Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.
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'----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 alpha DNA polymerases.
Morrison
A
A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Bell
J B
JB
Kunkel
T A
TA
Sugino
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004257:DNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D004257:DNA Polymerase II; D005092:Exonucleases