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

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.

Ecology and molecular targets of hypermutation in the global microbiome

“…These could represent either TR-VR sequences that accumulated mutations beyond the ones introduced by the DGR RT, or DGR RTs for which the incorporation of random nucleotides is not strictly associated with A residues. The small numbers of such "atypical" DGRs and their sparse distribution across the tree indicates however that the tendency of DGR RTs to incorporate random nucleotide specifically at template A-residues is both ancestral and conserved, thus most likely associated with biochemcial and/or structural constraints of the RT enzyme itself 11 .…”

“…Other examples of DGRs were subsequently characterized, with the best-studied instances, in Legionella and Treponema, targeting surface-displayed proteins 8,9 . All currently known DGRs seem to use the same molecular mechanism, known as mutagenic retrohoming, to generate hypervariation in the target protein 4,10,11 . Mechanistically, a DGR requires three main components: a reverse transcriptase (RT); a template region (TR), which in most cases is intergenic; and a variable region (VR), that is nearidentical to the TR and located within the coding sequence of the target protein.…”

“…The resulting sequence variant, i.e. TR-cDNA, is then integrated in the protein-coding gene, replacing the original VR sequence, through a yet-undefined homing mechanism 10,11 .…”

MyDGR: a server for identification and characterization of diversity-generating retroelements

“…The tertiary structure of Avd and mutational analysis revealed a strict correspondence between retrohoming and the interaction of Avd with RT, suggesting that the RT-Avd complex is important for DGR retrohoming (6). Handa et al (7) recently showed that a complex of the RT and Avd protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules. Additional sequence features of DGR systems include the IMH (initiation of mutagenic retrohoming) site (at the end of the VR) and the IMH* site found in the TR segment: IMH marks the 3’ boundary of A-to-N mutagenesis in the VR (8) and is often followed by a GC-rich inverted repeat required for efficient mutagenic retrohoming (9); and the IMH* in the TR segment differs from IMH and is not followed by an inverted repeat, thereby distinguishing the TR donor sequence from the recipient target DNA sequence (10).…”

Crystal structure of a Thermus aquaticus diversity-generating retroelement variable protein

“…This information is transferred from TR to VR , and during this process adenines within TR are specifically mutated to other bases. A recent study on the prototypical DGR of Bordetella bacteriophage indicates that the DGR RT in association with a second DGR protein, the accessory variability determinant (Avd), is necessary and sufficient for mutagenesis of adenines in TR [19]. Adenine-specific mutagenesis culminates in substitutions occurring only at amino acids that have adenines in their codons in TR .…”