Department of Chemistry & Biochemistry, 9500 Gilman Drive, La Jolla, CA, 92093-0375, USA.
Nucleic Acids Res. 2021 Jan 25;49(2):1033-1045. doi: 10.1093/nar/gkaa1240.
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.
多样性产生的 retroelements（DGRs）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。通过腺嘌呤诱变发生变异，其中 RNA 中的遗传信息被忠实逆转录到 cDNA，所有模板碱基除腺嘌呤外都是如此。我们通过由逆转录酶 bRT、Avd 蛋白和特定 RNA 组成的体外系统研究了典型的博德特氏菌噬菌体 DGR 中的腺嘌呤诱变决定因素。我们发现，bRT-Avd 复合物在逆转录过程中正确掺入的催化效率对于所有模板碱基都非常低，而腺嘌呤的效率最低。模板腺嘌呤上的错误掺入效率仅略低于正确掺入。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 氨基不敏感，但识别鸟嘌呤的 C6 羰基。我们还鉴定了两个预测为非特异性接触传入 dNTP 的 bRT 氨基酸，R74 和 I181，它们是腺嘌呤诱变的促进剂。我们的结果表明，bRT-Avd 的整体低催化效率与其进行腺嘌呤诱变的能力密切相关。
