Mutational analysis of the 3′→5′ proofreading exonuclease of Escherichia coli DNA polymerase III

The ε subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3′→5′ exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core....

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Bibliographic Details
Published in:Nucleic acids research 1998-09, Vol.26 (17), p.4005-4011
Main Authors: Taft-Benz, Sharon A., Schaaper, Roel M.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Codon, Terminator
Conserved Sequence
DNA Mutational Analysis
DNA Polymerase III - genetics
DNA Replication - genetics
DNA, Bacterial - biosynthesis
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli Proteins
Exodeoxyribonuclease V
Exodeoxyribonucleases - genetics
Genetic Complementation Test
Mutagenesis - genetics
Sequence Analysis, DNA
Sequence Homology, Amino Acid
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Summary:The ε subunit of Escherichia coli DNA polymerase III holoenzyme, the enzyme primarily responsible for the duplication of the bacterial chromosome, is a 3′→5′ exonuclease that functions as a proofreader for polymerase errors. In addition, it plays an important structural role within the pol III core. To gain further insight into how ε performs these joint structural and catalytic functions, we have investigated a set of 20 newly isolated dnaQ mutator mutants. The mutator effects ranged from strong (700–8000-fold enhancement) to moderate (6–20-fold enhancement), reflecting the range of proofreading deficiencies. Complementation assays revealed most mutators to be partially or fully dominant, suggesting that they carried an exonucleolytic defect but retained binding to the pol III core subunits. One allele, containing a stop codon 3 amino acids from the C-terminal end of the protein, was fully recessive. Sequence analysis of the mutants revealed mutations in the Exo I, Exo II and recently proposed Exo IIIε motifs, as well as in the intervening regions. Together, the data support the functional significance of the proposed motifs, presumably in catalysis, and suggest that the C-terminus of ε may be specifically involved in binding to the α (polymerase) subunit.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/26.17.4005