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 f...Continue Reading
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