Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212.
Department of Biochemistry, University of Missouri, Columbia, MO 65212.
Proc Natl Acad Sci U S A. 2017 Nov 21;114(47):E10187-E10195. doi: 10.1073/pnas.1715952114. Epub 2017 Nov 6.
Diversity-generating retroelements (DGRs) are molecular evolution machines that facilitate microbial adaptation to environmental changes. Hypervariation occurs via a mutagenic retrotransposition process from a template repeat () to a variable repeat () that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the phage DGR is primed by an adenine residue in RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is -independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of RNA for cDNA priming. Adenine-specific mutagenesis occurs during reverse transcription and does not involve dUTP incorporation, indicating it results from bRT-catalyzed misincorporation of standard deoxyribonucleotides. In vivo assays show that this hybrid RNA-cDNA molecule is required for mutagenic transposition, revealing a unique mechanism of DNA hypervariation for microbial adaptation.
多样性产生的 retroelements（DGRs）是促进微生物适应环境变化的分子进化机器。通过从模板重复（）到可变重复（）的诱变逆转录转位过程发生超变，导致腺嘌呤到随机核苷酸的转换。在这里，我们表明噬菌体 DGR 的逆转录是由 RNA 中的腺嘌呤残基引发的，并且依赖于 DGR 编码的逆转录酶（bRT）和辅助变异性决定因素（Avd），但不依赖于 bRT。我们还发现 bRT 的催化中心在 cDNA 引发的特定位点切割 RNA 中起着重要作用。逆转录过程中会发生腺嘌呤特异性诱变，不涉及 dUTP 的掺入，表明它是由 bRT 催化的标准脱氧核苷酸的错误掺入引起的。体内测定表明，这种杂交 RNA-DNA 分子是诱变转位所必需的，揭示了微生物适应的 DNA 超变的独特机制。
