title: Hydrolysis of 3'-terminal mispairs in vitro by the 3'----5' exonuclease of DNA polymerase delta permits subsequent extension by DNA polymerase alpha.
abstract: Purified DNA polymerase alpha, 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 alpha 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 alpha 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 alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells.
authors.affiliation: Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
authors.forename: F W
authors.initials: FW
authors.lastname: Perrino
authors.forename: L A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005821:Genetic Techniques; D013950:Thymus Gland / Q000201:enzymology
chemical_list: D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases