Skip to main content

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

Journal Article


Download full-text (Open Access)

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.

Publication Date


  • 2013

Citation


  • Ozawa, K., Horan, N. P., Robinson, A., Yagi, H., Hill, F. R., Jergic, S., Xu, Z., Loscha, K. V., Li, N., Tehei, M., Oakley, A. J., Otting, G., Huber, T. & Dixon, N. E. (2013). Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain. Nucleic Acids Research, 41 (10), 5354-5367.

Scopus Eid


  • 2-s2.0-84878571203

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1789&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/776

Has Global Citation Frequency


Number Of Pages


  • 13

Start Page


  • 5354

End Page


  • 5367

Volume


  • 41

Issue


  • 10

Place Of Publication


  • United Kingdom

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.

Publication Date


  • 2013

Citation


  • Ozawa, K., Horan, N. P., Robinson, A., Yagi, H., Hill, F. R., Jergic, S., Xu, Z., Loscha, K. V., Li, N., Tehei, M., Oakley, A. J., Otting, G., Huber, T. & Dixon, N. E. (2013). Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain. Nucleic Acids Research, 41 (10), 5354-5367.

Scopus Eid


  • 2-s2.0-84878571203

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=1789&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/776

Has Global Citation Frequency


Number Of Pages


  • 13

Start Page


  • 5354

End Page


  • 5367

Volume


  • 41

Issue


  • 10

Place Of Publication


  • United Kingdom