Nucleic Acids Research, Vol 26, Issue 17 4005-4011, Copyright © 1998 by Oxford University Press
SA Taft-Benz and RM Schaaper
The epsilon 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 epsilon 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 IIIepsilon 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 straightepsilon may be
specifically involved in binding to the alpha (polymerase) subunit.
ARTICLES
Mutational analysis of the 3'-->5' proofreading exonuclease of Escherichia coli DNA polymerase III
Laboratory of Molecular Genetics, National Institute of Environmental Health Science, PO Box 12233,Research Triangle Park, NC 27709, USA.
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