DGR mutagenic transposition occurs via hypermutagenic reverse transcription primed by nicked template RNA.

Abstract

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 (<i>TR</i>) to a variable repeat (<i>VR</i>) that results in adenine-to-random nucleotide conversions. Here we show that reverse transcription of the <i>Bordetella</i> phage DGR is primed by an adenine residue in <i>TR</i> RNA and is dependent on the DGR-encoded reverse transcriptase (bRT) and accessory variability determinant (Avd ), but is <i>VR</i>-independent. We also find that the catalytic center of bRT plays an essential role in site-specific cleavage of <i>TR</i> 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.