{"doi":"10.1016/j.jbc.2022.101996","title":"Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA","abstract":"The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer. The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer. The molecular processes involved in replicating DNA are complex, requiring the concerted action of multiple proteins (1Kornberg A. Baker T.A. DNA Replication.2nd ed. W.H. Freeman, NY1992Google Scholar, 2Hamdan S.M. Richardson C.C. Motors, switches, and contacts in the replisome.Annu. Rev. Biochem. 2009; 78: 205-243Crossref PubMed Scopus (135) Google Scholar). The replisome consists of the dynamic multiprotein assembly responsible for DNA replication. Despite vast differences in the number of components, the essential biochemical steps performed by the replisome are conserved throughout evolution (3Costa A. Hood I.V. Berger J.M. Mechanisms for initiating cellular DNA replication.Annu. Rev. Biochem. 2013; 82: 25-54Crossref PubMed Scopus (140) Google Scholar, 4Yao N.Y. O'Donnell M.E. The DNA replication machine: structure and dynamic function.Subcell. Biochem. 2021; 96: 233-258Crossref PubMed Scopus (5) Google Scholar). Bacteriophage T7 employs an economical replication system (2Hamdan S.M. Richardson C.C. Motors, switches, and contacts in the replisome.Annu. Rev. Biochem. 2009; 78: 205-243Crossref PubMed Scopus (135) Google Scholar, 5Kulczyk A.W. Richardson C.C. The replication system of bacteriophage T7.Enzymes. 2016; 39: 89-136Crossref PubMed Scopus (20) Google Scholar). Only four proteins are required to reconstitute coordinated synthesis of leading and lagging strands: the phage-encoded DNA polymerase gp5, the ssDNA-binding protein gp2.5, the primase-helicase gp4, and Escherichia coli thioredoxin Trx (Fig. 1A). The bifunctional primase-helicase gp4 lies at the center of the T7 replisome, structurally and functionally (6Kulczyk A.W. Moeller ","journal":"Journal of Biological Chemistry","year":2022,"id":268561,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9564,"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":930750,"name":"Seung‐Joo Lee","orcid":"0000-0002-0027-2554","position":1,"is_corresponding":false},{"id":931365,"name":"Noah J. Thompson","orcid":null,"position":2,"is_corresponding":false},{"id":307697,"name":"Jack D. Griffith","orcid":"0000-0002-8357-8689","position":3,"is_corresponding":false},{"id":176151,"name":"Charles C. Richardson","orcid":null,"position":4,"is_corresponding":false},{"id":833730,"name":"Alfredo J. Hernandez","orcid":"0000-0002-0903-8932","position":0,"is_corresponding":true}],"reference_count":57,"raw_metadata":null,"created_at":"2026-07-19T00:27:14.088663Z","pmid":"35500649","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":[]}