Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine.
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 base pairs with guanine and adenine as a function of pH using avian myeloblastosis reverse transcriptase. When B, F, and T were present as dNTP substrates, misincorporation efficiencies opposite G, normalized to incorporation of C opposite G, increased by about 20-, 13-, and 7-fold, respectively, as reaction pH increased from 7.0 to 9.5. Incorporation efficiencies to form the correct base pairs, B.A and F.A, normalized to T.A, decreased by 4- and 8-fold, respectively, with increasing pH. The effects of pH on misincorporation efficiencies were about 10-fold greater when B, F, and T were present as template bases. The relative misincorporation efficiencies of G opposite template B, F, and T, normalized to incorporation of A opposite B, F, and T, increased by about 430-, 370-, and 70-fold, respectively, as pH was increased from 6.5 to 9.5, while correct incorporation of A opposite template B and F decreased about 10-fold over the same pH range. Plots depicting incorrect and correct incorporation efficiencies versus pH were fit to a pH titration equation giving the fraction of ionized base as a function of pH. We conclude that avian myeloblastosis reverse transcriptase forms B.G and F.G mispairs in an ionized Watson-Crick conformation in preference to a neutral wobble structure containing favored keto tautomers of B or F. Although participation of disfavored enol tautomers in enzyme-catalyzed base mispair formation cannot be ruled out, the results are inconsistent with the "standard" disfavored tautomer model of mutagenesis. Instead, the data support a model in which ionization of halouracil bases is primarily responsible for B- and F-induced mutagenesis.
Yu
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Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340.
Eritja
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R
Bloom
L B
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Goodman
M F
MF
D001483:Base Sequence; D001973:Bromodeoxyuridine / Q000737:chemistry; D001976:Bromouracil / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D005467:Floxuridine / Q000737:chemistry; D005472:Fluorouracil / Q000737:chemistry*; D006147:Guanine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000737:chemistry
D007477:Ions; D009692:Nucleic Acid Heteroduplexes; D013942:Thymine Nucleotides; D005467:Floxuridine; D001976:Bromouracil; D006147:Guanine; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D001973:Bromodeoxyuridine; D005472:Fluorouracil