Eukaryotic Base Excision Repair: New Approaches Shine Light on Mechanism.

Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.

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.

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.

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.

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.

Transitions in DNA polymerase ß µs-ms dynamics related to substrate binding and catalysis.

DNA polymerase ß (pol ß) plays a central role in the DNA base excision repair pathway and also serves as an important model polymerase. Dynamic characterization of pol ß from methyl-TROSY 13C-1H multiple quantum CPMG relaxation dispersion experiments of Ile and Met sidechains and previous backbone relaxation dispersion measurements, reveals transitions in µs-ms dynamics in response to highly variable substrates. Recognition of a 1-nt-gapped DNA substrate is accompanied by significant backbone and sidechain motion in the lyase domain and the DNA binding subdomain of the polymerase domain, that may help to facilitate binding of the apoenzyme to the segments of the DNA upstream and downstream from the gap. Backbone µs-ms motion largely disappears after formation of the pol ß-DNA complex, giving rise to an increase in uncoupled µs-ms sidechain motion throughout the enzyme. Formation of an abortive ternary complex using a non-hydrolyzable dNTP results in sidechain motions that fit to a single exchange process localized to the catalytic subdomain, suggesting that this motion may play a role in catalysis.

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.

Revealing the role of the product metal in DNA polymerase ß catalysis.

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase ß, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.

Mapping Functional Substrate-Enzyme Interactions in the pol ß Active Site through Chemical Biology: Structural Responses to Acidity Modification of Incoming dNTPs.

We report high-resolution crystal structures of DNA polymerase (pol) ß in ternary complex with a panel of incoming dNTPs carrying acidity-modified 5'-triphosphate groups. These novel dNTP analogues have a variety of halomethylene substitutions replacing the bridging oxygen between Pß and Pγ of the incoming dNTP, whereas other analogues have alkaline substitutions at the bridging oxygen. Use of these analogues allows the first systematic comparison of effects of 5'-triphosphate acidity modification on active site structures and the rate constant of DNA synthesis. These ternary complex structures with incoming dATP, dTTP, and dCTP analogues reveal the enzyme's active site is not grossly altered by the acidity modifications of the triphosphate group, yet with analogues of all three incoming dNTP bases, subtle structural differences are apparent in interactions around the nascent base pair and at the guanidinium groups of active site arginine residues. These results are important for understanding how acidity modification of the incoming dNTP's 5'-triphosphate can influence DNA polymerase activity and the significance of interactions at arginines 183 and 149 in the active site.

Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State.

We examine the DNA polymerase ß (pol ß) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel ß,γ-CXY-dNTP analogues, including individual ß,γ-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log kpol versus p Ka4 of the leaving group bisphosphonic acids are used to interrogate the effects of the base identity, the dNTP analogue leaving group basicity, and the precise configuration of the C-X atom in R and S stereoisomers on the rate-determining step ( kpol). The dNTP analogues provide a range of leaving group basicity and steric properties by virtue of monohalogen, dihalogen, or methyl substitution at the carbon atom bridging the ß,γ-bisphosphonate that mimics the natural pyrophosphate leaving group in dNTPs. Brønsted plot relationships with negative slopes are revealed by the data, as was found for the dGTP and dTTP analogues, consistent with a bond-breaking component to the TS energy. However, greater multiplicity was shown in the linear free energy relationship, revealing an unexpected dependence on the nucleotide base for both A and C. Strong base-dependent perturbations that modulate TS relative to ground-state energies are likely to arise from electrostatic effects on catalysis in the pol active site. Deviations from a uniform linear Brønsted plot relationship are discussed in terms of insights gained from structural features of the prechemistry DNA polymerase active site.

Insertion of oxidized nucleotide triggers rapid DNA polymerase opening.

A novel mechanism is unveiled to explain why a pro-mutagenic nucleotide lesion (oxidized guanine, 8-oxoG) causes the mammalian DNA repair polymerase-ß (pol-ß) to rapidly transition to an inactive open conformation. The mechanism involves unexpected features revealed recently in time-lapse crystallography. Specifically, a delicate water network associated with a lesion-stabilizing auxilliary product ion Mg(p) triggers a cascade of events that leads to poor active site geometry and the rupture of crucial molecular interactions between key residues in both the anti(8-oxoG:C) and syn(8-oxoG:A) systems. Once the base pairs in these lesioned systems are broken, dislocation of both Asp192 (a metal coordinating ligand) and the oxoG phosphate group (PO4) interfere with the hydrogen bonding between Asp192 and Arg258, whose rotation toward Asp192 is crucial to the closed-to-open enzyme transition. Energetically, the lesioned open states are similar in energy to those of the corresponding closed complexes after chemistry, in marked contrast to the unlesioned pol-ß anti(G:C) system, whose open state is energetically higher than the closed state. The delicate surveillance system offers a fundamental protective mechanism in the cell that triggers DNA repair events which help deter insertion of oxidized lesions.

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.

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine.

Numerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability. Mammalian cells possess translesion DNA synthesis (TLS) pols that bypass DNA damage. The mechanism of TLS and repair of oxidized ribonucleotides remains to be elucidated. To address this, we analyzed the miscoding properties of the ribonucleotides riboguanosine (rG) and 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG) during TLS catalyzed by the human TLS pols κ and η in vitro The primer extension reaction catalyzed by human replicative pol α was strongly blocked by 8-oxo-rG. pol κ inefficiently bypassed rG and 8-oxo-rG compared with dG and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG), whereas pol η easily bypassed the ribonucleotides. pol α exclusively inserted dAMP opposite 8-oxo-rG. Interestingly, pol κ preferentially inserted dCMP opposite 8-oxo-rG, whereas the insertion of dAMP was favored opposite 8-oxo-dG. In addition, pol η accurately bypassed 8-oxo-rG. Furthermore, we examined the activity of the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 on the substrates, including rG and 8-oxo-rG. Both BER enzymes were completely inactive against 8-oxo-rG in DNA. However, OGG1 suppressed 8-oxo-rG excision by RNase H2, which is involved in the removal of ribonucleotides from DNA. These results suggest that the different sugar backbones between 8-oxo-rG and 8-oxo-dG alter the capacity of TLS and repair of 8-oxoguanine.

Hiding in Plain Sight: The Bimetallic Magnesium Covalent Bond in Enzyme Active Sites.

The transfer of phosphate groups is an essential function of many intracellular biological enzymes. The transfer is in many cases facilitated by a protein scaffold involving two closely spaced magnesium "ions". It has long been a mystery how these "ions" can retain their closely spaced positions throughout enzymatic phosphate transfer: Coulomb's law would dictate large repulsive forces between these ions at the observed distances. Here we show, however, that the electron density can be borrowed from nearby electron-rich oxygens to populate a bonding molecular orbital that is largely localized between the magnesium "ions". The result is that the Mg-Mg core of these phosphate transfer enzymes is surprisingly similar to a metastable [Mg2]2+ ion in the gas phase, an ion that has been identified experimentally and studied with high-level quantum-mechanical calculations. This similarity is confirmed by comparative computations of the electron densities of [Mg2]2+ in the gas phase and the Mg-Mg core in the structures derived from QM/MM studies of high-resolution X-ray crystal structures. That there is a level of covalent bonding between the two Mg "ions" at the core of these enzymes is a novel concept that enables an improved vision of how these enzymes function at the molecular level. The concept is broader than magnesium-other biologically relevant metals (e.g., Mn and Zn) can also form similar stabilizing covalent Me-Me bonds in both organometallic and inorganic crystals.

Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation.

We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.

Base excision repair of tandem modifications in a methylated CpG dinucleotide.

Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase ß, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase ß activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.

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.

DNA polymerase minor groove interactions modulate mutagenic bypass of a templating 8-oxoguanine lesion.

A major base lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG) that has ambiguous coding potential. Error-free DNA synthesis involves 8-oxoG adopting an anti-conformation to base pair with cytosine whereas mutagenic bypass involves 8-oxoG adopting a syn-conformation to base pair with adenine. Left unrepaired the syn-8-oxoG/dAMP base pair results in a G-C to T-A transversion. During base excision repair of this mispair, DNA polymerase (pol) ß is confronted with gap filling opposite 8-oxoG. To determine how pol ß discriminates between anti- and syn-8-oxoG, we introduced a point mutation (R283K) to alter insertion specificity. Kinetic studies demonstrate that this substitution results in an increased fidelity opposite 8-oxoG. Structural studies with R283K pol ß show that the binary DNA complex has 8-oxoG in equilibrium between anti- and syn-forms. Ternary complexes with incoming dCTP resemble the wild-type enzyme, with templating anti-8-oxoG base pairing with incoming cytosine. In contrast to wild-type pol ß, the ternary complex of the R283K mutant with an incoming dATP-analogue and templating 8-oxoG resembles a G-A mismatched structure with 8-oxoG adopting an anti-conformation. These results demonstrate that the incoming nucleotide is unable to induce a syn-8-oxoG conformation without minor groove DNA polymerase interactions that influence templating (anti-/syn-equilibrium) of 8-oxoG while modulating fidelity.

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.

Structure and mechanism of DNA polymerase ß.

DNA polymerase (pol) ß is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol ß has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol ß in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.