title: Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD.
abstract: The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics of PcrA activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state of the active helicase complex, its processivity and the mechanism of unwinding. By tethering either DNA or PcrA to a microscope coverslip surface, unwinding of both linear and natural circular plasmid DNA by PcrA/RepD was followed in real-time using total internal reflection fluorescence microscopy. Visualization was achieved using a fluorescent single-stranded DNA-binding protein. The single-molecule data show that PcrA, in combination with RepD, can unwind plasmid lengths of DNA in a single run, and that PcrA is active as a monomer. Although the average rate of unwinding was similar in single-molecule and bulk solution assays, the single-molecule experiments revealed a wide distribution of unwinding speeds by different molecules. The average rate of unwinding was several-fold slower than the PcrA translocation rate on single-stranded DNA, suggesting that DNA unwinding may proceed via a partially passive mechanism. However, the fastest dsDNA unwinding rates measured in the single-molecule unwinding assays approached the PcrA translocation speed measured on ssDNA.
authors.affiliation: MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
authors.forename: Liisa T
authors.initials: LT
authors.lastname: Chisty
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Toseland
authors.forename: Natalia
authors.initials: N
authors.lastname: Fili
authors.forename: Gregory I
authors.initials: GI
authors.lastname: Mashanov
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dillingham
authors.forename: Justin E
authors.initials: JE
authors.lastname: Molloy
authors.forename: Martin R
authors.initials: MR
authors.lastname: Webb
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D019921:Biotinylation; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D061925:Immobilized Nucleic Acids / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics*; D055503:Protein Multimerization; D021381:Protein Transport
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D061925:Immobilized Nucleic Acids; C058757:RepD protein, Staphylococcus aureus; C084217:pcrA protein, Bacteria; D004247:DNA; D004265:DNA Helicases
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