Determinants of adenine-mutagenesis in diversity-generating retroelements.
Determinants of adenine-mutagenesis in diversity-generating retroelements.
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在多样性生成逆转录元件中腺嘌呤突变的决定因素。
DOI：
10.1093/nar/gkaa1240
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发表时间：
2021-01-25
影响因子：
14.9
通讯作者：
Ghosh P
中科院分区：
文献类型：
--
作者：
Handa S;Reyna A;Wiryaman T;Ghosh P
多样性生成逆转录元件（DGR）在自然界中最大程度地改变蛋白质序列。这些元素由人类微生物组和微生物“暗物质”的成分编码。变异通过腺嘌呤诱变发生，其中RNA中的遗传信息被忠实地逆转录为除了腺嘌呤之外的所有模板碱基的cDNA。我们研究了腺嘌呤诱变的原型博德特氏菌噬菌体DGR通过在体外系统组成的逆转录酶bRT，Avd蛋白，和一个特定的RNA的决定因素。我们发现，在逆转录过程中的bRT-Avd复合物的正确掺入的催化效率是惊人的低，所有的模板碱基，最低的发生腺嘌呤。跨模板腺嘌呤的错误掺入的效率仅略低于正确掺入的效率。我们发现，C6，而不是N1或C2，嘌呤取代基是腺嘌呤诱变的关键决定因素。bRT-Avd对腺嘌呤的C6胺不敏感，但识别鸟嘌呤的C6羰基。我们还确定了两个bRT氨基酸预测非特异性接触传入的dNTPs，R74和I181，作为腺嘌呤诱变的启动子。我们的研究结果表明，整体低催化效率的bRT-Avd是密切相关的，其能力进行腺嘌呤诱变。
Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.
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影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
14.8
作者：
Kelley LA;Mezulis S;Yates CM;Wass MN;Sternberg MJ
通讯作者：
Sternberg MJ
影响因子：
29.7
作者：
Boehm T;McCurley N;Sutoh Y;Schorpp M;Kasahara M;Cooper MD
通讯作者：
Cooper MD
影响因子：
15
作者：
Harcourt EM;Ehrenschwender T;Batista PJ;Chang HY;Kool ET
通讯作者：
Kool ET
影响因子：
15.5
作者：
Benler, Sean;Cobian-Gueemes, Ana Georgina;Rohwer, Forest
通讯作者：
Rohwer, Forest