Sa. Taftbenz et Rm. Schaaper, MUTATIONAL ANALYSIS OF THE 3'-]5' PROOFREADING EXONUCLEASE OF ESCHERICHIA-COLI DNA-POLYMERASE-III, Nucleic acids research, 26(17), 1998, pp. 4005-4011
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 r
ole within the pol III core, To gain further insight into how epsilon
performs these joint structural and catalytic functions, we have inves
tigated a set of 20 newly isolated dnaQ mutator mutants, The mutator e
ffects ranged from strong (700-8000-fold enhancement) to moderate (6-2
0-fold enhancement), reflecting the range of proofreading deficiencies
. Complementation assays revealed most mutators to be partially or ful
ly 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, wa
s fully recessive. Sequence analysis of the mutants revealed mutations
in the fro I, fro II and recently proposed fro III epsilon. motifs, a
s well as in the intervening regions. Together, the data support the f
unctional significance of the proposed motifs, presumably in catalysis
, and suggest that the C-terminus of epsilon may be specifically invol
ved in binding to the alpha (polymerase) subunit.