Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease.
Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease.
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DOI：
10.1093/nar/gkm624
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发表时间：
2007
影响因子：
14.9
通讯作者：
Monnat RJ Jr
中科院分区：
文献类型：
--
作者：
Eklund JL;Ulge UY;Eastberg J;Monnat RJ Jr
我们使用酵母单杂交试验分离和表征的真核归巢核酸内切酶I-PpoI的变体，能够结合突变体，切割抗性I-PpoI目标或“归巢”位点DNA在体内。天然I-PpoI在体内和体外以高特异性识别并切割半回文15-bp靶位点。该靶位点存在于所有真核生物的28 S或等同大亚基rDNA基因中。能够结合突变靶位点DNA的I-PpoI变体在DNA-蛋白质界面中具有1至8个氨基酸取代。这些蛋白质的生物化学表征揭示了广泛的位点结合亲和力和位点歧视。三分之一的变异体能够切割靶位点DNA，但位点结合亲和力和位点切割之间没有系统的关系。几种变体的计算建模提供了对接触或邻近特定靶位点DNA碱基对的氨基酸取代如何确定I-PpoI位点结合亲和力和位点区分的机制性见解，并可能影响切割效率。
We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.
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影响因子：
14.9
作者：
LUKACSOVICH, T;YANG, D;WALDMAN, AS
通讯作者：
WALDMAN, AS
影响因子：
3.5
作者：
BOTSTEIN, D;FALCO, SC;DAVIS, RW
通讯作者：
DAVIS, RW
DOI：
10.1006/bbrc.1999.0152
发表时间：
1999-02-05
影响因子：
3.1
作者：
Monnat, RJ;Hackmann, AFM;Cantrell, MA
通讯作者：
Cantrell, MA
影响因子：
64.8
作者：
Ashworth, Justin;Havranek, James J.;Baker, David
通讯作者：
Baker, David
影响因子：
6.1
作者：
Lavery, Richard
通讯作者：
Lavery, Richard