Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment.

Abstract:

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.

Polymerases:

Topics:

Source / Purification, Fidelity, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Eco Pol IV Kobayashi S2002 Cloned or native Native organism
Eco Pol IV Kobayashi S2002 Frameshift Error Rate 0.00021 errors/bp Technique: M13mp2 forward mutation assay
Eco Pol IV Kobayashi S2002 Overall Error Rate 0.03 errors/bp
Eco Pol IV Kobayashi S2002 Overall Error Rate 0.036 errors/bp
Eco Pol IV Kobayashi S2002 Nucleotide Substitution Rate 5.1E-05 errors/bp Technique: M13mp2 forward mutation assay
Eco Pol IV Kobayashi S2002 Full length or truncated Full length
Eco Pol IV Kobayashi S2002 Processivity 50bp
Eco Pol IV Kobayashi S2002 Gap Filling Yes

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