Recognition of base-pairing by DNA polymerases during nucleotide incorporation: the properties of the mutagenic nucleotide dPTP alphaS.
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.
Harris
Victoria H
VH
Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield, UK S3 7HF.
Smith
Clifford L
CL
Cummins
W Jonathan
WJ
Hamilton
Alan L
AL
Hornby
David P
DP
Williams
David M
DM
D020029:Base Pairing; D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005453:Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism
D017931:DNA Primers; D003854:Deoxyribonucleotides; D011743:Pyrimidines; D013873:Thionucleotides; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III