Y265H mutator mutant of DNA polymerase beta. Proper teometric alignment is critical for fidelity

DNA polymerases have the unique ability to select a specific deoxynucleoside triphosphate from a pool of similarly structured substrates. One of these enzymes, DNA polymerase beta, offers a simple system to relate polymerase structure to the fidelity of DNA synthesis. In this study, a mutator DNA polymerase beta, Y265H, was identified using an in vivo genetic screen. Purified Y265H produced errors at a 40-fold higher frequency than the wild-type protein in a forward mutation assay. At 37 degrees C, transient kinetic analysis demonstrated that the alteration caused a 111-fold decrease in the maximum rate of polymerization and a 117-fold loss in fidelity for G misincorporation opposite template A. Our data suggest that the maximum rate of polymerization was reduced, because Y265H was dramatically impaired in its ability to perform nucleotidyl transfer in the presence of the correct nucleotide substrate. In contrast, at 20 degrees C, the mutant protein had a fidelity similar to wild-type enzyme. Both proteins at 20 degrees C demonstrate a rapid change in protein conformation, followed by a slow chemical step. These data suggest that proper geometric alignment of template, 3'-OH of the primer, magnesium ions, dNTP substrates, and the active site residues of DNA polymerase beta are important factors in polymerase fidelity and provide the first evidence that Tyr-265 is important for this alignment to occur properly in DNA polymerase beta.     

The E249K mutator mutant of DNA polymerase beta extends mispaired termini

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.     

The Asp285 variant of DNA polymerase beta extends mispaired primer termini via increased nucleotide binding

Endogenous DNA damage occurs at a rate of at least 20,000 lesions per cell per day. Base excision repair (BER) is a key pathway for maintaining genome stability. Several pol beta variants were identified as conferring resistance to 3'-azido-3'-deoxythymidine (AZT) in Escherichia coli (Kosa et al. (1999) J. Biol. Chem. 274, 3851-3858). Detailed biochemical studies on one of these AZT-resistant variants, His285 to Asp, have shown that the H285D variant of pol beta possesses pre-steady-state kinetics that are similar to the wild-type polymerase. In gap filling assays with 5-bp gapped DNA, H285D showed a slight mutator phenotype. In depth single turnover kinetic analysis revealed that H285D is much more efficient than wild-type pol beta at extending mispaired primer termini. This mispair extension property of H285D is attributed to a greatly increased binding to the next correct nucleotide in the presence of a mispair. This change in K d(dNTP),app is not accompanied by a change in k pol; values for k pol are the same for both H285D and wild-type. Close examination of available structural data, as well as molecular modeling, has shown that residue 285 is able to make several stabilizing contacts in the fingers domain of the polymerase, and the introduction of a negatively charged side chain could have important effects on the enzyme. It is postulated that the loss of the contact between His285, Lys289, and Ile323 is responsible for the ability of H285D to extend mispairs through disruption of contacts near the C-terminal end of pol beta and propagation into the nucleotide binding pocket.     

Hydrophobic interactions in the hinge domain of DNA polymerase beta are important but not sufficient for maintaining fidelity of DNA synthesis

We previously described a general mutator form of mammalian DNA polymerase beta containing a cysteine substitution for tyrosine 265. Residue 265 localizes to a hydrophobic hinge region predicted to mediate a polymerase conformational change that may aid in nucleotide selectivity. In this study we tested the hypothesis that van der Waals and hydrophobic contacts between Y265 and neighboring residues are important for DNA synthesis fidelity and catalysis, by altering interactions in the hinge domain via substitution at position 265. Consistent with the importance of hydrophobic interactions, we found that phenylalanine, leucine, and tryptophan substitutions did not alter significantly the steady-state catalytic efficiency of DNA synthesis, relative to wild type, while the polar serine substitution decreased catalytic efficiency 6-fold. However, we found that all substitutions other than phenylalanine increased the error frequency, relative to wild type, in the order serine > tryptophan = leucine. Therefore, maintenance of the hydrophobicity of residue 265 was not sufficient for maintaining fidelity of DNA synthesis. We conclude that while hydrophobic interactions in the hinge domain are important for fidelity, additional factors such as electrostatic and van der Waals interactions contributed by the tyrosine 265 aromatic ring are required to retain wild-type fidelity.     

Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.     

The roles of Tyr391 and Tyr619 in RB69 DNA polymerase replication fidelity

In the family-B DNA polymerase of bacteriophage RB69, the conserved aromatic palm-subdomain residues Tyr391 and Tyr619 interact with the last primer-template base-pair. Tyr619 interacts via a water-mediated hydrogen bond with the phosphate of the terminal primer nucleotide. The main-chain amide of Tyr391 interacts with the corresponding template nucleotide. A hydrogen bond has been postulated between Tyr391 and the hydroxyl group of Tyr567, a residue that plays a key role in base discrimination. This hydrogen bond may be crucial for forcing an infrequent Tyr567 rotamer conformation and, when the bond is removed, may influence fidelity. We investigated the roles of these residues in replication fidelity in vivo employing phage T4 rII reversion assays and an rI forward assay. Tyr391 was replaced by Phe, Met and Ala, and Tyr619 by Phe. The Y391A mutant, reported previously to decrease polymerase affinity for incoming nucleotides, was unable to support DNA replication in vivo, so we used an in vitro fidelity assay. Tyr391F/M replacements affect fidelity only slightly, implying that the bond with Tyr567 is not essential for fidelity. The Y391A enzyme has no mutator phenotype in vitro. The Y619F mutant displays a complex profile of impacts on fidelity but has almost the same mutational spectrum as the parental enzyme. The Y619F mutant displays reduced DNA binding, processivity, and exonuclease activity on single-stranded DNA and double-stranded DNA substrates. The Y619F substitution would disrupt the hydrogen bond network at the primer terminus and may affect the alignment of the 3' primer terminus at the polymerase active site, slowing chemistry and overall DNA synthesis.     

The enhanced DNA replication fidelity of a mutant herpes simplex virus type 1 DNA polymerase is mediated by an improved nucleotide selectivity and reduced mismatch extension ability

We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.     

Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket

Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.     

A DNA polymerase beta mutator mutant with reduced nucleotide discrimination and increased protein stability

DNA polymerase beta (pol beta) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol beta (M282Lbeta) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on alpha-helix N of pol beta. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lbeta has a 7.5-fold higher mutation frequency than wild-type pol beta; M282Lbeta commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lbeta. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lbeta ultimately lead to an overall reduction in polymerase fidelity.     

Rapid segmental and subdomain motions of DNA polymerase beta

DNA polymerase (pol) beta is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between alpha-helix N and the nascent base pair. Kinetic and structural characterization of pol beta has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and alpha-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of alpha-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol beta were determined. The results revealed that the segmental motion of alpha-helix N was rapid ( approximately 1 ns) and far more rapid than the step that limits chemistry. Binding of Mg(2+) and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in alpha-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within alpha-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of alpha-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol beta deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors.     

Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.     

Active site mutation in DNA polymerase gamma associated with progressive external ophthalmoplegia causes error-prone DNA synthesis

Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mtDNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase gamma (pol gamma). Since Tyr-955 is a highly conserved residue critical for nucleotide recognition among family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (k(cat)) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (K(m)). The Y955C derivative is 2-fold less accurate for base pair substitutions than wild-type pol gamma despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10-100-fold. The error-prone DNA synthesis observed for the Y955C pol gamma is consistent with the accumulation of mtDNA mutations in patients with PEO.     

Highly organized but pliant active site of DNA polymerase beta: compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses

To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg(258) is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe(272), Asn(279), and Arg(283)) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg(258)) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.     

Local deformations revealed by dynamics simulations of DNA polymerase Beta with DNA mismatches at the primer terminus

Nanosecond dynamics simulations for DNA polymerase beta (pol beta)/DNA complexes with three mismatched base-pairs, namely GG, CA, or CC (primer/template) at the DNA polymerase active site, are performed to investigate the mechanism of polymerase opening and how the mispairs may affect the DNA extension step; these trajectories are compared to the behavior of a pol beta/DNA complex with the correct GC base-pair, and assessed with the aid of targeted molecular dynamics (TMD) simulations of all systems from the closed to the open enzyme state. DNA polymerase conformational changes (subdomain closing and opening) have been suggested to play a critical role in DNA synthesis fidelity, since these changes are associated with the formation of the substrate-binding pocket for the nascent base-pair. Here we observe different large C-terminal subdomain (thumb) opening motions in the simulations of pol beta with GC versus GG base-pairs. Whereas the conformation of pol beta in the former approaches the observed open state in the crystal structures, the enzyme in the latter does not. Analyses of the motions of active-site protein/DNA residues help explain these differences. Interestingly, rotation of Arg258 toward Asp192, which coordinates both active-site metal ions in the closed "active" complex, occurs rapidly in the GG simulation. We have previously suggested that this rotation is a key slow step in the closed to open transition. TMD simulations also point to a unique pathway for Arg258 rotation in the GG mispair complex. Simulations of the mismatched systems also reveal distorted geometries in the active site of all the mispair complexes examined. The hierarchy of the distortions (GG>CC>CA) parallels the experimentally deduced inability of pol beta to extend these mispairs. Such local distortions would be expected to cause inefficient DNA extension and polymerase dissociation and thereby might lead to proofreading by an extrinsic exonuclease. Thus, our studies on the dynamics of pol beta opening in mismatch systems provide structural and dynamic insights to explain experimental results regarding inefficient DNA extension following misincorporation; these details shed light on how proofreading may be invoked by the abnormal active-site geometry.     

Human DNA polymerase iota promiscuous mismatch extension

Human DNA polymerase iota is a low-fidelity template copier that preferentially catalyzes the incorporation of the wobble base G, rather than the Watson-Crick base A, opposite template T (Tissier, A., McDonald, J. P., Frank, E. G., and Woodgate, R. (2000) Genes Dev. 14, 1642-1650; Johnson, R. E., Washington, M. T., Haracska, L., Prakash, S., and Prakash, L. (2000) Nature 406, 1015-1019; Zhang, Y., Yuan, F., Wu, X., and Wang, Z. (2000) Mol. Cell. Biol. 20, 7099-7108). Here, we report on its ability to extend all 12 possible mispairs and 4 correct pairs in different sequence contexts. Extension from both matched and mismatched primer termini is generally most efficient and accurate when A is the next template base. In contrast, extension occurs less efficiently and accurately when T is the target template base. A striking exception occurs during extension of a G:T mispair, where the enzyme switches specificity, "preferring" to make a correct A:T base pair immediately downstream from an originally favored G:T mispair. Polymerase iota generates a variety of single and tandem mispairs with high frequency, implying that it may act as a strong mutator when copying undamaged DNA templates in vivo. Even so, its limited ability to catalyze extension from a relatively stable primer/template containing a "buried" mismatch suggests that polymerase iota-catalyzed errors are confined to short template regions.     

Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization

The mitochondrial DNA polymerase from embryos of Drosophila melanogaster has been examined with regard to template-primer utilization, processivity, and fidelity of nucleotide polymerization. The enzyme replicates predominantly single-stranded and double-stranded DNAs: the rate of DNA synthesis is greatest on the gapped homopolymeric template poly(dA).oligo(dT), while the highest substrate specificity is observed on single-stranded DNA templates of natural DNA sequence. Kinetic experiments and direct physical analysis of DNA synthetic products indicate that the Drosophila DNA polymerase gamma polymerizes nucleotides by a quasi-processive mechanism. The mitochondrial enzyme demonstrates a high degree of accuracy in nucleotide incorporation which is nearly identical with that of the replicative DNA polymerase alpha from Drosophila embryos. Thus, the catalytic properties of the near-homogeneous Drosophila DNA polymerase gamma are consistent with the in vivo requirements for mitochondrial DNA synthesis as described in a variety of animal systems.     

A thymine isostere in the templating position disrupts assembly of the closed DNA polymerase beta ternary complex

The high fidelity of the DNA polymerization process is critically important for the stability of the cellular genome. The role of template and incoming nucleotide base pairing in polymerase fidelity has recently been explored by the use of nucleotide isosteres, which preserve the steric but not the electronic properties of the corresponding bases. The DNA repair enzyme, DNA polymerase beta (Pol beta), is among the most discriminating, being inactive when the thymine isostere difluorotoluene (DFT) is present in the templating base position. To explore the physical basis for this inactivity, we have performed NMR studies on [methyl-13C]methionine-labeled Pol beta complexed with double-hairpin DNA, used to model the gapped nucleotide substrate, and having either a thymine or a DFT isostere at the templating base position. The six methionine residues distributed throughout the enzyme provide useful conformational probes of the lyase and polymerase domains and subdomains. Analysis of the proton shift of Met282 that results from formation of an abortive Pol beta-gapped DNA-dATP complex is consistent with an open to closed conformational change of the enzyme predicted from crystal structures. In contrast, the same resonance is nearly unshifted when a ternary complex is formed from dATP and gapped DNA in which a DFT isostere replaces thymine at the templating base position. Alternatively, the resonances of Met191 and Met155, located in the catalytic subdomain, show perturbations upon formation of the abortive ternary complex, which are qualitatively similar, but significantly weaker, than the changes observed when thymine is present at the templating base position. The changes in the Met155 and Met191 methyl resonances are in fact more similar to those observed in the binary Pol beta-dATP complex. These studies demonstrate that the block in catalysis is directly related to the absence of the set of conformational transitions that include the "open" to "closed" transition monitored by Met282.