Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.

Abstract

We have identified an amino-proximal sequence motif, Phe-Asp-lle-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, aphidicolinsensitive 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 Il 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 ofE. coiU DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3' -5' proofreading exonuclease activity. None ofthese residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila a DNA polymerases.Class B DNA polymerasest are related through a series of conserved amino acid sequences that occur in the order IV, II, VI, III, I, V, with region I being the most highly conserved (2). Regions II, III, I, and V are implicated in DNA polymerase functions (3)(4)(5)(6)(7)(8). The location of the 3' -* 5' exonuclease activity domain has been sought through amino acid sequence similarity with the apparently unrelated E. coli DNA polymerase I. Crystallographic and mutational evidence has determined that the 3' -*5' exonuclease active site of E. coli DNA polymerase I is composed of a group of carboxylate residues (D355, E357, D424, and D501) clustering around two metal ions that coordinate the 3'-terminal phosphate and a second group of residues (L361, F473, and Y497) located around the terminal base and ribose positions (9, 10). It has been proposed that residues D355 and E357 are conserved in the left part of region IV ofaphidicolin-sensitive DNA polymerases (11-13). Similarly, residues D424, Y497, and D501 were suggested to be conserved in the right part of, and distal to, region IV (4,12,13). Mutation to alanine of three of the relevant carboxylate residues in bacteriophage 029 DNA polymerase leads to exonuclease-deficient DNA polymerase activity (13). Though related to other aphidicolinsensitive DNA polymerases, 429 DNA polymerase does not have a well-conserved region IV (2) and may not be a good test case for alignments involving this region. With coliphage T4 DNA polymerase, which does have a well-conserved region IV (2, 11, 12), mutation to alanine ofresidues D189 and E191, predicted to correspond to the E. coli polymerase residues D355 and E357, does not lead to exonuclease deficiency, calling into question the concept of a generally conserved 3'-* 5' exonuclease active sit...

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

“…28) using a heterologous kanMX cassette as described previously (29). The chromosomal POL2 allele of this strain was replaced with the pol2-4 allele as described previously (11). The pol2-4 mutation results in the FDIET 3 FAIAT amino acid replacement in the Exo I motif of Pol ⑀.…”

“…18). The wild type allele of the POL2 gene in these strains was replaced by the pol2-4 allele as described previously (11). Ura ϩ revertants of these pol2-4 bik1::ura3-24 strains were obtained, yielding pol2-4 mutants with the wild type URA3 gene in two orientations relative to ARS306.…”

“…Both Pol ␦ and Pol ⑀ have intrinsic 3Ј 3 5Ј exonuclease activities that correct polymerase errors during replication (9,10). Mutations in POL3 or POL2 inactivating the exonucleases result in a mutator phenotype (11,12).…”

“…Both Pol ␦ and Pol ⑀ have intrinsic 3Ј 3 5Ј exonuclease activities that correct polymerase errors during replication (9, 10). Mutations in POL3 or POL2 inactivating the exonucleases result in a mutator phenotype (11,12).To accommodate roles for Pol ␦ and Pol ⑀ at a replication fork, it was proposed that Pol ⑀ elongates the leading DNA strand, while Pol ␦ elongates Okazaki fragments on the lagging DNA strand (13). Other models for the roles of Pol ␦ and Pol ⑀ in chromosomal replication have also been considered (14, 15).…”

Self-synthesizing DNA transposons in eukaryotes

“…6, which is published as supporting information on the PNAS web site). These motifs include the Exo I (defined by the DXE consensus), Exo II (Nx 3 F͞YD), and Exo III (Yx 3 D) motifs that constitute a catalytic core of the exonuclease conserved in all proofreading POLBs (21)(22)(23), and the S͞TLx 2 h motif also conserved in the POLB exonuclease domain (24) (see Fig. 6).…”