DNA polymerases accurately replicate DNA by incorporating mostly correct dNTPs opposite any given template base. We have identified the chemical features of purine dNTPs that human pol α uses to discriminate between right and wrong dNTPs. Removing N-3 from guanine and adenine, two high-fidelity bases, significantly lowers fidelity. Analogously, adding the equivalent of N-3 to low-fidelity benzimidazole-derived bases (i.e., bases that pol α rapidly incorporates opposite all four natural bases) and to generate 1-deazapurines significantly strengthens the ability of pol α to identify the resulting 1-deazapurines as wrong. Adding the equivalent of the purine N-1 to benzimidazole or to 1-deazapurines significantly decreases the rate at which pol α polymerizes the resulting bases opposite A, C, and G while simultaneously enhancing polymerization opposite T. Conversely, adding the equivalent of adenine's C-6 exocyclic amine (N-6) to 1- and 3-deazapurines also enhances polymerization opposite T but does not significantly decrease polymerization opposite A, C, and G. Importantly, if the newly inserted bases lack N-1 and N-6, pol α does not efficiently polymerize the next correct dNTP, whereas if it lacks N-3, one additional nucleotide is added and then chain termination ensues. These data indicate that pol α uses two orthogonal screens to maximize its fidelity. During dNTP polymerization, it uses a combination of negative (N-1 and N-3) and positive (N-1 and N-6) selectivity to differentiate between right and wrong dNTPs, while the shape of the base pair is essentially irrelevant. Then, to determine whether to add further dNTPs onto the just added nucleotide, pol α appears to monitor the shape of the base pair at the primer 3′-terminus. The biological implications of these results are discussed.
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