Pt. Pham et al., THE BASE SUBSTITUTION AND FRAMESHIFT FIDELITY OF ESCHERICHIA-COLI DNA-POLYMERASE III HOLOENZYME IN-VITRO, The Journal of biological chemistry, 273(36), 1998, pp. 23575-23584
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.