Mismatched and Matched dNTP Incorporation by DNA Polymerase β Proceed via Analogous Kinetic Pathways

Polymerase fidelity, at its most basic level, is determined by the free energy difference between the highest energy barriers along the correct and incorrect dNTP1 incorporation pathways. Since fidelity requires such a comparison, an ever-increasing gap between the structural and mechanistic information available for matched dNTP incorporation compared with that concerning mismatched dNTP incorporation renders the molecular mechanism of DNA polymerase fidelity unclear (1,2). The thermodynamic instability inherent to polymerase mismatched ternary complexes makes them difficult to crystallize. For this reason, we do not yet have available a structure representing a truly functional prechemistry closed mismatched ternary complex (E′DnN, Scheme 1), although complexes depicting mispairs within the active site (3,4) and mismatch extension (5,6) for various polymerases have been resolved. Likewise, kinetic data for mismatched dNTP incorporations by many DNA polymerases have been reported (7−13), yet commonly used kinetic approaches prevent characterization of individual steps in the mismatched dNTP incorporation pathway. Scheme 1 The Ile260 residue of Pol β is located in the hydrophobic hinge region between the palm and the C-terminal fingers subdomain (14,15). Several Pol β variants with alterations in residues lining this hinge region have demonstrated compromised fidelity (16−20). The mutator activity of I260Q was first identified by a genetic screen (21), and subsequent kinetic characterization showed that while this mutant possessed parameters of DNA binding similar to that of wild-type, it interestingly showed loose binding discrimination of mismatched dNTP substrates, and thus a low fidelity (22). These characteristics make the I260Q mutant of specific interest in fidelity studies of Pol β, in order to further understand how one mutation can so drastically alter the mismatch discrimination profile of this enzyme. Stopped-flow analyses of Pol β, using either 2-aminopurine (2-AP), a fluorescent analogue of adenine and guanine, or Pol βʼs sole Trp325 residue, have been very important in advancing Pol β mechanism studies (23−26). Both probes serve as independent reporters of global enzyme−DNA conformational changes which occur during the catalysis of dNTP incorporation into DNA. While monitoring the fluorescence emission of either 2-AP or Trp325 in stopped-flow, the resultant trace observed during correct dNTP incorporation possesses two phases of fluorescence change. Intricate studies involving a variety of chemical probes have been conducted in order to determine the nature of these two phases. The collective findings of these studies support a model in which the fast fluorescence transition is attributed to dNTP-induced subdomain conformational change (step 2, Scheme 1) to form the ternary closed complex prior to chemistry (step 4, Scheme 1), and the slow fluorescence transition is attributed to subdomain reopening (step 6, Scheme 1) after the rate-limiting chemical step. This work aims to apply our previously established stopped-flow fluorescence methods, as well as steady-state fluorescence spectroscopy to explore two specific aspects of polymerase fidelity: (i) the mechanism by which WT Pol β discriminates against incorrect dNTP substrate to maintain a moderately high fidelity, and (ii) the deviation of this mechanism in generation of a lower fidelity variant by a single I260Q mutation. Such topics are significant in developing a full understanding of the unique means by which DNA polymerases are able to choose a correct dNTP substrate, whose identity is ever-changing with each round of catalysis, from a pool four nucleotides. Moreover, while it is interesting to comprehend how a polymerase accomplishes this feat to achieve a certain fidelity in DNA replication, it is equally interesting to investigate how a single residue mutation can alter its fidelity. Here, we focus on the kinetic pathway of misincorporation, while another recent paper using small-angle X-ray scattering (SAXS) and molecular modeling lends complementary insights into structural aspects of misincorporation (27). The results of our analyses suggest that Pol β incorporates both correct and mismatched dNTP via analogous kinetic mechanisms differing at the transition state of the chemical step. Furthermore, the divergence in fidelity between WT and I260Q is consequent of differences in ability to stabilize the mismatched ternary complex and chemical transition state.