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

Nicholas Chim,John C Chaput,Kefan Yang,Anh M Trinh,Roman A Meza

doi:10.1038/s41467-021-22937-z

Nicholas Chim, John C Chaput + Show 3 more

Open Access

https://doi.org/10.1038/s41467-021-22937-z

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Journal: Nature Communications	Publication Date: May 11, 2021
Citations: 14	License type: open-access

Affiliation: University of California, Irvine

Abstract

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.

Highlights

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
In the solution-catalyzed structure, the templating base is held outside the active site pocket through a stacking interaction with Y719 (Supplementary Fig. 2a), while the in crystallo-catalyzed structure shows the base occupying a well-defined hydrophobic pocket formed between the O and O1 helices of the finger subdomain, as first described by Beese and colleagues (Supplementary Fig. 2b)[17]
In contrast to the canonical model where translocation is thought to be the last step in the catalytic cycle, time-lapsed images capturing the order and transition between intermediates demonstrate that translocation precedes phosphodiester bond formation in the mechanism of DNA synthesis

Summary

Introduction

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. If we assume, based on the position of the templating base, that the solution-catalyzed structure defines an earlier intermediate in the reaction pathway (Supplementary Fig. 2), such as the starting point of the catalytic cycle, and that the in crystallo-catalyzed structure captures a subsequent intermediate, additional steps would still be required for the templating base to enter the active site so that it can form a Watson-Crick base pair with the incoming dNTP substrate.

Results

Conclusion