Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference.
Perpetuating the homing endonuclease life cycle: identification of mutations that modulate and change I-TevI cleavage preference.
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DOI：
10.1093/nar/gkw614
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发表时间：
2016-09-06
影响因子：
14.9
通讯作者：
Edgell DR
中科院分区：
文献类型：
--
作者：
Roy AC;Wilson GG;Edgell DR
归巢内切酶是具有序列耐受性的DNA内切酶，作为可移动的遗传元件。归巢内切酶切割具有多个核苷酸取代的底物的能力表明其具有高度的适应性，因为改变或调节切割偏好需要相对较少的氨基酸取代。在这里，使用靶向噬菌体T4胸苷酸合成酶基因的GIY-YIG归巢内切酶I-TevI的定向进化实验，我们很容易分离出显著拓宽I-TevI切割偏好的变异，以及微调切割偏好的变异。通过结合取代，我们观察到在一些未被野生型酶切割的底物上的切割效率提高了~ 10,000倍，这与切割位点信息含量的读出减少有关。引人注目的是，我们能够将I-TevI的切割偏好改变为针对胸苷酸合成酶基因中不同切割位点的同分异构体I-BmoI的切割偏好，从而概括了该归巢内切酶家族中切割偏好的进化。我们的研究结果确定了一种分离具有不同切割偏好的GIY-YIG核酸酶结构域的策略，并提供了对归巢内切酶如何通过促进转位到不同靶点来逃避饱和靶点群体中的死端生命周期的见解。
Homing endonucleases are sequence-tolerant DNA endonucleases that act as mobile genetic elements. The ability of homing endonucleases to cleave substrates with multiple nucleotide substitutions suggests a high degree of adaptability in that changing or modulating cleavage preference would require relatively few amino acid substitutions. Here, using directed evolution experiments with the GIY-YIG homing endonuclease I-TevI that targets the thymidylate synthase gene of phage T4, we readily isolated variants that dramatically broadened I-TevI cleavage preference, as well as variants that fine-tuned cleavage preference. By combining substitutions, we observed an ∼10 000-fold improvement in cleavage on some substrates not cleaved by the wild-type enzyme, correlating with a decrease in readout of information content at the cleavage site. Strikingly, we were able to change the cleavage preference of I-TevI to that of the isoschizomer I-BmoI which targets a different cleavage site in the thymidylate synthase gene, recapitulating the evolution of cleavage preference in this family of homing endonucleases. Our results define a strategy to isolate GIY-YIG nuclease domains with distinct cleavage preferences, and provide insight into how homing endonucleases may escape a dead-end life cycle in a population of saturated target sites by promoting transposition to different target sites.
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影响因子：
48
作者：
Huber W;Carey VJ;Gentleman R;Anders S;Carlson M;Carvalho BS;Bravo HC;Davis S;Gatto L;Girke T;Gottardo R;Hahne F;Hansen KD;Irizarry RA;Lawrence M;Love MI;MacDonald J;Obenchain V;Oleś AK;Pagès H;Reyes A;Shannon P;Smyth GK;Tenenbaum D;Waldron L;Morgan M
通讯作者：
Morgan M
影响因子：
14.9
作者：
Kleinstiver BP;Fernandes AD;Gloor GB;Edgell DR
通讯作者：
Edgell DR
DOI：
10.1073/pnas.1117984109
发表时间：
2012-05-22
影响因子：
11.1
作者：
Kleinstiver, Benjamin P.;Wolfs, Jason M.;Edgell, David R.
通讯作者：
Edgell, David R.
影响因子：
5.7
作者：
Lambert, Abigail R.;Hallinan, Jazmine P.;Stoddard, Barry L.
通讯作者：
Stoddard, Barry L.
影响因子：
11.4
作者：
BRYK, M;QUIRK, SM;BELFORT, M
通讯作者：
BELFORT, M