Fidelity and Error Specificity of the α Catalytic Subunit of Escherichia coli DNA Polymerase III

• Published in: The Journal of biological chemistry Vol. 271; no. 31; pp. 18947 - 18953
• Main Authors: Mo, Jin-Yao, Schaaper, Roel M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.08.1996
• Subjects: Bacterial Proteins - genetics; Base Sequence; DNA Polymerase III - chemistry; DNA Polymerase III - genetics; DNA Polymerase III - metabolism; DNA Replication; DNA, Bacterial - biosynthesis; DNA, Bacterial - genetics; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins; Lac Operon; Lac Repressors; Molecular Sequence Data; Mutation; Protein Conformation; Repressor Proteins - genetics; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.31.18947

Escherichia coli DNA polymerase III holoenzyme is the replicative enzyme primarily responsible for the duplication of the E. coli 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 first step in understanding the mechanisms responsible for the high fidelity of this process, we have purified the polymerase III α catalytic subunit, free of exonuclease activity, and analyzed its fidelity in vitro. We employed a newly developed gap-filling assay using the N-terminal 250 bases of the lacI gene as a forward mutational target. When synthesizing across this target, α 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 with a pyrimidine 5′-neighbor. These results suggest that the α subunit (i) has a relatively low ability to extend from misincorporated bases, accounting for the low level of observed base substitutions, and (ii) has a relatively high capability of extension after misalignment of a misincorporated base on the next (complementary) template base, accounting for the high level of frameshift mutations. This model is supported by an experiment in which α subunit was required to initiate DNA synthesis from a terminal mispair in a sequence context that allowed slippage on the next template base. Among the products of this reaction, frameshifts outnumbered base pair substitutions by greater than 70-fold. A comparison to in vivo mutational spectra suggests that the pol III accessory factors may play a major role in modulating the fidelity of DNA synthesis.