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

• Published in: Nucleic acids research Vol. 49; no. 2; pp. 1046 - 1064
• Main Authors: Tompkins, Kassidy J, Houtti, Mo, Litzau, Lauren A, Aird, Eric J, Everett, Blake A, Nelson, Andrew T, Pornschloegl, Leland, Limón-Swanson, Lidia K, Evans, Robert L, Evans, Karen, Shi, Ke, Aihara, Hideki, Gordon, Wendy R
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 25-01-2021
• Subjects: Amino Acid Motifs; Amino Acid Sequence; BASIC BIOLOGICAL SCIENCES; Circoviridae - enzymology; Conserved Sequence; Crystallography, X-Ray; Deoxyribonuclease I - chemistry; Deoxyribonuclease I - metabolism; DNA Helicases - chemistry; DNA Helicases - metabolism; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - metabolism; Gene Library; Massively Parallel (Deep) Sequencing; Models, Molecular; Molecular Docking Simulation; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic Acid Enzymes; Nucleic Acid Enzymology; Plant Viruses - enzymology; Protein Binding; Protein Conformation; Protein Engineering - methods; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Replication Origin; Sequence Alignment; Sequence Homology, Amino Acid; Single-Strand Specific DNA and RNA Endonucleases - chemistry; Single-Strand Specific DNA and RNA Endonucleases - metabolism; Substrate Specificity; Trans-Activators - chemistry; Trans-Activators - metabolism; Viral Proteins - chemistry; Viral Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkaa1248

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.