{"doi":"10.1093/nar/gkaa1248","title":"Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering","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.","journal":"Nucleic Acids Research","year":2020,"id":62373,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":52,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9531,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":329584,"name":"Mo Houtti","orcid":"0000-0003-0902-0940","position":1,"is_corresponding":false},{"id":329585,"name":"L.A. Litzau","orcid":"0000-0003-4541-2925","position":2,"is_corresponding":false},{"id":329586,"name":"Eric J. Aird","orcid":"0000-0002-4873-042X","position":3,"is_corresponding":false},{"id":329587,"name":"Blake A. Everett","orcid":"0000-0001-6014-9163","position":4,"is_corresponding":false},{"id":225249,"name":"Andrew T. Nelson","orcid":"0000-0002-2591-0428","position":5,"is_corresponding":false},{"id":329588,"name":"Leland Pornschloegl","orcid":"0000-0002-3136-1197","position":6,"is_corresponding":false},{"id":330458,"name":"Lidia K Limón-Swanson","orcid":null,"position":7,"is_corresponding":false},{"id":329589,"name":"Robert L. Evans","orcid":"0000-0002-1690-9927","position":8,"is_corresponding":false},{"id":329590,"name":"Karen Evans","orcid":"0000-0001-6646-7090","position":9,"is_corresponding":false},{"id":103757,"name":"Ke Shi","orcid":"0000-0003-4175-3714","position":10,"is_corresponding":false},{"id":103759,"name":"Hideki Aihara","orcid":"0000-0001-7508-6230","position":11,"is_corresponding":false},{"id":329591,"name":"Wendy R. Gordon","orcid":"0000-0001-7696-5560","position":12,"is_corresponding":false},{"id":329583,"name":"Kassidy J. Tompkins","orcid":"0000-0003-0427-2671","position":0,"is_corresponding":true}],"reference_count":77,"raw_metadata":null,"created_at":"2026-07-18T21:10:11.612909Z","pmid":"33410911","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}