{"doi":"10.3389/fmicb.2020.01960","title":"Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases","abstract":"Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (2-4 bp). Here, we focus on MDREs that have an SRA and /or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3′ stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I (N(8 10)/N(8-9)), and Sau43800I (N(8-9)/N(7-8)). EcoWI and Bsp305I are more specific for PT modified DNA in Mg2+ buffer, and promiscuous with Mn2+. Ksp11411I is more PT specific with Ni2+. EcoWI and Ksp11411I cleave fully- and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependence has biological function: EcoWI expressing cells restrict dnd+ GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs).","journal":"Frontiers in Microbiology","year":2020,"id":84171,"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":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9559,"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":432176,"name":"H. Czapinska","orcid":"0000-0002-5163-2855","position":1,"is_corresponding":false},{"id":432177,"name":"Alexey Fomenkov","orcid":"0000-0002-2463-5946","position":2,"is_corresponding":false},{"id":432178,"name":"В. А. Потапов","orcid":"0000-0002-3151-6726","position":3,"is_corresponding":false},{"id":433189,"name":"Daniel F. Heiter","orcid":null,"position":4,"is_corresponding":false},{"id":336454,"name":"Bo Cao","orcid":"0000-0002-4933-6424","position":5,"is_corresponding":false},{"id":289582,"name":"Peter C. Dedon","orcid":"0000-0003-0011-3067","position":6,"is_corresponding":false},{"id":432179,"name":"Matthias Bochtler","orcid":"0000-0001-7884-4463","position":7,"is_corresponding":false},{"id":432180,"name":"Shuang-yong Xu","orcid":"0000-0001-6293-8481","position":8,"is_corresponding":false},{"id":433188,"name":"Thomas Lutz","orcid":null,"position":0,"is_corresponding":true}],"reference_count":37,"raw_metadata":null,"created_at":"2026-07-18T21:54:50.827092Z","pmid":"33013736","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":[]}