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

Significance Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacteria, archaea, and their viruses. DGRs use a reverse transcriptase (RT)-mediated mechanism to diversify protein-encoding genes to facilitate adaptation of their hosts to changing environments. Here, we demonstrate that the Bordetella phage DGR-encoded RT uses the 3′-OH of a nicked template RNA to initiate reverse transcription, during which random nucleotides are incorporated when adenine residues in the template are copied into complementary DNA (cDNA). We further show that this mutated, covalently linked RNA-cDNA molecule is required for DGR-mediated sequence diversification, revealing a mechanism of accelerated evolution with broad practical applications.