Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

• Type of Material: Article
• Authors: Sumit Handa, University of California San Diego; Yong Jiang, Emory University; Sijia Tao, Emory University; Robert Foreman, University of California San Diego; Raymond F Schinazi, Emory University; Jeff F. Miller, University of California Los Angeles; Partho Ghosh, University of California San Diego
• Language: English
• Date: 2018-10-12
• Publisher: Oxford University Press (OUP): Policy C - Option B
• Publication Version: Final Published Version
• Copyright Statement: © 2018, Oxford University Press
• License: Creative Commons Attribution-NonCommercial 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gky620
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 46
• Issue: 18
• Start Page: 9711
• End Page: 9725
• Grant/Funding Information: National Institutes of Health (NIH) [R01 AI096838 to J.F.M. and P.G.]. Funding for open access charge: NIH [R01 AI096838].
• Keywords: RNA; LECTIN FOLD; DIVERSITY-GENERATING RETROELEMENTS; Biochemistry & Molecular Biology; 5' END; READ ALIGNMENT; GROUP-II INTRON; Life Sciences & Biomedicine; REVERSE-TRANSCRIPTASE; Science & Technology; SINGLE-STRANDED-DNA
• Research Categories: Biology, Microbiology; Chemistry, Biochemistry

Abstract

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.