title: DNA polymerase 3'→5' exonuclease activity: Different roles of the beta hairpin structure in family-B DNA polymerases.
abstract: Proofreading by the bacteriophage T4 and RB69 DNA polymerases requires a β hairpin structure that resides in the exonuclease domain. Genetic, biochemical and structural studies demonstrate that the phage β hairpin acts as a wedge to separate the primer-end from the template strand in exonuclease complexes. Single amino acid substitutions in the tip of the hairpin or deletion of the hairpin prevent proofreading and create "mutator" DNA polymerases. There is little known, however, about the function of similar hairpin structures in other family B DNA polymerases. We present mutational analysis of the yeast (Saccharomyces cerevisiae) DNA polymerase δ hairpin. Deletion of the DNA polymerase δ hairpin (hpΔ) did not significantly reduce DNA replication fidelity; thus, the β hairpin structure in yeast DNA polymerase δ is not essential for proofreading. However, replication efficiency was reduced as indicated by a slow growth phenotype. In contrast, the G447D amino acid substitution in the tip of the hairpin increased frameshift mutations and sensitivity to hydroxyurea (HU). A chimeric yeast DNA polymerase δ was constructed in which the T4 DNA polymerase hairpin (T4hp) replaced the yeast DNA polymerase δ hairpin; a strong increase in frameshift mutations was observed and the mutant strain was sensitive to HU and to the pyrophosphate analog, phosphonoacetic acid (PAA). But all phenotypes - slow growth, HU-sensitivity, PAA-sensitivity, and reduced fidelity, were observed only in the absence of mismatch repair (MMR), which implicates a role for MMR in mediating DNA polymerase δ replication problems. In comparison, another family B DNA polymerase, DNA polymerase ɛ, has only an atrophied hairpin with no apparent function. Thus, while family B DNA polymerases share conserved motifs and general structural features, the β hairpin has evolved to meet specific needs.
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
authors.forename: Hariyanto
authors.initials: H
authors.lastname: Darmawan
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
authors.forename: Melissa
authors.initials: M
authors.lastname: Harrison
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Electronic address: Linda.Reha-Krantz@ualberta.ca.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Reha-Krantz
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D053843:DNA Mismatch Repair; D004252:DNA Mutational Analysis; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D004271:DNA, Fungal; C491232:POL3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases
keywords: DNA polymerase evolution; DNA polymerase mutations in cancer; DNA polymerase proofreading; Hydroxyurea sensitivity; Mismatch repair and checkpoint response; Phosphonoacetic acid sensitivity