Uncoupling DNA translocation and helicase activity in PcrA: direct evidence for an active mechanism

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3'-tailed DNA duplex reveal a contact region that covers a signific ant region of the substrate both in the presence and absence of a non-hydro lysable analogue of ATP, ADPNP, However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By c ombining this information with that obtained from crystal structures of Pcr A complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and singl e-stranded DNA translocation activities intact, These mutants are all locat ed in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support a n 'active' mechanism for PcrA that involves two distinct ATP-dcpendent proc esses: destabilization of the duplex DNA ahead of the enzyme that is couple d to DNA translocation along the single strand product.