A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode
Horan, Nicholas P.
Mason, Claire E.
Robinson, Andrew J.
Goudsmits, Joris M H
Beck, Jennifer L.
Van Oijen, Antoine M.
Huber, Thomas L.
Dixon, Nicholas E.
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Permanent link to this recordhttp://hdl.handle.net/10754/562660
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AbstractProcessive DNA synthesis by the αÉ"θ core of the Escherichia coli Pol III replicase requires it to be bound to the β 2 clamp via a site in the α polymerase subunit. How the É" proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of É". Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in É" that, in conjunction with the site in α, maintains a closed state of the αÉ"θ-β 2 replicase in the polymerization mode of DNA synthesis. The É"-β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states. © 2013 European Molecular Biology Organization.
SponsorsWe thank Michelle Blayney and Linda Jessop for preliminary ESI-MS data. This work was supported by grants from the Australian Research Council, including Fellowships to KO, TH, and NED, and by a KAUST Faculty Initiated Collaborative grant to SMH and NED.
PubMed Central IDPMC3642676
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