Eukaryotic DNA Polymerase Amino Acid Sequence Required for 3' → 5' Exonuclease Activity

• Published in: Proceedings of the National Academy of Sciences - PNAS 1991-11, Vol.88 (21), p.9473-9477
• Main Authors: Morrison, Alan, Bell, Juliette B., Kunkel, Thomas A., Sugino, Akio
• Format: Article
• Language: English
• Subjects: Active sites; Adenoviruses; Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; DNA; DNA Mutational Analysis; DNA Polymerase II - chemistry; DNA Polymerase II - metabolism; Enzymes; Enzymes and enzyme inhibitors; Exonucleases - chemistry; Exonucleases - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Fungal; Genetic mutation; Molecular Sequence Data; Oligonucleotides - chemistry; Plasmids; Polymerase Chain Reaction; Regional identity; Saccharomyces cerevisiae - enzymology; Sequence Alignment; Structure-Activity Relationship; Transferases; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.21.9473

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3' → 5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3' → 5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3' rightarrow 5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3' → 5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila α DNA polymerases.