Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits ?, epsilon, ? and ? reveals a highly flexible arrangement of the proofreading domain.

ABSTRACT: A complex of the three (???) core subunits and the ?2 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 ? (polymerase) and the C-terminal segment of ? (proofreading exonuclease) subunits shows that ? is attached to ? at a site far from the polymerase active site. Both ? and ? contain clamp-binding motifs (CBMs) that interact simultaneously with ?2 in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable ???:?2 complexes. Nuclear magnetic resonance experiments with reconstituted ???:?2 demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ? with its ?-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 ?-PHP domain confirm the conformational variability of the tether. Structural models of the ???:?2 replicase complex with primer-template DNA combine all available structural data.

SUBMITTER: Ozawa K 

PROVIDER: S-EPMC3664792 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature