Role of the 2-amino group of purines during dNTP polymerization by human DNA polymerase alpha.


We used a series of dNTP analogues in conjunction with templates containing modified bases to elucidate the role that N(2) of a purine plays during dNTP polymerization by human DNA polymerase alpha. Removing N(2) from dGTP had small effects during correct incorporation opposite C but specifically increased misincorporation opposite A. Adding N(2) to dATP and related analogues had small and variable effects on the efficiency of polymerization opposite T. However, the presence of N(2) greatly enhanced polymerization of these dATP analogues opposite a template C. The ability of N(2) to enhance polymerization opposite C likely results from formation of a hydrogen bond between the purine N(2) and pyrimidine O(2). Even in those cases where formation of a wobble base pair, tautomerization, and/or protonation of the base pair between the incoming dNTP and template base cannot occur (e.g., 2-pyridone.purine (or purine analogue) base pairs), N(2) enhanced formation of the base pair. Importantly, N(2) had similar effects on dNTP polymerization both when added to the incoming purine dNTP and when added to the template base being replicated. The mechanistic implications of these results regarding how pol alpha discriminates between right and wrong dNTPs are discussed.




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