Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine

J Biol Chem. 1993 Jul 25;268(21):15935-43.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Bromodeoxyuridine / chemistry
  • Bromouracil / chemistry*
  • DNA / chemistry*
  • Floxuridine / chemistry
  • Fluorouracil / chemistry*
  • Guanine / chemistry*
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Ions
  • Molecular Sequence Data
  • Nucleic Acid Heteroduplexes*
  • RNA-Directed DNA Polymerase / metabolism
  • Templates, Genetic
  • Thymine Nucleotides / chemistry

Substances

  • Ions
  • Nucleic Acid Heteroduplexes
  • Thymine Nucleotides
  • Floxuridine
  • Bromouracil
  • Guanine
  • DNA
  • RNA-Directed DNA Polymerase
  • Bromodeoxyuridine
  • Fluorouracil