Open AccessMolecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering
Author(s) -
Kassidy J. Tompkins,
Mo Houtti,
L.A. Litzau,
Eric J. Aird,
Blake A. Everett,
Andrew T. Nelson,
Leland Pornschloegl,
Lidia K. Limón-Swanson,
III Evans,
Karen Evans,
Ke Shi,
Hideki Aihara,
Wendy J. Gordon
Publication year - 2021
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkaa1248
Subject(s) - biology , endonuclease , computational biology , geminiviridae , genome , dna , genetics , capsid , rolling circle replication , homing endonuclease , restriction enzyme , bioconjugation , gene , dna replication , biochemistry , begomovirus
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.