PMID:15352874
| Citation |
Lehtinen, DA and Perrino, FW (2004) Dysfunctional proofreading in the Escherichia coli DNA polymerase III core. Biochem. J. 384:337-48 |
|---|---|
| Abstract |
The epsilon-subunit contains the catalytic site for the 3'-->5' proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the alpha-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the epsilon mutants were purified, and these proteins exhibited 3'-->5' exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type epsilon protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 epsilon mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified epsilon mutants with the alpha- and theta;-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active epsilon511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The epsilon511 mutant associated tightly with the alpha-subunit, but the exonuclease activity of epsilon511 was not stimulated in the alpha-epsilon511 complex. Addition of the theta;-subunit to generate the alpha-epsilon511-theta; DNA pol III core partially restored stimulation of the epsilon511 exonuclease, indicating a role for the theta;-subunit in co-ordinating the alpha-epsilon polymerase-exonuclease interaction. The alpha-epsilon511-theta; DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the alpha-epsilon511-theta; complex. Thus the epsilon511 mutant has wild-type 3'-->5' exonuclease activity and associates physically with the alpha- and theta;-subunits to generate a proofreading-defective DNA pol III enzyme. |
| Links |
PubMed PMC1134117 Online version:10.1042/BJ20040660 |
| Keywords |
Alleles; Catalytic Domain/physiology; Computer Simulation; DNA Polymerase III/genetics; DNA Polymerase III/metabolism; DNA Polymerase III/physiology; Escherichia coli K12/enzymology; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Escherichia coli Proteins/physiology; Exonucleases/genetics; Exonucleases/metabolism; Exonucleases/physiology; Multienzyme Complexes/genetics; Multienzyme Complexes/metabolism; Multienzyme Complexes/physiology; Mutagenesis/genetics; Mutagenesis/physiology; Mutation/genetics; Mutation/physiology; Protein Subunits/genetics; Protein Subunits/metabolism |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.