THE BASE SUBSTITUTION AND FRAMESHIFT FIDELITY OF ESCHERICHIA-COLI DNA-POLYMERASE III HOLOENZYME IN-VITRO

We have investigated the in vitro fidelity of Escherichia coil DNA pol ymerase III holoenzyme from a wildtype and a proofreading-impaired mut D5 strain. Exonuclease assays showed the mutD5 holoenzyme to have a 30 -50-fold reduced 3'-->5'-exonuclease activity. Fidelity was assayed du ring gap-filling synthesis across the lacI(d) forward mutational targe t. The error rate for both enzymes was lowest at low dNTP concentratio ns (10-50 mu M) and highest at high dNTP concentration (1000 mu M) The mutD5 proofreading defect increased the error rate by only 3-5-fold. Both enzymes produced a high level of (-1)-frameshift mutations in add ition to base substitutions. The base substitutions were mainly C-->T, G-->T, and G-->C, but cNTP pool imbalances suggested that these may r eflect misincorporations opposite damaged template bases and that, ins tead, T-->C, G-->A, and C-->T transitions represent the normal polymer ase III-mediated base base mispairs. The frequent (-1)-frameshift muta tions do not result from direct slippage but may be generated via a me chanism involving ''misincorporation plus slippage.'' Measurements of the fidelity of wild-type and mutD5 holoenzyme during M13 in vivo repl ication revealed significant differences between the in vivo and in vi tro fidelity with regard to both the frequency of frameshift errors an d the extent of proofreading.