N-6-methoxyadenine-pyrimidine base pairs as substrates for the mismatch repair system of Escherichia coli

The availability of nucleoside analogues with ambiguous base-pairing proper ties would be of considerable value in molecular biology. We have incorpora ted deoxynebularine [9-(2-deoxy-beta -D-ribofuranosyl)purine, P], deoxyinos ine [9-(2-deoxy-beta -D-ribofuranosyl)-6-hydroxypurine, I] and [9-(2-deoxy- beta -D-ribofuranosyl)-6-methoxyaminopurine, (MeO)A] into hexadecamer oligo deoxyribonucleotides and tested their behaviour in DNA-DNA hybridisations i n vitro, as well as in oligonucleotide-directed mutagenesis experiments in vivo. The results showed that P behaved as an adenine analogue in all assay s. Oligonucleotide duplexes containing I/C or I/T base pairs displayed simi lar thermal stabilities in DNA-DNA hybridisation experiments, however, duri ng DNA synthesis in vitro and in vivo, hypoxanthine behaved strictly as a g uanine analogue. Only (MeO)A was truly ambiguous in all assays. The H-1 NMR spectrum of the nucleoside demonstrated the existence of two distinct taut omeric forms in a ratio of ca 8 : 2, implying that the base might pair with both C and T. Indeed, within the context of synthetic hexadecamer duplexes , (MeO)A/C and (MeO)A/T pairs brought about a similar thermal destabilisati on, with the former base pair being only marginally less favoured. When use d as hybridisation probes on single-stranded M13 DNA, the (MeO)A-containing hexadecamer oligonucleotides were shown to bind with similar efficiencies to target sequences containing either C or T opposite the analogue. Interes tingly, when (MeO)A is in the template strand during DNA replication, the p olymerase III holoenzyme of E. coli reads it predominantly as a G, which in dicates that (MeO)A exists in B-DNA mostly as the anti-imino tautomer.