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bioRxiv · 10.1101/2020.09.01.278671

Molecular underpinnings of ssDNA specificity by Rep HUH endonucleases and implications for HUH-tag multiplexing and engineering

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.
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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 for 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 engineering of only four amino acids in 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.

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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.. 2020-09-01. Molecular underpinnings of ssDNA specificity by Rep HUH endonucleases and implications for HUH-tag multiplexing and engineering. https://doi.org/10.1101/2020.09.01.278671

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