TY - JOUR
T1 - Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering
AU - Tompkins, Kassidy J.
AU - Houtti, Mo
AU - Litzau, Lauren A.
AU - Aird, Eric J.
AU - Everett, Blake A.
AU - Nelson, Andrew T.
AU - Pornschloegl, Leland
AU - Limón-Swanson, Lidia K.
AU - Evans, Robert L.
AU - Evans, Karen
AU - Shi, Ke
AU - Aihara, Hideki
AU - Gordon, Wendy R.
N1 - Publisher Copyright:
© 2021 The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2021/1/25
Y1 - 2021/1/25
N2 - 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.
AB - 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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U2 - 10.1093/nar/gkaa1248
DO - 10.1093/nar/gkaa1248
M3 - Article
C2 - 33410911
AN - SCOPUS:85100325765
SN - 0305-1048
VL - 49
SP - 1046
EP - 1064
JO - Nucleic acids research
JF - Nucleic acids research
IS - 2
ER -