Recognition of base-pairing by DNA polymerases during nucleotide incorporation: the properties of the mutagenic nucleotide dPTPαS
2003
The highly mutagenic nucleoside dP (6-(2-deoxy-s-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 dPTPαS 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.
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