title: Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, epsilon, θ and β reveals a highly flexible arrangement of the proofreading domain.
abstract: A complex of the three (αεθ) core subunits and the β2 sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β2 complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β2 replicase complex with primer-template DNA combine all available structural data.
authors.affiliation: School of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Horan
authors.forename: Andrew
authors.initials: A
authors.lastname: Robinson
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yagi
authors.forename: Flynn R
authors.initials: FR
authors.lastname: Hill
authors.forename: Slobodan
authors.initials: S
authors.lastname: Jergic
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Xu
authors.forename: Karin V
authors.initials: KV
authors.lastname: Loscha
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.forename: Moeava
authors.initials: M
authors.lastname: Tehei
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Oakley
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
mesh_terms: D000595:Amino Acid Sequence; D004258:DNA Polymerase III / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; C074904:DNA polymerase III, alpha subunit; C101139:beta subunit, DNA polymerase III; C505238:holE protein, E coli; D004258:DNA Polymerase III; C468316:dnaQ protein, E coli; D005090:Exodeoxyribonucleases