Human DNA polymerase alpha uses a combination of positive and negative selectivity to polymerize purine dNTPs with high fidelity

Abstract

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 alpha 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 alpha rapidly incorporates opposite all four natural bases) and to generate 1-deazapurines significantly strengthens the ability of pol alpha 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 alpha 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 alpha 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 alpha 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 alpha appears to monitor the shape of the base pair at the primer 3'-terminus. The biological implications of these results are discussed.

Figure 1

The structures, names, and numbering schemes of the dNTPs discussed. a: Generic structure of benzimidazole and purine 2′-deoxyribofuranosyl-5′-triphosphates, including numbering schemes for the two aromatic systems. b: Analog and natural dNTPs discussed.

Figure 2

The four primer-template sequences used in this study to determine incorporation rates by human pol α. The template base immediately opposite the incoming nucleotide is in bold.

Figure 3

Effect of adding N-3 to the low fidelity bases benzimidazole (dBTP) and 4-methylbenzimidazole (dZTP) to generate 1-deazapurine (d1DPTP) and 1-deaza-6-methylpurine (dQTP). The open bars in the graph are the average value by which pol α discriminates against polymerization of each analogue opposite A, C, T, and G relative to polymerization of a correct dNTP.

Figure 4

Adding N-1 to benzimidazole (dBTP) or 1-deaza-6-methylpurine (dQTP) increases polymerization opposite T and decreases polymerization opposite A, C, and G. The open bars in the graph are the average value by which pol α discriminates against polymerization of each analogue opposite A, C, and G relative to polymerization of a correct dNTP, while the black bars show how much slower pol α polymerizes each analogue opposite T as compared to dATP.

Figure 5

Generating d6F₃PTP by adding N-1 and N-3 to d4F₃BTP decreases polymerization opposite A, C, and G while maintaining polymerization opposite T. The open bars in the graph are the average value by which pol α discriminates against polymerization of each analogue opposite A, C, and G relative to polymerization of a correct dNTP, while the black bars show how much slower pol α polymerizes each analogue opposite T as compared to dATP.

Figure 6

Effect of adding the equivalent of adenine’s exocyclic amine at C-6 (N-6). The open bars in the graph are the average value by which pol α discriminates against polymerization of each analogue opposite A, C, and G relative to polymerization of a correct dNTP, while the black bars show how much slower pol α polymerizes each analogue opposite T as compared to dATP.

Figure 7

Read-through comparison of the analogues by human pol α. Assays contained 1 nM PrimerX-template (where X represents the analogue at the primer 3′-terminus), 0.5 nM pol α, and 25 μM of the noted dNTPs.

Figure 8

Plot of base hydrophobicity (logP) versus fidelity of base-dNTP opposite templates A, C, and G (The average of the three discrimination numbers, taken from Table 2).

Figure 9

Shown is a representation of an incoming dATP and a template T inside the active site of RB69 (adapted from the data in PDB 1IG9). We replaced the dTTP:A base-pair with a dATP:T base-pair while maintaining the location of the sugar-phosphates of each nucleotide. Only selected residues are shown for simplicity.