A THYMIDINE TRIPHOSPHATE SHAPE ANALOG LACKING WATSON-CRICK PAIRING ABILITY IS REPLICATED WITH HIGH SEQUENCE SELECTIVITY

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with th ymidine, has been proposed as a probe for the importance of hydrogen b onds in biological systems, Consistent with its lack of strong H-bond donors or accepters, F is shown here by thermal denaturation studies t o pair very poorly and with no significant selectivity among natural b ases in DNA oligonucleotides. We report the synthesis of the 5'-tripho sphate derivative of 1 and the study of its ability to be inserted int o replicating DNA strands by the Klenow fragment (KF, exo(-) mutant) o f Escherichia coli DNA polymerase I, We find that this nucleotide deri vative (dFTP) is a surprisingly good substrate for KF; steady-state me asurements indicate it is inserted into a template opposite adenine wi th efficiency (V-max/K-m) only 40-fold lower than dTTP, Moreover, it i s inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed fur dTTP, Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A i n the inverted A-F pair is not, Combined with data from studies with F in the template strand, the results show that KF can efficiently repl icate a base pair (A-F/F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity, The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believe d and that nucleotide/template shape complementarity may play a more i mportant role than previously believed.