Autonomous 3 '-> 5 ' exonucleases can proofread for DNA polymerase beta from rat liver

Autonomous 3' --> 5' exonucleases are not bound covalently to DNA polymeras es but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28 +/- 2 k Da) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the cours e of reduplication of the primed DNA of bacteriophage (phi X174 amber 3 in vitro. The extent of correction increases together with the rise in 3' --> 5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase sug gests that exonucleolytic proofreading could augment the accuracy of DNA sy nthesis by two orders of magnitude. These results are not explained by exon ucleolytic degradation of the primers ("no synthesis-no errors"), since sim ilar data are obtained with the use of the primers 15 or 150 nucleotides lo ng in the course of a fidelity assay of DNA polymerases, both alpha and bet a, in the presence of various concentrations of 3' --> 5' exonuclease. (C) 2000 Elsevier Science B.V. All rights reserved.