We have utilized an electrophoretic assay of misincorporation to investigate the possibility that ionization of 5-bramouracil (BU) may play a role in its mispairing during DNA synthesis in vitro. We examined the effects of increasing pH on the relative rates of formation of BU•G and T.G mispairs during chain elongation catalyzed by various DNA polymerases. For the Klenow fragment of Escherichia coli DNA polymerase I, increasing pH facilitated BU•G mispair formation (relative to T.G mispairing) when BU was present in the template strand. This effect showed a strong dependence on sequence context. Increasing pH had little effect on the relative rate of misincorporation of BrdUMP versus dTMP (at template G) by the Klenow polymerase. Misincorporation opposite template BU residues catalyzed by Maloney murine leukemia virus DNA polymerase and DNA polymerase β (Novikoff hepatoma) also increased with pH, but for these two enzymes, there was no apparent dependence on sequence context. With T4 DNA polymerase and E. coli DNA polymerase III holoenzyme, a similar occurrence of BU.G and T.G mispairing during polymerization was observed, whether BU was present in the template or in the incoming nucleotide, and there was little effect of pH. The results reported here are consistent with a mispairing mechanism for template BU wherein the anionic form of the base mispairs with G.
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