Importance of terminal base pair hydrogen-bonding in 3 '-end proofreading by the Klenow fragment of DNA polymerase I

We describe studies aimed at evaluating the physical factors governing the rate of 3'-end proofreading by the Klenow fragment of E. coli DNA polymeras e I. Two nonpolar deoxynucleoside isosteres containing 2,4-difluorotoluene (F) and 4-methylbenzimidazole (Z), which are non-hydrogen-bonding shape mim ics of thymine and adenine, respectively, are used to investigate the effec ts of base pair geometry and stability on the rate of this exonuclease acti vity. Steady-state kinetics measurements show that complementary T.A base p airs at the end of a primer-template duplex are edited 14-40-fold more slow ly than mismatches. By contrast, a 3'-end T residue in a T.Z pair is edited at a rate equivalent to that of natural base mismatches despite the fact t hat it resembles a T.A pair in structure. Similarly, the A in an A.F pair i s edited as rapidly as a mismatched pair despite its close structural mimic ry of an A.T pair. Interestingly, when the base pairs are reversed and F or Z is located at the 3'-end, they are edited more slowly, possibly implicat ing specific interactions between the exonuclease domain and the base of th e nucleotide being edited, Finally, thermal denaturation studies are carrie d out to investigate the relationship between editing and the ease of unwin ding of the duplex. The rapid editing of bases opposite F or Z residues at the duplex terminus seems to correlate well with the stability of these bas e pairs when placed in a context resembling a primer-template duplex. In ge neral, the rate of 3'-end editing appears to be governed by the rate of fra ying of the DNA terminal pair, and base pair geometry appears to have littl e effect.