Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA |
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Authors: | Alfredo J. Hernandez Seung-Joo Lee Noah J. Thompson Jack D. Griffith Charles C. Richardson |
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Affiliation: | 1.Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA;2.Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, The University of North Carolina-Chapel Hill, Chapel Hill, North Carolina, USA |
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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. |
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Keywords: | DNA helicase, enzyme mutation, protein– DNA interaction, bacteriophage, viral protein, multifunctional enzyme, mass spectrometry, site-directed mutagenesis |
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