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 lowfidelity benzimidazole-derived bases (i.e., bases that pol α rapidly incorporates opposite all 4 natural bases) and to generate 1-deazapurines significantly increases 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 † This work was supported by grants to RDK from the NIH (GM54194 and TW007372-01) and the Army Research Office (W911NF-05-1-0172), to MH from the Ministry of Education of the Czech Republic (Centre of Biomolecules and Complex Molecular Systems, LC 512), and to JE from the Deutsche Forschungsgemeinschaft (SFB 579).

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo−) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.

The Sulfolobus solfataricus Y-family DNA polymerase Dpo4 is a model for translesion replication and has been used in the analysis of individual steps involved in catalysis. The role of conformational changes has not been clear. Introduction of Trp residues into the Trp-devoid wild-type protein provided fluorescence probes of these events, particularly in the case of mutants T239W and N188W. With both mutants, a rapid increase in Trp fluorescence was observed only in the case of normal base pairing (G:C), was saturable with respect to dCTP concentration, and occurred in the absence of phosphodiester bond formation. A subsequent decrease in the Trp fluorescence occurred when phosphodiester bond formation was permitted, and these rates were independent of the dCTP concentration. This step is relatively slow and is attributed to a conformational relaxation step occurring after pyrophosphate release, which was measured and shown to be fast in a separate experiment. The measured rate of release of DNA from Dpo4 was rapid and is not rate-limiting. Overall, the measurements provide a kinetic scheme for Dpo4 different than generally accepted for replicative polymerases or proposed for Dpo4 and other Y-family polymerases: the initial enzyme⅐DNA⅐dNTP complex undergoes a rapid (18 s ؊1 ), reversible (21 s ؊1 ) conformational change, followed by relatively rapid phosphodiester bond formation (11 s ؊1 ) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2 s ؊1 ) and then rapid DNA release, yielding an overall steady-state k cat of <1 s ؊1 .Replicative DNA polymerases insert dNTPs with high efficiency and fidelity but lose much of this capacity when they encounter DNA lesions that do not closely resemble the four canonical bases (A, T, C, or G), leading to deleterious miscoding and/or blocks to further polymerization (1). In the past decade the slower and less efficient Y-family and other translesion DNA polymerases have been characterized and found to often replace replicative polymerases at these blocked junctions (2). Based on biochemical and structural analysis, a popular consensus is that abnormal substrate geometry forms the basis for this "switch" to a translesion polymerase (3-5). Whereas replicative DNA polymerases use numerous residues to bind tightly to properly shaped substrates to bring about rapid and processive polymerization, Y-family polymerases make fewer proteinsubstrate contacts and allow aberrant shapes within their active sites (6).Whether these two major enzyme classes catalyze polymerization similarly is not yet clear. Generally it is believed that replicative polymerases bind DNA tightly, followed by binding a correct dNTP (sorted from the mixture of all four that the DNA polymerase encounters) that instigates an "induced-fit" conformational change to form an active ternary complex leading to high efficiency and fidelity polymerization, e.g. (7). Subsequent to catalysis, a "relaxing" of the complex and the release of PP i 4 both must occur, follow...