A high security double lock and key mechanism in HUH relaxases controls oriT-processing for plasmid conjugation.
A high security double lock and key mechanism in HUH relaxases controls oriT-processing for plasmid conjugation.
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Huh松弛的高安全性双重锁定和关键机制控制质粒结合的Orit加工。
DOI：
10.1093/nar/gku741
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发表时间：
2014
影响因子：
14.9
通讯作者：
de la Cruz F
中科院分区：
文献类型：
--
作者：
Carballeira JD;González-Pérez B;Moncalián G;de la Cruz F
在接合转移中，松弛酶起到DNA选择筛的作用。大多数质粒松弛酶属于hH内切酶家族。TrwC是R388质粒的松弛酶，是HH松弛酶家族的原型，也包括F质粒的TraI。在本文中，我们证明了TrwC通过高度安全的双锁和密钥机制来处理其靶NIC位点。它既受TrwC-DNA分子间相互作用的控制，也受几个NIC核苷酸之间的分子内DNA相互作用的控制。用简并寡核苷酸文库进行系统诱变，确定TrwC与DNA相互作用的序列特异图。特异图揭示了基于R388的接合的最低NIC序列要求。X射线结晶学观察到，一些NIC位序列变体仍然能够在TrwC加工所需的NIC位形成U形。此外，纯化的TrwC松弛酶有效地切割了含有这些突变序列的ssDNA和dsDNA底物。由于TrwC能够催化含有NIC位点的DNA分子中的DNA整合，因此NIC位点功能活性序列变体的特征应该可以提高松弛酶介导的潜在靶序列在任何靶基因组中整合的搜索质量。
Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC–DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in a nic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxase-mediated integration in any target genome.
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影响因子：
5.6
作者：
Boer, R;Russi, S;de la Cruz, F
通讯作者：
de la Cruz, F
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
4.7
作者：
Gonzalez-Perez, Blanca;Daniel Carballeira, Jose;de la Cruz, Fernando
通讯作者：
de la Cruz, Fernando
打破并连接单链的DNA：嗯，核酸内切酶超家族。
DOI：
10.1038/nrmicro3067
发表时间：
2013-08
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Chandler M;de la Cruz F;Dyda F;Hickman AB;Moncalian G;Ton-Hoang B
通讯作者：
Ton-Hoang B
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH