DIFFERENTIAL DISCRIMINATION OF DNA-POLYMERASES FOR VARIANTS OF THE NONSTANDARD NUCLEOBASE PAIR BETWEEN XANTHOSINE AND 2,4-DIAMINOPYRIMIDINE, 2 COMPONENTS OF AN EXPANDED GENETIC ALPHABET

Mammalian DNA polymerases alpha and epsilon, the Klenow fragment of Es cherichia coli DNA polymerase I and HIV-1 reverse transcriptase (RT) w ere examined for their ability to incorporate components of an expande d genetic alphabet in different forms. Experiments were performed with templates containing 2'-deoxyxanthosine (dX) or 2'-deoxy-7-deazaxanth osine (c(7)dX), both able to adopt a hydrogen bonding acceptor-donor-a cceptor pattern on a purine nucleus (puADA). Thus these heterocycles a re able to form a non-standard nucleobase pair with 2,4-diaminopyrimid ine (pyDAD) that fits the Watson-Crick geometry, but is joined by a no n-standard hydrogen bonding pattern. HIV-1 RT incorporated d(pyDAD)TP opposite dX with a high efficiency that was largely independent of pH. Specific incorporation opposite c(7)dX was significantly lower and al so independent of pH. Mammalian DNA polymerases alpha and epsilon from calf thymus and the Klenow fragment from E.coli DNA polymerase I fail ed to incorporate d(pyDAD)TP opposite c(7)dX.