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It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase activity, but without follow-up studies. Here we report that the 3'-esterase activity is intrinsic to the Thermococcus sp . 9°N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3'-ester with a fluorophore. We first show that 3'-esterified dNTP can be incorporated into a template-primer DNA, and solve the crystal structures of the reaction intermediates and products. Then we show that the reaction can occur continuously, modulated by active site residues Tyr409 and Asp542. Finally, we use 5'-FAM-labeled primer and esterified dNTP with a dye to show that the reaction can proceed to ca. 450 base pairs, and that the intermediates of many individual steps can be identified. The results demonstrate the feasibility of a 3'-editing based DNA sequencing method that could find practical applications after further optimization., Competing Interests: Competing interestsThe authors declare no competing interests.

Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving "prebinding" of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol μ is a unique member of the X-family with multiple functions and with unusual Mn 2+ preference. Here we report that Pol μ not only prebinds MdNTP in a catalytically active conformation but also exerts a Mn 2+ over Mg 2+ preference at this early stage of catalysis, for various functions: incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the nonhomologous end joining (NHEJ) repair, incorporation of dNTP to an ssDNA, and incorporation of an 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn 2+ over Mg 2+ preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol μ. Overall, this work provides the structural and mechanistic basis for the long-standing puzzle in the Mn 2+ preference of Pol μ and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

DNA polymerases are required for DNA replication and DNA repair in all of the living organisms. Different DNA polymerases are responsible different stages of DNA metabolism, and many of them are multifunctional enzymes. It was generally assumed that the different reactions are catalyzed by the same enzyme molecule. In addition to 1:1 DNA polymerase.DNA complex reported by crystallization studies, 2:1 and higher order DNA polymerase.DNA complexes have been identified in solution studies by various biochemical and biophysical approaches. Further, abundant evidences for the DNA polymerase-DNA interactions in several DNA polymerases suggested that the 2:1 complex represents the more active form. This review describes the current status of this emerging subject and explores their potential in vitro and in vivo functional significance, particularly for the 2:1 complexes of mammalian DNA polymerase beta (Pol beta), the Klenow fragment of E. coli DNA polymerase I (KF), and T4 DNA polymerase., ((c) 2008 Wiley-Liss, Inc.)

A sequential ordered substrate binding established previously for several DNA polymerases is generally extended to all DNA polymerases, and the characterization of novel polymerases is often based on the assumption that the enzymes should productively bind DNA substrate first, followed by template-directed dNTP binding. The comprehensive kinetic study of DNA polymerase X (Pol X) from African swine fever virus reported here is the first analysis of the substrate binding order performed for a low-fidelity DNA polymerase. A classical steady-state kinetic approach using substrate analogue inhibition assays demonstrates that Pol X does not follow the bi-bi ordered mechanism established for other DNA polymerases. Further, using isotope-trapping experiments and stopped-flow fluorescence assays, we show that Pol X can bind Mg (2+).dNTPs in a productive manner in the absence of DNA substrate. We also show that DNA binding to Pol X, although rapid, may not always be productive. Furthermore, we show that binding of Mg (2+).dNTP to Pol X facilitates subsequent formation of the catalytically competent Pol X.DNA.dNTP ternary complex, whereas DNA binding prior to dNTP binding brings the enzyme into a nonproductive conformation where subsequent nucleotide substrate binding is hindered. Together, our results suggest that Pol X prefers an ordered sequential mechanism with Mg (2+).dNTP as the first substrate.

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol beta-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol beta-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5'-dRP lyase domain of the second Pol beta molecule with the active site of the 1 : 1 Pol beta-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5'-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol beta-DNA complex enhances the function of Pol beta.

After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.

The structural specificity that translesion DNA polymerases often show for a particular class of lesions suggests that the predominant criterion of selection during their evolution has been the capacity for lesion tolerance and that the error-proneness they display when copying undamaged templates may simply be a byproduct of this adaptation. Regardless of selection criteria/evolutionary history, at present both of these properties coexist in these enzymes, and both properties confer a fitness advantage. The repair polymerase, Pol X, encoded by the African swine fever virus (ASFV) is one of the most error-prone polymerases known, leading us to previously hypothesize that it may work in tandem with the exceptionally error-tolerant ASFV DNA ligase to effect viral mutagenesis. Here, for the first time, we test whether the error-proneness of Pol X is coupled with a capacity for lesion tolerance by examining its ability to utilize the types of damaged DNA and dNTP substrates that are expected to be relevant to ASFV. We (i) test Pol X's ability to both incorporate opposite to and extend from ubiquitous oxidative purine (7,8-dihydro-8-oxoguanine), oxidative pyrimidine (5,6-dihydroxy-5,6-dihydrothymine), and noncoding (AP site) lesions, in addition to 5,6-dihydrothymine, (ii) determine the catalytic efficiency and dNTP specificity of Pol X when catalyzing incorporation opposite to, and when extending from, 7,8-dihydro-8-oxoguanine in a template/primer context, and (iii) quantitate Pol X-catalyzed incorporation of the damaged nucleotide 8-oxo-dGTP opposite to undamaged templates in the context of both template/primer and a single-nucleotide gap. Our findings are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.

We previously demonstrated that the DNA repair system encoded by the African swine fever virus (ASFV) is both extremely error-prone during the single-nucleotide gap-filling step (catalyzed by ASFV DNA polymerase X) and extremely error-tolerant during the nick-sealing step (catalyzed by ASFV DNA ligase). On the basis of these findings we have suggested that at least some of the diversity known to exist among ASFV isolates may be a consequence of mutagenic DNA repair, wherein damaged nucleotides are replaced with undamaged but incorrect nucleotides by Pol X and the resultant mismatched nicks are sealed by ASFV DNA ligase. Recently, this hypothesis appeared to be discredited by Salas and co-workers [(2003) J. Mol. Biol. 326, 1403-1412], who reported the fidelity of Pol X to be, on average, 2 orders of magnitude higher than what we previously published. In an effort to address this discrepancy and provide a definitive conclusion about the fidelity of Pol X, herein we examine the fidelity of Pol X-catalyzed single-nucleotide gap-filling in both the steady state and the pre-steady state under a diverse array of assay conditions (varying pH and ionic strength) and within different DNA sequence contexts. These studies corroborate our previously published data (demonstrating the low fidelity of Pol X to be independent of assay condition/sequence context), do not reproduce the data of Salas et al., and therefore confirm Pol X to be one of the most error-prone polymerases known. These results are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.

Presented here is the development a semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. The utility of structure-based combinatorial protein engineering (SCOPE) was demonstrated by its application to two distantly related members of the X-family of DNA polymerases: rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (Pol X). These proteins share similar folds but have low sequence identity, and differ greatly in both size and activity. "Equivalent" subdomain elements of structure were designed on the basis of the tertiary structure of Pol beta and the corresponding regions of Pol X were inferred from homology modeling and sequence alignment analysis. Libraries of chimeric genes with up to five crossovers were synthesized in a series of PCR reactions by employing hybrid oligonucleotides that code for variable connections between structural elements. Genetic complementation in Escherichia coli enabled identification of several novel DNA polymerases with enhanced phenotypes. Both the composition of structural elements and the manner in which they were linked were shown to be essential for this property, indicating the importance of these aspects of design.