Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase

Crystal structures and biochemical analyses of PcrA helicase provide eviden ce for a model for processive DNA unwinding that involves coupling of singl e-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The D NA tracking model invokes ATP-dependent flipping of bases between several p ockets on the enzyme formed by conserved aromatic amino acid residues. We h ave used site-directed mutagenesis to confirm the requirement of ail of the se residues for helicase activity. We also demonstrate that the duplex unwi nding defects correlate with an inability of certain mutant proteins to tra nslocate effectively on ssDNA, Moreover, the results define an essential tr iad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.