Exonucleolytic proofreading by calf thymus DNA polymerase delta.

Publisher Website
Google Scholar
Abstract
The fidelity of DNA synthesis by calf thymus DNA polymerase .delta. (pol .delta.) in vitro has been determined using an M13lacZ.alpha. nonsense codon reversion assay. Pol .delta. is highly accurate, producing on average <1 single-base substitution error for each 106 nucleotides polymerized. This accuracy is 10- and 500-fold greater than that of DNA polymerases .alpha. and .beta., respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3'' to 5'' exonuclease associated with pol .delta.. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by pol .delta. are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of pol .delta.. These results suggest that 3'' to 5'' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.