Varied molecular interactions at the active sites of several DNA polymerases: Nonpolar nucleoside isosteres as probes

We describe a survey of protein-DNA interactions with seven different DNA p olymerases and reverse transcriptases, carried out with nonpolar nucleoside isosteres F (a thymidine analog) and Z and Q (deoxyadenosine analogues). P revious results have shown that Z and F can be efficiently replicated oppos ite each other by the exonuclease-free Klenow fragment of DNA polymerase I from Escherichia coli (KF-), although both of them lack Watson-Crick II-bon ding ability. We find that exonuclease-inactive T7 DNA polymerase (T7(-)), Thermus aquaticus DNA polymerase (Taq), and HIV-reverse transcriptase (HIV- RT) synthesize the nonnatural base pairs A-F, F-A, F-Z, and Z-F with high e fficiency, similarly to KF-. Steady-state kinetics were also measured for T 7(-) and the efficiency of insertion is very similar to that of KF-; intere stingly, the replication selectivity with this pair is higher for T7- than KF-, possibly due to a tighter active site. A second group comprised of cal f thymus DNA polymerase ct (Pol cc) and avian myeloblastosis virus reverse transcriptase (AMV-RT) was able to replicate the A-F and F-A base pairs to some extent but not the F-Z and the Z-F base pairs. Most of the insertion w as recovered when Z was replaced by the nucleoside Q (9-methyl-1-H-imidazo[ (4,5)-b]pyridine), which is analogous to Z but possesses a minor groove acc eptor nitrogen. This strongly supports the existence of an energetically im portant hydrogen-bonded interaction between the polymerase and the minor gr oove at the incipient base pair for these enzymes. A third group, formed by human DNA polymerase beta (Pol beta) and Moloney murine leukemia virus rev erse transcriptase (MMLV-RT), failed to replicate the F-Z and Z-F base pair s. No insertion recovery was observed when Z was replaced by Q, possibly in dicating that hydrogen bonds are needed at both the template and the tripho sphate sites. The results point out the importance of DNA minor groove inte ractions at the incipient base pair for the activity of some polymerases, a nd demonstrate the variation in these interactions from enzyme to enzyme.