Selective infidelity in diversity-generating retroelements

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Diversity-generating retroelements (DGRs) are unique and unparalleled generators of massive protein sequence diversity. These elements are prevalent in the microbial ‘dark matter’, which appear to comprise a major fraction of microbial life, and are widespread in the human virome and microbiome. The only other example in the natural world of massive protein sequence variation occurs in the vertebrate adaptive immune system, in which variation enables the recognition of novel targets and consequent adaptation to dynamic environments. A similar benefit appears to be provided by DGRs. DGRs diversify proteins through a fundamentally different mechanism than the vertebrate immune system. In DGRs, diversification arises from genetic information being transmitted unfaithfully for one specific base, adenine, and faithfully for the others. This occurs during reverse transcription of genetic information from RNA to cDNA, and the specificity to adenine shapes the pattern of protein functional variation. This selective infidelity to adenines is the central hallmark feature of DGRs. Selectivity infidelity is unique in biology and how it occurs unknown. We have made a significant breakthrough on this problem by reconstituting specific infidelity in vitro for the prototypical Bordetella bacteriophage DGR. We have found that the DGR reverse transcriptase bRT in complex with the DGR accessory variability determinant (Avd) protein is necessary and sufficient to synthesize adenine- mutagenized cDNA. Our results indicate that bRT-Avd and the DGR RNA combine to form a functional and structured ribonucleoprotein (RNP) particle. We are on the verge of uncovering the molecular and atomic details underlying selective infidelity through the following specific aims: (1) Visualize bRT-Avd/RNA complexes in different functional states by cryo-electron microscopy; (2) Identify the nucleobase and protein determinants responsible for selective infidelity; and (3) Determine the basis for position- specific modulation of specific infidelity. Our proposed studies will provide fundamental advances in understanding DGRs. Additionally, because of the uniqueness of selective infidelity, these studies will also likely provide novel insights into mechanisms that control fidelity in other reverse transcriptases (e.g., HIV RT) and nucleotide polymerases in general.