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

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Bacterial replicative DNA polymerases possess a Polymerase and Histidinol Phosphatase (PHP) domain of unclear function.
• Some bacterial PHP domains act as metal-dependent nucleases for proofreading, but E. coli Pol III's PHP domain lacks key residues for activity.

Purpose of the Study:

• To investigate the function and structural role of the PHP domain in E. coli DNA polymerase III.
• To explore the evolutionary relationship between PHP domain metal-binding loss and proofreading exonuclease presence.

Main Methods:

• Genomic searches to identify coevolutionary patterns.
• Site-directed mutagenesis to restore metal binding in the PHP domain.
• X-ray crystallography to determine the structure of the metal-bound mutant.
• Protein unfolding studies to assess polymerase stability and activity.

Main Results:

• Genomic data suggests PHP domain metal-binding loss coevolved with separate proofreading exonucleases.
• The E. coli Pol III PHP domain structure is highly conserved despite lacking metal-coordinating residues.
• Restoration of metal binding was achieved with three point mutations, confirmed by crystal structure.
• Mutations in the PHP domain's degenerate metal-binding site reduced overall Pol III stability and activity.

Conclusions:

• The conserved PHP domain in bacterial replicative polymerases has variable metal-binding and proofreading capabilities, implying non-enzymatic functions.
• The PHP domain is a significant structural component of Pol III, influencing its stability and catalytic activity.