Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation

Motif III is one of the seven protein motifs that are characteristic of sup erfamily I helicases. To investigate its role in the helicase mechanism we have introduced a variety of mutations at three of the most conserved amino acid residues (Q254, W259 and R260). Biochemical characterisation of the r esulting proteins shows that mutation of motif III affects both ATP hydroly sis and single-stranded DNA binding. We propose that amino acid residue Q25 4 acts as a gamma-phosphate sensor at the nucleotide binding pocket transmi tting conformational changes to the DNA binding site, since the nature of t he charge on this residue appears to control the degree of coupling between ATPase and helicase activities. Residues W259 and R260 both participate in direct DNA binding interactions that are critical for helicase activity.