title: A structural role for the PHP domain in E. coli DNA polymerase III.
abstract: 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 non-enzymatic 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Tiago
authors.initials: T
authors.lastname: Barros
authors.forename: Joel
authors.initials: J
authors.lastname: Guenther
authors.forename: Brian
authors.initials: B
authors.lastname: Kelch
authors.forename: Jordan
authors.initials: J
authors.lastname: Anaya
authors.forename: Arjun
authors.initials: A
authors.lastname: Prabhakar
authors.forename: Mike
authors.initials: M
authors.lastname: O'Donnell
authors.forename: John
authors.initials: J
authors.lastname: Kuriyan
authors.forename: Meindert H
authors.initials: MH
authors.lastname: Lamers
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; D008670:Metals; D004258:DNA Polymerase III