Department of Chemistry & Biochemistry, University of California, San Diego, CA 92093, USA.
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA.
Nucleic Acids Res. 2018 Oct 12;46(18):9711-9725. doi: 10.1093/nar/gky620.
Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.
多样性产生的 retroelements（DGRs）通过诱变 retrohoming 产生无与伦比的蛋白质序列变异水平。序列信息从不变的模板区域（TR）通过 RNA 中间体转移到蛋白质编码可变区。在转移过程中，腺嘌呤的选择性保真度是来自不同细菌、古细菌和微生物病毒的 DGRs 的标志。我们在体外重现了典型的博德特氏菌噬菌体 DGR 的选择性保真度。DGR 逆转录酶 bRT 和五聚体辅助变异性决定因子（Avd）蛋白的复合物以及 DGR RNA 是合成模板引发的、共价连接的 RNA-cDNA 分子所必需的，这与体内观察到的情况一致。我们确定 RNA-cDNA 分子是分支的，最有可能通过 2'-5'磷酸二酯键连接。腺嘌呤诱变是 bRT-Avd 复合物的固有特性，该复合物在逆转录 DGR 或非 DGR RNA 模板的腺嘌呤时表现出前所未有的混杂性。相比之下，bRT-Avd 的延续性严格依赖于模板，仅在 DGR RNA 上发生。这种限制主要是由于 TR 下游的非编码片段，该片段特异性结合 Avd 并为延续性聚合创造了特权位点。限制在 DGR RNA 上可能会保护宿主基因组免受损伤。这些结果定义了一种新的大规模序列多样化途径的早期步骤。
