{"doi":"10.1101/2022.05.05.490830","title":"The iron-sulfur cluster is critical for DNA binding by human DNA polymerase ε","abstract":"ABSTRACT DNA polymerase ε (Polε) is a key enzyme for DNA replication in eukaryotes. It is attached to a helicase and performs DNA synthesis on the leading strand. Recently it was shown that the catalytic domain of yeast Polε (Polε CD ) contains a [4Fe-4S] cluster located at the base of the processivity domain (P-domain) and coordinated by four conserved cysteines. In this work, we have shown that human Polε CD (hPolε CD ) expressed in bacterial cells also contains an iron-sulfur cluster. In comparison, recombinant hPolε CD produced in insect cells contains an eight-fold-lower level of iron. Interestingly, the iron content correlates with the level of DNA-binding molecules, which suggests an important role of the iron-sulfur cluster in hPolε interaction with DNA. Indeed, mutation of two conserved cysteines that coordinate the cluster abolished template:primer binding and, therefore, DNA polymerase and proofreading exonuclease activities. We propose that the cluster regulates the conformation of the P-domain, which, like a gatekeeper, controls access to a DNA-binding cleft for a template:primer. In addition, we performed kinetic and binding studies of hPolε CD . The binding studies demonstrated low affinity of hPolε CD to DNA and a strong effect of salt concentration on stability of the hPolε CD /DNA complex. Pre-steady-state kinetic studies have shown a maximal polymerization rate constant of 51.5 s -1 and a relatively low affinity to incoming dNTP with an apparent K D of 105 μM. This work provides notable insight into the role of a [4Fe-4S] cluster in Polε function.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":308462,"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":0.9578,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":560961,"name":"Andrey G. Baranovskiy","orcid":"0000-0002-8017-6932","position":1,"is_corresponding":false},{"id":936071,"name":"Lucia M. Morstadt","orcid":"0000-0002-4972-6086","position":2,"is_corresponding":false},{"id":936072,"name":"Nigar D. Babayeva","orcid":"0000-0002-4410-1327","position":3,"is_corresponding":false},{"id":575874,"name":"Elena I. Stepchenkova","orcid":"0000-0002-5854-8701","position":4,"is_corresponding":false},{"id":560962,"name":"T.H. Tahirov","orcid":"0000-0002-1238-0069","position":5,"is_corresponding":false},{"id":936070,"name":"Alisa E. Lisova","orcid":"0000-0002-3647-6460","position":0,"is_corresponding":true}],"reference_count":27,"raw_metadata":null,"created_at":"2026-07-19T00:33:03.486542Z","pmid":null,"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":[]}