FIDELITY AND ERROR SPECIFICITY OF THE ALPHA-CATALYTIC SUBUNIT OF ESCHERICHIA-COLI DNA-POLYMERASE-III

Escherichia coil DNA polymerase III holoenzyme is the replicative enzy me primarily responsible for the duplication of the E. coil chromosome . This process occurs with high accuracy, less than 10(-9) to 10(-10) errors being committed per base pair per round of replication. As a fi rst step in understanding the mechanisms responsible for the high fide lity of this process, we have purified the polymerase III alpha cataly tic subunit, free of exonuclease activity, and analyzed its fidelity i n vitro. We employed a newly developed gap-filling assay using the N-t erminal 250 bases of the loci gene as a forward mutational target. Whe n synthesizing across this target, alpha subunit produced mutations at a frequency of 0.6%. DNA sequencing revealed that the mutants created in vitro consisted mostly of frameshift mutations, although some base substitutions were also observed, The frameshifts, occurring at more than 120-fold above the background, consisted largely of -1 deletions. Among them, about 80% were the deletion of a purine template base wit h a pyrimidine 5'-neighbor, These results suggest that the alpha subun it (i) has a relatively low ability to extend from misincorporated bas es, accounting for the low level of observed base substitutions, and ( ii) has a relatively high capability of extension after misalignment o f a misincorporated base on the next (complementary) template base, ac counting for the high level of frameshift mutations. This model is sup ported by an experiment in which alpha subunit was required to initiat e DNA synthesis from a terminal mispair in a sequence context that all owed slippage on the next template base. Among the products of this re action, frameshifts outnumbered base pair substitutions by greater tha n 70-fold. A comparison to in vivo mutational spectra suggests that th e pol III accessory factors may play a major role in modulating the fi delity of DNA synthesis.