NEBULARINE (9-2'-DEOXY-BETA-D-RIBOFURANOSYLPURINE) HAS THE TEMPLATE CHARACTERISTICS OF ADENINE IN-VIVO AND IN-VITRO

Nebularine (9-beta-D-ribofuranosylpurine; Nb) is a naturally occurring nucleoside with structural features suggestive of a universal base. H owever, previous observations based on thermal melting characteristics of oligonucleotides suggested that Nb formed stable pairs only with t hymine. To determine the template characteristics of Nb, we constructe d M13 viral single-stranded DNA (ssDNA) molecules bearing a single sit e-specific deoxynebularine (9-2'-deoxy-beta-D-ribofuranosylpurine) res idue. The ssDNA constructs were transfected into Escherichia coli cell s to determine the specificity of base insertion opposite Nb, as well as to determine the effect of Nb on the replicability of the transfect ed DNA. Base insertion opposite Nb, analyzed by a multiplex sequencing technology, suggests that Nb has the template characteristics of aden ine. Analysis of DNA replicability, measured as transfection efficienc y, indicates that Nb does not block DNA replication. UV irradiation of host cells before transfection did not significantly affect survival or base insertion specificity within the limits of multiplex sequencin g technology employed, suggesting that inducible mutagenic phenomena a ppear to have only minor effects on translesion synthesis across Nb. I n addition, in vitro DNA elongation experiments on oligonucleotide tem plates using E. coli DNA polymerase I (Klenow fragment) as the model p olymerase showed that Nb templates for T, but not for other bases unde r the tested conditions. The data reported in this communication under score the importance of base-pair geometry as a specificity-determinan t during base insertion by replicative polymerases. (C) 1997 Elsevier Science B.V.