Mismatch extension by Escherichia coli DNA polymerase III holoenzyme.


The in vitro fidelity of Escherichia coli DNA polymerase III ...
The in vitro fidelity of Escherichia coli DNA polymerase III holoenzyme (HE) is characterized by an unusual propensity for generating (-1)-frameshift mutations. Here we have examined the capability of HE isolated from both a wild-type and a proofreading-impaired mutD5 strain to polymerize from M13mp2 DNA primer-templates containing a terminal T(template).C mismatch. These substrates contained either an A or a G as the next (5') template base. The assay allows distinction between: (i) direct extension of the terminal C (producing a base substitution), (ii) exonucleolytic removal of the C, or (iii), for the G-containing template, extension after misalignment of the C on the next template G (producing a (-1)-frameshift). On the A-containing substrate, both HEs did not extend the terminal C (<1%); instead, they exonucleolytically removed it (>99%). In contrast, on the G-containing substrate, the MutD5 HE yielded 61% (-1)-frameshifts and 6% base substitutions. The wild-type HE mostly excised the mispaired C from this substrate before extension (98%), but among the 2% mutants, (-1)-frameshifts exceeded base substitutions by 20 to 1. The preference of polymerase III HE for misalignment extension over direct mismatch extension provides a basis for explaining the in vitro (-1)-frameshift specificity of polymerase III HE.




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