title: A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode.
abstract: Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the β2 clamp via a site in the α polymerase subunit. How the ε 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 ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ-β2 replicase in the polymerization mode of DNA synthesis. The ε-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.
authors: School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia.
authors: Slobodan
authors: S
authors: Jergic
authors: Nicholas P
authors: NP
authors: Horan
authors: Mohamed M
authors: MM
authors: Elshenawy
authors: Claire E
authors: CE
authors: Mason
authors: Thitima
authors: T
authors: Urathamakul
authors: Kiyoshi
authors: K
authors: Ozawa
authors: Andrew
authors: A
authors: Robinson
authors: Joris M H
authors: JM
authors: Goudsmits
authors: Yao
authors: Y
authors: Wang
authors: Xuefeng
authors: X
authors: Pan
authors: Jennifer L
authors: JL
authors: Beck
authors: Antoine M
authors: AM
authors: van Oijen
authors: Thomas
authors: T
authors: Huber
authors: Samir M
authors: SM
authors: Hamdan
authors: Nicholas E
authors: NE
authors: Dixon
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004258:DNA Polymerase III / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid
chemical_list: D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; C074904:DNA polymerase III, alpha subunit; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III; C468316:dnaQ protein, E coli