A structural role for the PHP domain in E. coli DNA polymerase III

Barros, Tiago; Guenther, Joel; Kelch, Brian; Anaya, Jordan; Prabhakar, Arjun; O’Donnell, Mike; Kuriyan, John; Lamers, Meindert H.

doi:10.1186/1472-6807-13-8

A structural role for the PHP domain in E. coli DNA polymerase III

Journal Article · Tue May 14 00:00:00 EDT 2013 · BMC Structural Biology (Online)

DOI:https://doi.org/10.1186/1472-6807-13-8· OSTI ID:1626547

Barros, Tiago ^[1]; Guenther, Joel ^[2]; Kelch, Brian ^[2]; Anaya, Jordan ^[2]; Prabhakar, Arjun ^[2]; O’Donnell, Mike ^[3]; Kuriyan, John ^[4]; Lamers, Meindert H. ^[2]

Univ. of California, Berkeley, CA (United States). Dept. of Chemistry. Dept. of Molecular and Cell Biology. Howard Hughes Medical Inst.; DOE/OSTI
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry. Dept. of Molecular and Cell Biology. Howard Hughes Medical Inst.
Rockefeller Univ., New York, NY (United States). Howard Hughes Medical Inst. Lab. of DNA Replication
Univ. of California, Berkeley, CA (United States). Dept. of Chemistry. Dept. of Molecular and Cell Biology. Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Accelerator & Fusion Research Division. Physical Biosciences Division

Background: In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Results: Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. Conclusions: While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional nonenzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1626547

Journal Information:: BMC Structural Biology (Online), Journal Name: BMC Structural Biology (Online) Journal Issue: 1 Vol. 13; ISSN 1472-6807

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Biophysics
DNA polymerase III
DNA replication
PHP domain
Proofreading exonuclease

A structural role for the PHP domain in E. coli DNA polymerase III

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References (50)

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