Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.01.278671v1?rss=1 Authors: Tompkins, K. J., Houtti, M., Litzau, L. A., Aird, E. J., Everett, B. A., Nelson, A. T., Pornschloegl, L., Limon-Swanson, L. K., Evans, R. L., Evans, K., Shi, K., Aihara, H., Gordon, W. R. Abstract: Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay termed HUH-seq to interrogate subtleties in Rep specificity, and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed us to engineer a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications. Copy rights belong to original authors. Visit the link for more info