{"doi":"10.1101/2024.02.15.580553","title":"Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB","abstract":"ABSTRACT Mitomycin C (MMC) repair factor A ( mrfA ) and factor B ( mrfB ), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis . Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg 2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ- family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg 2+ -dependent 3’-5’ DNA exonuclease. We show that Leu113 aids in coordinating the 3’ end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC- induced DNA adducts. GRAPHICAL ABSTRACT","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":494459,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9274,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1305543,"name":"Lia M Munson","orcid":null,"position":1,"is_corresponding":false},{"id":277673,"name":"Jayakrishnan Nandakumar","orcid":"0000-0001-9146-2785","position":2,"is_corresponding":false},{"id":471594,"name":"Lyle A. Simmons","orcid":"0000-0002-9600-7623","position":3,"is_corresponding":false},{"id":349978,"name":"Kelly A. Manthei","orcid":"0000-0003-3874-8228","position":0,"is_corresponding":true}],"reference_count":62,"raw_metadata":null,"created_at":"2026-07-19T02:09:11.736910Z","pmid":"38405983","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":[]}