Mismatch Extension by Escherichia coli DNA Polymerase III Holoenzyme

• Published in: The Journal of biological chemistry 1999-02, Vol.274 (6), p.3705-3710
• Main Authors: Pham, Phuong T., Olson, Matthew W., McHenry, Charles S., Schaaper, Roel M.
• Format: Article
• Language: English
• Subjects: Bacteriophage M13 - genetics; Base Pair Mismatch; DNA Polymerase III - metabolism; Escherichia coli; Frameshift Mutation; Holoenzymes - metabolism; Templates, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.6.3705

The in vitro fidelity ofEscherichia 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 (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.