HYDROLYSIS OF 3'-TERMINAL MISPAIRS INVITRO BY THE 3'-]5' EXONUCLEASE OF DNA POLYMERASE-DELTA PERMITS SUBSEQUENT EXTENSION BY DNA POLYMERASE-ALPHA

Purified DNA polymerase α, the major replicating enzyme found in mammalian cells, lacks an associated 3′ → 5′ proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase α cannot remove mispaired 3′-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase α in vivo but dissociates from it during purification. Using this assay, we purified a 3′ → 5′ exonuclease from calf thymus that preferentially hydrolyzes mispaired 3′-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase α. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase α and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase δ. In related studies, we showed that the 3′ → 5′ exonuclease of authentic DNA polymerase δ, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase α. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases α and δ, functioning at the site of DNA replication in mammalian cells. © 1990, American Chemical Society. All rights reserved.