Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine

摘要: To test whether ionized base pairs influence polymerase-catalyzed misinsertion rates, we measured the efficiency of forming 5-bromouracil (B), 5-fluorouracil (F), and thymine with guanine adenine as a function pH using avian myeloblastosis reverse transcriptase. When B, F, T were present dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation C increased by about 20-, 13-, 7-fold, respectively, reaction from 7.0 9.5. Incorporation form correct pairs, B.A F.A, T.A, decreased 4- 8-fold, increasing pH. The effects on 10-fold greater when template bases. relative G T, A 430-, 370-, 70-fold, was 6.5 9.5, while B F over same range. Plots depicting incorrect versus fit titration equation giving fraction We conclude that transcriptase forms B.G F.G mispairs in an Watson-Crick conformation preference neutral wobble structure containing favored keto tautomers or F. Although participation disfavored enol enzyme-catalyzed mispair formation cannot be ruled out, results are inconsistent "standard" tautomer model mutagenesis. Instead, data support which ionization halouracil bases is primarily responsible for B- F-induced