PROOFREADING 3'-]5' EXONUCLEASES ISOLATED FROM RAT-LIVER NUCLEI

Mammalian nuclear DNA polymerases alpha and beta are known to be devoi d of the editing 3' --> 5' exonucleolytic activity. Presumably this ac tivity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3' --> 5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-50) have been isolated from rat liver nuclei and puri fied to near homogeneity. They are shown to excise mismatched nucleoti des from poly[d(A-T)] template, respectively, 10-fold and 2-fold faste r than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fideli ty of in-vitro reproduction of the primed DNA from bacteriophage phiX1 74 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polym erase activities being similar. Extrapolation of in-vitro DNA-replicat ion fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading au gments the accuracy of DNA synthesis by 2-3 orders of magnitude.