Fidelity and error specificity of the alpha catalytic subunit of Escherichia coli DNA polymerase III.

Abstract:

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 alpha 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, 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 with a pyrimidine 5'-neighbor. These results suggest that the alpha 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 alpha 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.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Fidelity, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol alpha Mo JY1996 3-5' Exonuclease (proofreading) No
Human Pol alpha Mo JY1996 Cloned or native Cloned in E. coli
Human Pol alpha Mo JY1996 Frameshift Error Rate 0.00031 errors/bp Technique: Reversion
Human Pol alpha Mo JY1996 Frameshift Error Rate 4E-05 errors/bp Technique: Forward mutational
Human Pol alpha Mo JY1996 Overall Error Rate 0.003 errors/bp Technique: M13mp2 forward mutation assay
Human Pol alpha Mo JY1996 Overall Error Rate 5E-05 errors/bp Technique: Forward mutational
Human Pol alpha Mo JY1996 Nucleotide Substitution Rate 1.3E-06 errors/bp Technique: Reversion
Human Pol alpha Mo JY1996 Nucleotide Substitution Rate 1.1E-05 errors/bp Technique: Forward mutational
Human Pol alpha Mo JY1996 Full length or truncated Full length
Human Pol alpha Mo JY1996 Specific Activity 4.5E+06 units/mg
Human Pol alpha Mo JY1996 Gap Filling Yes

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