The mechanism of DNA synthesis has been inferred from static structures, but the
absence of temporal information raises longstanding questions about the order of
events in one of life's most central processes. Here we follow the reaction
pathway of a replicative DNA polymerase using time-resolved X-ray
crystallography to elucidate the order and transition between intermediates. In
contrast to the canonical model, the structural changes observed in the
time-lapsed images reveal a catalytic cycle in which translocation precedes
catalysis. The translocation step appears to follow a push-pull mechanism where
the O-O1 loop of the finger subdomain acts as a pawl to facilitate
unidirectional movement along the template with conserved tyrosine residues 714
and 719 functioning as tandem gatekeepers of DNA synthesis. The structures
capture the precise order of critical events that may be a general feature of
enzymatic catalysis among replicative DNA polymerases.
