CHARACTERIZATION OF BACILLUS-STEAROTHERMOPHILUS PCRA HELICASE - EVIDENCE AGAINST AN ACTIVE ROLLING MECHANISM

PcrA from Bacillus stearothermophilus is a DNA helicase for which, des pite the availability of a crystal structure, there is very little bio chemical information. We show that the enzyme has a broad nucleotide s pecificity, even being able to hydrolyse ethenonucleotides, and is abl e to couple the hydrolysis to unwinding of DNA substrates, In common w ith the Escherichia coli helicases Rep and UvrD, PcrA is a 3'-5' helic ase but at high protein concentrations it can also displace a substrat e 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 D NA, The enzyme shows a specificity for the DNA substrate in gel mobili ty 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 r olling model for 3'-5' helicase activity.