Recognition of base-pairing by DNA polymerases during nucleotide incorporation: the properties of the mutagenic nucleotide dPTP alphaS

Org Biomol Chem. 2003 Jun 21;1(12):2070-4. doi: 10.1039/b302011h.


The highly mutagenic nucleoside dP (6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) is a bicyclic analogue of N4-methoxy-2'-deoxycytidine. It exists as a mixture of its imino and amino tautomers in solution with a ratio of about 10:1 based on its tautomeric constant. The bicyclic nature of the heterocycle P restrains the amino substituent in an anti conformation and permits effective Watson-Crick base-pairing using either tautomer. The specificity of incorporation of dP by the 3'-5'-exonuclease-free Klenow fragment of DNA polymerase I (exo-free Klenow) has been studied using the 5'-(1-thio)triphosphate dPTP alphaS in combination with phosphorothioate-specific sequencing of the DNA products. The method provides a convenient qualitative assay for studying nucleotide incorporation and reveals for the first time a potential role for the minor tautomeric forms of the natural DNA bases in base misinsertion (substitution mutagenesis) during replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Pairing
  • Base Sequence
  • DNA Polymerase I / metabolism*
  • DNA Primers / chemistry
  • DNA Replication
  • Deoxyribonucleotides / chemistry
  • Deoxyribonucleotides / metabolism
  • Exodeoxyribonucleases / metabolism
  • Fluorescence
  • Molecular Sequence Data
  • Mutagenesis
  • Nucleic Acid Conformation
  • Pyrimidines / chemistry*
  • Pyrimidines / metabolism
  • Thionucleotides / chemistry*
  • Thionucleotides / metabolism


  • DNA Primers
  • Deoxyribonucleotides
  • Pyrimidines
  • Thionucleotides
  • DNA Polymerase I
  • Exodeoxyribonucleases
  • exodeoxyribonuclease III