Target Site Recognition by a Diversity-Generating Retroelement
Target Site Recognition by a Diversity-Generating Retroelement
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DOI：
10.1371/journal.pgen.1002414
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发表时间：
2011-12-01
期刊：
影响因子：
4.5
通讯作者：
Miller, Jeff F.
中科院分区：
文献类型：
--
作者：
Guo, Huatao;Tse, Longping V.;Miller, Jeff F.
Diversity-generating retroelements（DGRs）是广泛分布于细菌、噬菌体和质粒基因组中的体内序列多样化机器。它们的功能是通过诱变归巢将大量靶向多样性引入编码蛋白质的DNA序列。腺嘌呤残基在从供体模板重复序列（TR）到受体可变重复序列（VR）的逆转录转座过程中转化为随机核苷酸。使用博德特氏菌噬菌体BPP-1元件作为原型，我们已经表征了DGR靶位点功能的要求。尽管VR上游的序列是不对称的，但有效的反向归巢需要一个紧接VR下游的24 bp序列，该序列含有短的反向重复序列。反向重复序列形成发夹或十字形结构，突变分析表明，虽然茎的结构很重要，但其序列可以变化。相反，循环具有序列依赖性功能。结构特异性核酸酶消化证实了DNA发夹/十字形的存在，并且标记共转化测定表明其影响效率，但不影响cDNA整合的位点。与其他噬菌体DGRs的比较表明，相似的结构是靶序列的保守特征。使用卡那霉素抗性决定簇作为报告基因，我们发现IMH和发夹/十字形形成区的移植足以将DGR多样化机制靶向异源基因。除了进一步了解DGR的retrohoming，我们的研究结果表明，DGR可能提供独特的工具，通过体内DNA多样化的蛋白质进化。
Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.