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

Autor:	Tompkins KJ; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Houtti M; Department of Computer Science and Engineering, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Litzau LA; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Aird EJ; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Everett BA; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Nelson AT; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Pornschloegl L; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Limón-Swanson LK; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Evans RL; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Evans K; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Shi K; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Aihara H; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA., Gordon WR; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Twin Cities, Minneapolis, MN 55455, USA.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2021 Jan 25; Vol. 49 (2), pp. 1046-1064.
DOI:	10.1093/nar/gkaa1248
Abstrakt:	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. (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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