIN)-caused specificity transition. Here we investigated mutant TspGWI N473A, containing a single amino acid (aa) substitution in the NPPY motif of the MTase. Even though the aa substitution is located within the MTase polypeptide segment, DNA cleavage and modification are almost completely abolished, indicating that the REase and MTase are intertwined. Remarkably, the TspGWI N473A REase functionality can be completely reconstituted by the addition of SIN. We hypothesize that SIN binds specifically to the enzyme and restores the DNA cleavage-competent protein tertiary structure. This indicates the significant role of allosteric effectors in DNA cleavage in Thermus sp. enzymes. This is the first case of REase mutation suppression by an S-adenosylmethionine (SAM) cofactor analogue. Moreover, the TspGWI N473A clone strongly affects E. coli division control, acting as a ‘selfish gene’. The mutant lacks the competing MTase activity and therefore might be useful for applications in DNA manipulation. Here we present a case study of a novel strategy for REase activity/specificity alteration by a single aa substitution, based on the bioinformatic analysis of active motif locations, combining (a) aa sequence engineering (b) the alteration of protein enzymatic properties, and (c) the use of cofactor–analogue cleavage reconstitution and stimulation.
IL-3A病毒感染小球藻二/三碱基限制性内切酶R.CviJI的克隆及应用。
影响因子：
3.5
作者：
Skowron,PM;Swaminathan,N;McMaster,K;George,D;VanEtten,JL;Mead,DA
通讯作者：
Mead,DA