Watching right and wrong nucleotide insertion captures hidden polymerase fidelity checkpoints.

Efficient and accurate DNA synthesis is enabled by DNA polymerase fidelity checkpoints that promote insertion of the right instead of wrong nucleotide. Erroneous X-family polymerase (pol) λ nucleotide insertion leads to genomic instability in double strand break and base-excision repair. Here, time-lapse crystallography captures intermediate catalytic states of pol λ undergoing right and wrong natural nucleotide insertion. The revealed nucleotide sensing mechanism responds to base pair geometry through active site deformation to regulate global polymerase-substrate complex alignment in support of distinct optimal (right) or suboptimal (wrong) reaction pathways. An induced fit during wrong but not right insertion, and associated metal, substrate, side chain and pyrophosphate reaction dynamics modulated nucleotide insertion. A third active site metal hastened right but not wrong insertion and was not essential for DNA synthesis. The previously hidden fidelity checkpoints uncovered reveal fundamental strategies of polymerase DNA repair synthesis in genomic instability.

Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair.

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) µ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol µ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol µ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol µ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide.

Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase µ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

Preferential DNA Polymerase ß Reverse Reaction with Imidodiphosphate.

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase ß chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pß and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.

DNA polymerase ß nucleotide-stabilized template misalignment fidelity depends on local sequence context.

DNA polymerase ß has two DNA-binding domains that interact with the opposite sides of short DNA gaps. These domains contribute two activities that modify the 5' and 3' margins of gapped DNA during base excision repair. DNA gaps greater than 1 nucleotide (nt) pose an architectural and logistical problem for the two domains to interact with their respective DNA termini. Here, crystallographic and kinetic analyses of 2-nt gap-filling DNA synthesis revealed that the fidelity of DNA synthesis depends on local sequence context. This was due to template dynamics that altered which of the two template nucleotides in the gap served as the coding nucleotide. We observed that, when a purine nucleotide was in the first coding position, DNA synthesis fidelity was similar to that observed with a 1-nt gap. However, when the initial templating nucleotide was a pyrimidine, fidelity was decreased. If the first templating nucleotide was a cytidine, there was a significantly higher probability that the downstream template nucleotide coded for the incoming nucleotide. This dNTP-stabilized misalignment reduced base substitution and frameshift deletion fidelities. A crystal structure of a binary DNA product complex revealed that the cytidine in the first templating site was in an extrahelical position, permitting the downstream template nucleotide to occupy the coding position. These results indicate that DNA polymerase ß can induce a strain in the DNA that modulates the position of the coding nucleotide and thereby impacts the identity of the incoming nucleotide. Our findings demonstrate that "correct" DNA synthesis can result in errors when template dynamics induce coding ambiguity.

Molecular basis for the faithful replication of 5-methylcytosine and its oxidized forms by DNA polymerase ß.

DNA methylation is an epigenetic mark that regulates gene expression in mammals. One method of methylation removal is through ten-eleven translocation-catalyzed oxidation and the base excision repair pathway. The iterative oxidation of 5-methylcytosine catalyzed by ten-eleven translocation enzymes produces three oxidized forms of cytosine: 5-hydroxmethylcytosine, 5-formylcytosine, and 5-carboxycytosine. The effect these modifications have on the efficiency and fidelity of the base excision repair pathway during the repair of opposing base damage, and in particular DNA polymerization, remains to be elucidated. Using kinetic assays, we show that the catalytic efficiency for the incorporation of dGTP catalyzed by human DNA polymerase ß is not affected when 5-methylcytosine, 5-hydroxmethylcytosine, and 5-formylcytosine are in the DNA template. In contrast, the catalytic efficiency of dGTP insertion decreases ∼20-fold when 5-carboxycytosine is in the templating position, as compared with unmodified cytosine. However, DNA polymerase fidelity is unaltered when these modifications are in the templating position. Structural analysis reveals that the methyl, hydroxymethyl, and formyl modifications are easily accommodated within the polymerase active site. However, to accommodate the carboxyl modification, the phosphate backbone on the templating nucleotide shifts ∼2.5 Å to avoid a potential steric/repulsive clash. This altered conformation is stabilized by lysine 280, which makes a direct interaction with the carboxyl modification and the phosphate backbone of the templating strand. This work provides the molecular basis for the accommodation of epigenetic base modifications in a polymerase active site and suggests that these modifications are not mutagenically copied during base excision repair.

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, ß-C-Fapy•dGTP, with DNA polymerase ß. The crystallographic snapshots and kinetic data indicate that binding of ß-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions ß-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of ß-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase ß has evolved to hinder Fapy•dGTP insertion.

Time-lapse crystallography snapshots of a double-strand break repair polymerase in action.

DNA polymerase (pol) µ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol µ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PPi. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol µ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) µ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.

Modulating the DNA polymerase ß reaction equilibrium to dissect the reverse reaction.

DNA polymerases catalyze efficient and high-fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, that removes the DNA primer terminus and generates deoxynucleoside triphosphates. Because pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase ß and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that this reaction was limited by a nonchemical step. Use of a pyrophosphate analog, in which the bridging oxygen is replaced with an imido group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect, indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium favoring the reverse reaction. These results highlight the importance of the bridging atom between the ß- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium.

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse.

DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is "balanced," as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3' of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase ß active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol ß, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

Substrate-induced DNA polymerase ß activation.

DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase ß, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain.

Substrate rescue of DNA polymerase ß containing a catastrophic L22P mutation.

DNA polymerase (pol) ß is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol ß variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol ß containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol ß and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca(2+) and a complementary dNTP indicates that the fraction of pol ß(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol ß indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol ß provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the "inactive" variant interferes with DNA repair.

Steady-state, pre-steady-state, and single-turnover kinetic measurement for DNA glycosylase activity.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k(off)). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k(off)). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Observing a DNA polymerase choose right from wrong.

DNA polymerase (pol) ß is a model polymerase involved in gap-filling DNA synthesis utilizing two metals to facilitate nucleotidyl transfer. Previous structural studies have trapped catalytic intermediates by utilizing substrate analogs (dideoxy-terminated primer or nonhydrolysable incoming nucleotide). To identify additional intermediates during catalysis, we now employ natural substrates (correct and incorrect nucleotides) and follow product formation in real time with 15 different crystal structures. We are able to observe molecular adjustments at the active site that hasten correct nucleotide insertion and deter incorrect insertion not appreciated previously. A third metal binding site is transiently formed during correct, but not incorrect, nucleotide insertion. Additionally, long incubations indicate that pyrophosphate more easily dissociates after incorrect, compared to correct, nucleotide insertion. This appears to be coupled to subdomain repositioning that is required for catalytic activation/deactivation. The structures provide insights into a fundamental chemical reaction that impacts polymerase fidelity and genome stability.

Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase ß (pol ß) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol ß. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,ß)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol ß, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol ß binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

Amino acid substitution in the active site of DNA polymerase ß explains the energy barrier of the nucleotidyl transfer reaction.

DNA polymerase ß (pol ß) is a bifunctional enzyme widely studied for its roles in base excision DNA repair, where one key function is gap-filling DNA synthesis. In spite of significant progress in recent years, the atomic level mechanism of the DNA synthesis reaction has remained poorly understood. Based on crystal structures of pol ß in complex with its substrates and theoretical considerations of amino acids and metals in the active site, we have proposed that a nearby carboxylate group of Asp256 enables the reaction by accepting a proton from the primer O3'group, thus activating O3'as the nucleophile in the reaction path. Here, we tested this proposal by altering the side chain of Asp256 to Glu and then exploring the impact of this conservative change on the reaction. The D256E enzyme is more than 1000-fold less active than the wild-type enzyme, and the crystal structures are subtly different in the active sites of the D256E and wild-type enzymes. Theoretical analysis of DNA synthesis by the D256E enzyme shows that the O3'proton still transfers to the nearby carboxylate of residue 256. However, the electrostatic stabilization and location of the O3' proton transfer during the reaction path are dramatically altered compared with wild-type. Surprisingly, this is due to repositioning of the Arg254 side chain in the Glu256 enzyme active site, such that Arg254 is not in position to stabilize the proton transfer from O3'. The theoretical results with the wild-type enzyme indicate an early charge reorganization associated with the O3' proton transfer, and this does not occur in the D256E enzyme. The charge reorganization is mediated by the catalytic magnesium ion in the active site.

Effect of ß,γ-CHF- and ß,γ-CHCl-dGTP halogen atom stereochemistry on the transition state of DNA polymerase ß.

Recently, we synthesized the first individual ß,γ-CHX-dGTP diastereomers [(R)- or (S)-CHX, where X is F or Cl] and determined their structures in ternary complexes with DNA polymerase ß (pol ß). We now report stereospecificity by pol ß on the mixed ß,γ-CHX diastereomer pairs using nuclear magnetic resonance and on the separate diastereomers using transient kinetics. For both the F and Cl diastereomers, the R isomer is favored over the S isomer for G·C correct incorporation, with stereospecificities [(k(pol)/K(d))(R)/(k(pol)/K(d))(S)] of 3.8 and 6.3, respectively, and also for G·T misincorporation, with stereospecificities of 11 and 7.8, respectively. Stereopreference for the (R)-CHF-dGTP diastereomer was abolished for k(pol) but not K(d) with mutant pol ß (R183A). These compounds constitute a new class of stereochemical probes for active site interactions involving halogen atoms. As Arg183 is unique in family X pols, the design of CXY deoxyribonucleotide analogues to enhance interaction is a possible strategy for inhibiting BER selectively in cancer cells.

Stereospecific formation of a ternary complex of (S)-α,ß-fluoromethylene-dATP with DNA pol ß.

The influence of water: crystallization of (R/S)-α,ß-CHF-dATP with the preorganized pol ß-DNA complex shows that (S)-α,ß-CHF-dATP is preferentially bound to the active site with the C=F fluorine proximal to a structural water bound to Asp276.

Binary complex crystal structure of DNA polymerase ß reveals multiple conformations of the templating 8-oxoguanine lesion.

Oxidation of genomic DNA forms the guanine lesion 7,8-dihydro-8-oxoguanine (8-oxoG). When in the template base position during DNA synthesis the 8-oxoG lesion has dual coding potential by virtue of its anti- and syn-conformations, base pairing with cytosine and adenine, respectively. This impacts mutagenesis, because insertion of adenine opposite template 8-oxoG can result in a G to T transversion. DNA polymerases vary by orders of magnitude in their preferences for mutagenic vs. error-free 8-oxoG lesion bypass. Yet, the structural basis for lesion bypass specificity is not well understood. The DNA base excision repair enzyme DNA polymerase (pol) ß is presented with gap-filling synthesis opposite 8-oxoG during repair and has similar insertion efficiencies for dCTP and dATP. We report the structure of pol ß in binary complex with template 8-oxoG in a base excision repair substrate. The structure reveals both the syn- and anti-conformations of template 8-oxoG in the confines of the polymerase active site, consistent with the dual coding observed kinetically for this enzyme. A ternary complex structure of pol ß with the syn-8-oxoG:anti-A Hoogsteen base pair in the closed fully assembled preinsertion active site is also reported. The syn-conformation of 8-oxoG is stabilized by minor groove hydrogen bonding between the side chain of Arg283 and O8 of 8-oxoG. An adjustment in the position of the phosphodiester backbone 5'-phosphate enables 8-oxoG to adopt the syn-conformation.