| Abstract
| - The mechanism by which purine−purine mispairs are formed and extended was examinedwith the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreadingexonuclease activity inactivated. The structures of the purine−purine mispairs were examined by comparingthe kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanineat four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminalbase pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotideis in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, thekpol/Kd values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G weredecreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispairformation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the synconformation and the template base in the anti conformation. During mispair extension, the only decreasein kpol/Kd was associated with the G/G base pair in which 7-deazaguanine was in the template strand.These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647−5659] in which ahydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 duringG/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is inthe anti conformation during mispair extension. These results suggest that purine−purine mispairs areformed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is inthe anti conformation. During extension, however, the conformation of the primer terminus changes to ananti configuration while the template base may be in either the syn or anti conformations.
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