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

Publication Type:

Journal Article


Nucleic Acids Res, Volume 49, Issue 2, p.1046-1064 (2021)


Amino Acid Motifs, Amino Acid Sequence, Circoviridae, Conserved Sequence, Crystallography, X-Ray, Deoxyribonuclease I, DNA Helicases, DNA, Single-Stranded, Gene Library, Models, Molecular, Molecular Docking Simulation, Molecular Sequence Data, Nucleic Acid Conformation, Plant Viruses, Protein Binding, Protein Conformation, Protein Engineering, Recombinant Fusion Proteins, Replication Origin, Sequence Alignment, Sequence Homology, Amino Acid, Single-Strand Specific DNA and RNA Endonucleases, Substrate Specificity, Trans-Activators, Viral Proteins


<p>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.</p>

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