Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A‐family of polymerases than in the B‐family of replicative polymerases. To address this issue, we have determined the X‐ray crystal structures of phi29 DNAP, a member of the protein‐primed subgroup of the B‐family of polymerases, complexed with primer‐template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre‐ and post‐translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A‐family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single‐stranded template DNA in very different ways.