MUTATIONAL ANALYSIS OF THE 3'-]5' PROOFREADING EXONUCLEASE OF ESCHERICHIA-COLI DNA-POLYMERASE-III

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.