Following replicative DNA synthesis by time-resolved X-ray crystallography

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.

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Fig: Time-ordered images capture the initiation step of DNA synthesis: X-ray crystal structures of polymerase intermediates observed between 0–120 min (left column). Cartoon overlays and polder maps contoured at 2–4 σ (blue boxes). Red arrow indicates conformational changes between structures. The transitions are labeled: (1) movement of the DNA duplex, (2) opening of the O-O1 loop to form a hydrophobic pocket, (3) closing of the O-O1 loop and (4) chemical bond formation. Color scheme: 5’ templating base (red), Y714 and Y719 (purple), 3’ nucleotide of DNA primer (orange), dATP (blue), earlier reaction time (gray), and later reaction time (yellow). Abbreviations: O (O helix), O1 (O1 helix), T (template), P (primer), Y (tyrosine), D (aspartate), S (serine), I (isoleucine), and G (glycine).

The structures capture the precise order of critical events that may be a general feature of enzymatic catalysis among replicative DNA polymerases.

Chim, N., Meza, R.A., Trinh, A.M. et al. Following replicative DNA synthesis by time-resolved X-ray crystallography. Nat Commun 12,2641 (2021). https://doi.org/10.1038/s41467-021-22937-z

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