6WE0

Wheat dwarf virus Rep domain complexed with a single-stranded DNA 10-mer comprising the cleavage site

Summary for 6WE0

• Entry DOI: 10.2210/pdb6we0/pdb
• Descriptor: Replication-associated protein, DNA (5'-D(*TP*AP*AP*TP*AP*TP*TP*AP*CP*C)-3'), MANGANESE (II) ION, ... (4 entities in total)
• Functional Keywords: huh-tag, huh motif, replicase, viral protein, single stranded dna, ssdna, ssdna binding, replication, dna binding protein, replication-dna complex, replication/dna
• Biological source: Wheat dwarf virus; More
• Total number of polymer chains: 2
• Total formula weight: 18817.30

Authors

Tompkins, K.,Litzau, L.A.,Shi, K.,Nelson, A.,Evans III, R.L.,Gordon, W.R. (deposition date: 2020-04-01, release date: 2020-12-16, Last modification date: 2023-10-18)

Primary citation

Tompkins, K.J.,Houtti, M.,Litzau, L.A.,Aird, E.J.,Everett, B.A.,Nelson, A.T.,Pornschloegl, L.,Limon-Swanson, L.K.,Evans, R.L.,Evans, K.,Shi, K.,Aihara, H.,Gordon, W.R.
Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.
Nucleic Acids Res., 49:1046-1064, 2021

PubMed 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.

PubMed: 33410911
DOI: 10.1093/nar/gkaa1248

Experimental method

X-RAY DIFFRACTION (1.8 Å)