Mechanism of tandem-repeat DNA synthesis by an antiviral reverse transcriptase | bioRxiv
Mechanism of tandem-repeat DNA synthesis by an antiviral reverse transcriptaseDefense-associated reverse transcriptases (DRTs) employ DNA synthesis to protect bacteria against phage infection. We previously showed that DRT10, a tripartite system comprising an RT, a noncoding RNA (ncRNA), and a SLATT effector protein, catalyzes protein-primed, tandem-repeat DNA synthesis in a mechanism strikingly analogous to eukaryotic telomerase. However, the structural basis by which the RT-ncRNA complex directs repeat addition processivity and controls repeat length remains unknown. Here we present cryo-EM reconstructions of two evolutionarily diverse DRT10 RT-ncRNA systems that reveal an unanticipated 2:1 architecture, wherein two RT monomers bind opposite sides of a single, pseudo-symmetric ncRNA. Biochemical experiments demonstrate that each RT monomer reverse transcribes the template encoded on its respective side of the ncRNA, but only one generates the long repetitive product, with the template sequence defined by the distance between two flanking stem-loop anchors. Together with earlier studies of DRT2, DRT3, and DRT9, our findings identify a conserved mechanistic logic underlying ncRNA-templated tandem-repeat synthesis across Class 2 UG antiviral systems, despite vastly different architectural solutions.
### Competing Interest Statement
Columbia University has filed a patent application related to this work. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits. The remaining authors declare no competing interests.www.biorxiv.org