Hydrolysis of 3'-terminal Mispairs in Vitro by the 3'----5' Exonuclease of DNA Polymerase Delta Permits Subsequent Extension by DNA Polymerase Alpha

Biochemistry. 1990 Jun 5;29(22):5226-31. doi: 10.1021/bi00474a002.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cattle
  • DNA Polymerase II / metabolism*
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism*
  • Exonucleases / isolation & purification
  • Exonucleases / metabolism*
  • Genetic Techniques
  • Thymus Gland / enzymology

Substances

  • DNA Polymerase II
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase
  • Exonucleases