title: The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis.
abstract: The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
authors.forename: Victoria H
authors.initials: VH
authors.lastname: Harris
authors.forename: Clifford L
authors.initials: CL
authors.lastname: Smith
authors.forename: W
authors.initials: W
authors.lastname: Jonathan Cummins
authors.forename: Alan L
authors.initials: AL
authors.lastname: Hamilton
authors.forename: Harry
authors.initials: H
authors.lastname: Adams
authors.forename: Mark
authors.initials: M
authors.lastname: Dickman
authors.forename: David P
authors.initials: DP
authors.lastname: Hornby
authors.forename: David M
authors.initials: DM
authors.lastname: Williams
mesh_terms: D000225:Adenine / Q000737:chemistry; D020029:Base Pairing; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D003853:Deoxyribonucleosides / Q000737:chemistry*; D003854:Deoxyribonucleotides / Q000737:chemistry; D006147:Guanine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry*; D010078:Oxazines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*
chemical_list: C115248:2'-deoxy-ribofuranosyl-(3,4-dihydro-6H,8H-pyrimido(4,5-c)(1,2)oxazin-7-one); C097986:6-(2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido(4,5-c)(1,2)oxazin-7-one 5'-triphosphate; D017931:DNA Primers; D003853:Deoxyribonucleosides; D003854:Deoxyribonucleotides; D009692:Nucleic Acid Heteroduplexes; D010078:Oxazines; D011743:Pyrimidines; C062254:6H,8H-3,4-dihydropyrimido(4,5-c)(1,2)oxazin-7-one; D006147:Guanine; D004247:DNA; D004256:DNA Polymerase I; D000225:Adenine