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.

Revealing an Internal Stabilization Deficiency in the DNA Polymerase ß K289M Cancer Variant through the Combined Use of Chemical Biology and X-ray Crystallography.

The human DNA polymerase (pol) ß cancer variant K289M has altered polymerase activity in vitro, and the structure of wild-type pol ß reveals that the K289 side chain contributes to a network of stabilizing interactions in a C-terminal region of the enzyme distal to the active site. Here, we probed the capacity of the K289M variant to tolerate strain introduced within the C-terminal region and active site. Strain was imposed by making use of a dGTP analogue containing a CF2 group substitution for the ß-γ bridging oxygen atom. The ternary complex structure of the K289M variant displays an alteration in the C-terminal region, whereas the structure of wild-type pol ß is not altered in the presence of the dGTP CF2 analogue. The alteration in the K289M variant impacts the active site, because the enzyme in the ternary complex fails to adopt the normal open to closed conformational change and assembly of the catalytically competent active site. These results reveal the importance of the K289-mediated stabilizing network in the C-terminal region of pol ß and suggest an explanation for why the K289M cancer variant is deficient in polymerase activity even though the position 289 side chain is distal to the active site.

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.

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.

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.

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.

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.

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.

Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid.

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF(2)-substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF(2)-substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF(2) groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol beta) binding pocket by obtaining a 2.5 A resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg(2+) interaction over Watson-Crick hydrogen bonding was found and discussed.

Molecular insights into DNA polymerase deterrents for ribonucleotide insertion.

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase ß discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn(2+) for Mg(2+) decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2' of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase ß utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

ß,γ-CHF- and ß,γ-CHCl-dGTP diastereomers: synthesis, discrete 31P NMR signatures, and absolute configurations of new stereochemical probes for DNA polymerases.

Deoxynucleoside 5'-triphosphate analogues in which the ß,γ-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base-selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of ß,γ-CXY dNTP diastereomers: (S)- and (R)-ß,γ-CHCl-dGTP (12a-1/12a-2) and (S)- and (R)-ß,γ-CHF-dGTP (12b-1/12b-2). Central to their preparation was conversion of the prochiral parent bisphosphonic acids to the P,C-dimorpholinamide derivatives 7 of their (R)-mandelic acid monoesters, which provided access to the individual diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by preparative HPLC. Selective acidic hydrolysis of the P-N bond then afforded "portal" diastereomers, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H(2) (Pd/C) gave the four individual diastereomeric nucleotides 12, which were characterized by (31)P, (1)H, and (19)F NMR spectroscopy and by mass spectrometry. After treatment with Chelex-100 to remove traces of paramagnetic ions, at pH ~10 the diastereomer pairs 12a,b exhibit discrete P(α) and P(ß)(31)P resonances. The more upfield P(α) and more downfield P(ß) resonances (and also the more upfield (19)F NMR resonance in 12b) are assigned to the R configuration at the P(ß)-CHX-P(γ) carbons on the basis of the absolute configurations of the individual diastereomers as determined from the X-ray crystallographic structures of their ternary complexes with DNA and polymerase ß.

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.

Base excision repair and design of small molecule inhibitors of human DNA polymerase ß.

Base excision repair (BER) can protect a cell after endogenous or exogenous genotoxic stress, and a deficiency in BER can render a cell hypersensitive to stress-induced apoptotic and necrotic cell death, mutagenesis, and chromosomal rearrangements. However, understanding of the mammalian BER system is not yet complete as it is extraordinarily complex and has many back-up processes that complement a deficiency in any one step. Due of this lack of information, we are unable to make accurate predictions on therapeutic approaches targeting BER. A deeper understanding of BER will eventually allow us to conduct more meaningful clinical interventions. In this review, we will cover historical and recent information on mammalian BER and DNA polymerase ß and discuss approaches toward development and use of small molecule inhibitors to manipulate BER. With apologies to others, we will emphasize results obtained in our laboratory and those of our collaborators.

Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase beta.

Based on a recent ternary complex crystal structure of human DNA polymerase beta with a G:A mismatch in the active site, we carried out a theoretical investigation of the catalytic mechanism of incorrect nucleotide incorporation using molecular dynamics simulation, quantum mechanics, combined quantum mechanics, and molecular mechanics methods. A two-stage mechanism is proposed with a nonreactive active-site structural rearrangement prechemistry step occurring before the nucleotidyl transfer reaction. The free energy required for formation of the prechemistry state is found to be the major factor contributing to the decrease in the rate of incorrect nucleotide incorporation compared with correct insertion and therefore to fidelity enhancement. Hence, the transition state and reaction barrier for phosphodiester bond formation after the prechemistry state are similar to that for correct insertion reaction. Key residues that provide electrostatic stabilization of the transition state are identified.

DNA polymerase beta substrate specificity: side chain modulation of the "A-rule".

Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA polymerase (pol) beta, a family X polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol beta insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol beta bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.

Halogenated beta,gamma-methylene- and ethylidene-dGTP-DNA ternary complexes with DNA polymerase beta: structural evidence for stereospecific binding of the fluoromethylene analogues.

Beta,gamma-fluoromethylene analogues of nucleotides are considered to be useful mimics of the natural substrates, but direct structural evidence defining their active site interactions has not been available, including the influence of the new chiral center introduced at the CHF carbon, as in beta,gamma-fluoromethylene-dGTP, which forms an active site complex with DNA polymerase beta, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. We report X-ray crystallographic results for a series of beta,gamma-CXY dGTP analogues, where X,Y = H, F, Cl, Br, and/or CH(3). For all three R/S monofluorinated analogues examined (CHF, 3/4; CCH(3)F, 13/14; CClF 15/16), a single CXF-diastereomer (3, 13, 16) is observed in the active site complex, with the CXF fluorine atom at a approximately 3 A (bonding) distance to a guanidinium N of Arg183. In contrast, for the CHCl, CHBr, and CHCH(3) analogues, both diasteromers (6/7, 8/9, 10/11) populate the dGTP site in the enzyme complex about equally. The structures of the bound dichloro (5) and dimethyl (12) analogue complexes indicate little to no steric effect on the placement of the bound nucleotide backbone. The results suggest that introduction of a single fluorine atom at the beta,gamma-bridging carbon atom of these dNTP analogues enables a new, stereospecific interaction within the preorganized active site complex that is unique to fluorine. The results also provide the first diverse structural data set permitting an assessment of how closely this class of dNTP analogues mimics the conformation of the parent nucleotide within the active site complex.

DNA polymerase structure-based insight on the mutagenic properties of 8-oxoguanine.

An aerobic environment burdens DNA polymerase substrates with oxidized substrates (DNA and nucleotide pools). A major promutagenic lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG). Guanine oxidation alters the hydrogen bonding properties of the base and glycosidic-preference of the nucleotide. The favored glycosidic syn-conformation exposes the Hoogsteen edge of the base for hydrogen bonding with adenine during DNA synthesis. The cell has recognized the threat of this lesion and has evolved an intricate surveillance system to provide DNA polymerases with unmodified substrates. Failure to do so leads to transversion mutations. Since the mutagenic properties of the base are dictated by the anti-syn-conformation of the nucleotide, the molecular interactions of 8-oxoG in the confines of the DNA polymerase active site are expected to influence its coding potential. Recent structural characterization of DNA polymerases from several families with this lesion in the nascent base pair binding pocket has provided insight to the mutagenic properties of this modified nucleotide. These structures reveal that flexibility around the template-binding pocket can permit 8-oxoG to assume an anti- or syn-conformation and code for cytosine or adenine incorporation, respectively. In contrast, the binding pocket for the incoming nucleotide does not have this flexibility so that 8-oxodGTP insertion opposite cytosine is strongly discouraged.

Structure of a DNA polymerase abortive complex with the 8OG:dA base pair at the primer terminus.

Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions. The (syn)8OG:dA base pair is indistinguishable from the cognant base pair and can be extended by DNA polymerases with reduced efficiency. To examine the structural basis of this reduced efficiency, we sought to obtain the structure of the "product" complex of DNA polymerase (pol) ß with the (syn)8OG:dA base pair at the primer terminus by soaking the binary complex crystals with a hydrolysable dCTP analogue complementary to the template base G. Crystallographic refinement of the structure revealed that the adenine of the (syn)8OG:dA base pair had been expelled from the primer terminus and a dCMP was inserted opposite 8OG in a reverse orientation; another uninserted molecule of the analogue was bound to the templating base G. This leads to an abortive complex that could form the basis of oxidatively-induced pol ß stalling.

The X family portrait: structural insights into biological functions of X family polymerases.

The mammalian family X DNA polymerases (DNA polymerases beta, lambda, mu, and TdT) contribute to base excision repair and double-strand break repair by virtue of their ability to fill short gaps in DNA. Structural information now exists for all four of these enzymes, making this the first mammalian polymerase family whose structural portrait is complete. Here we consider how distinctive structural features of these enzymes contribute to their biological functions in vivo.