title: Characterisation of Bacillus stearothermophilus PcrA helicase: evidence against an active rolling mechanism.
abstract: PcrA from Bacillus stearothermophilus is a DNA helicase for which, despite the availability of a crystal structure, there is very little biochemical information. We show that the enzyme has a broad nucleotide specificity, even being able to hydrolyse ethenonucleotides, and is able to couple the hydrolysis to unwinding of DNA substrates. In common with the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helicase but at high protein concentrations it can also displace a substrate with a 5' tail. However, in contrast to Rep and UvrD, we do not see any evidence for dimerisation of the protein even in the presence of DNA. The enzyme shows a specificity for the DNA substrate in gel mobility assays, with the preferred substrate being one with both single and double stranded regions of DNA. We propose that these data, together with existing structural evidence, support an inchworm rather than a rolling model for 3'-5' helicase activity.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: L E
authors.initials: LE
authors.lastname: Bird
authors.forename: J A
authors.initials: JA
authors.lastname: Brannigan
authors.forename: H S
authors.initials: HS
authors.lastname: Subramanya
authors.forename: D B
authors.initials: DB
authors.lastname: Wigley
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006868:Hydrolysis; D009711:Nucleotides / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D009711:Nucleotides; C084217:pcrA protein, Bacteria; D000251:Adenosine Triphosphatases; D004265:DNA Helicases