Changing the recognition site of a conjugative relaxase by rational design
Changing the recognition site of a conjugative relaxase by rational design
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DOI：
10.1002/biot.200800184
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发表时间：
2009-04-01
影响因子：
4.7
通讯作者：
de la Cruz, Fernando
中科院分区：
文献类型：
--
作者：
Gonzalez-Perez, Blanca;Daniel Carballeira, Jose;de la Cruz, Fernando
TrwC是一种松弛酶蛋白，在细菌与R388的接合过程中启动和完成DNA加工。TrwC识别供体细胞中的特定DNA序列(25个核苷酸)：NIC位点。例如，NIC中的单个转换C24G避免了TrwC对DNA的处理。使用这个简单的模型，我们的目标是获得TrwC特异性可以改变的原则证据。参考DNA-TrwC复合体的几种结构，设计了一个聚焦饱和突变文库(NNK)，使氨基酸Lys262随机化，因为其侧链似乎立体地阻碍了野生型TrwC对C24G NIC突变的识别。使用细菌接合作为体内选择系统，发现了几个TrwC变异体，显示出底物特异性的变化。这些变异体也在竞争性测试中进行了测试
TrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA-TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using bacterial conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to