The mechanism of DNA synthesis has been inferred from static structures, but the absence of temporal information raises questions about the order of events in one of life’s most central processes. In this talk, I will describe how a series of control experiments led us to investigate the catalytic cycle of a replicative DNA polymerase I member by time-resolved X-ray crystallography. Time-lapsed structures obtained for the initiation and elongation cycles of DNA synthesis reveal that translocation precedes chemical bond formation in the mechanism of DNA synthesis. 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 two conserved tyrosine residues functioning as tandem gatekeepers of DNA synthesis. Further studies are now needed to determine if this mechanism of translocation is a general property of replicative DNA polymerases.

Visualizing the catalytic cycle of DNA synthesis by time-resolved x-ray crystallography

ACS Spring Meeting 2021

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