Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ~1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease.     

Critical amino acids in human DNA polymerases η and κ involved in erroneous incorporation of oxidized nucleotides

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Polη and Polκ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Polη was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Polη) to 7 : 1 (R61K). Similarly, Tyr112 in Polκ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polκ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Polη, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Polη and Polκ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.     

Promiscuous mismatch extension by human DNA polymerase lambda

DNA polymerase lambda (Pol λ) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol λ to extend mismatched primer-termini, either on ‘open’ template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5′-phosphate. Interestingly, Pol λ extended mismatches with an average efficiency of ≈10⁻² relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were ≈260-fold higher than on template-primer molecules. A crystal structure of Pol λ in complex with a single-nucleotide gap containing a dG·dGMP mismatch at the primer-terminus (2.40 Å) suggests that, at least for certain mispairs, Pol λ is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol λ suggests a potential role as a ‘mismatch extender’ during non-homologous end joining (NHEJ), and possibly during translesion synthesis.     

Use of damaged DNA and dNTP substrates by the error-prone DNA polymerase X from African swine fever virus

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

The Steric Gate of DNA Polymerase ι Regulates Ribonucleotide Incorporation and Deoxyribonucleotide Fidelity

Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A “steric gate” pol ι mutant is considerably more active in the presence of Mn²⁺ compared with Mg²⁺ and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be “at risk” for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations.     

Mismatched dNTP incorporation by DNA polymerase beta does not proceed via globally different conformational pathways

Understanding how DNA polymerases control fidelity requires elucidation of the mechanisms of matched and mismatched dNTP incorporations. Little is known about the latter because mismatched complexes do not crystallize readily. In this report, we employed small-angle X-ray scattering (SAXS) and structural modeling to probe the conformations of different intermediate states of mammalian DNA polymerase β (Pol β) in its wild-type and an error-prone variant, I260Q. Our structural results indicate that the mismatched ternary complex lies in-between the open and the closed forms, but more closely resembles the open form for WT and the closed form for I260Q. On the basis of molecular modeling, this over-stabilization of mismatched ternary complex of I260Q is likely caused by formation of a hydrogen bonding network between the side chains of Gln²⁶⁰, Tyr²⁹⁶, Glu²⁹⁵ and Arg²⁵⁸, freeing up Asp¹⁹² to coordinate MgdNTP. These results argue against recent reports suggesting that mismatched dNTP incorporations follow a conformational path distinctly different from that of matched dNTP incorporation, or that its conformational closing is a major contributor to fidelity.     

Interaction between DNA Polymerase λ and Anticancer Nucleoside Analogs

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase λ (Pol λ) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol λ. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol λ efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol λ could modulate the cytotoxic effect of AraC.     

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda

DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn⁺⁺ and Mg⁺⁺ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed.     

The active site of TthPolX is adapted to prevent 8-oxo-dGTP misincorporation

Full genome sequencing of bacterial genomes has revealed the presence of numerous genes encoding family X DNA polymerases. These enzymes play a variety of biological roles and, accordingly, display often striking functional differences. Here we report that the PolX from the heat-stable organism Thermus thermophilus (TthPolX) inserts the four dNTPs with strong asymmetry. We demonstrate that this behaviour is related to the presence of a single divergent residue in the active site of TthPolX. Mutation of this residue (Ser²⁶⁶) to asparagine, the residue present in most PolXs, had a strong effect on TthPolX polymerase activity, increasing and equilibrating the insertion efficiencies of the 4 dNTPs. Moreover, we show that this behaviour correlates with the ability of TthPolX to insert 8-oxo-dGMP. Although the wild-type enzyme inefficiently incorporates 8-oxo-dGMP, the substitution of Ser²⁶⁶ to asparagine resulted in a dramatic increase in 8-oxo-dGMP incorporation opposite dA. These results suggest that the presence of a serine at position 266 in TthPolX allows the enzyme to minimize the formation of dA:8-oxo-dGMP at the expense of decreasing the insertion rate of pyrimidines. We discuss the structural basis for these effects and the implications of this behaviour for the GO system (BER of 8-oxo-dG lesions).     

The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases δ and η

A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols δ and η. Yeast Pol δ and yeast Pol η both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol η is 10-fold more efficient than Pol δ, and following bypass Pol η switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol η is at least 10-fold more accurate than yeast Pol δ during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol η in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol η. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol η. The fact that yeast and mammalian Pol η have intrinsically different catalytic properties has potential biological implications.     

Segregation of Replicative DNA Polymerases during S Phase: DNA POLYMERASE ?, BUT NOT DNA POLYMERASES α/δ, ARE ASSOCIATED WITH LAMINS THROUGHOUT S PHASE IN HUMAN CELLS*

DNA polymerases (Pol) α, δ, and ϵ replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ϵ being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G₁/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ϵ were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G₁/S arrest and early in S phase, Pol α, δ, and ϵ were associated with the same nucleoprotein complexes, whereas in late S phase Pol ϵ and Pol α/δ were largely associated with distinct complexes. At G₁/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ϵ, not Pol α/δ, remained associated with lamins. Consistently, Pol ϵ, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ϵ and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ϵ, to post-replicative processes such as translesion synthesis or post-replicative repair.     

Frequency and patterns of ribonucleotide incorporation around autonomously replicating sequences in yeast reveal the division of labor of replicative DNA polymerases

Ribonucleoside triphosphate (rNTP) incorporation in DNA by DNA polymerases is a frequent phenomenon that results in DNA structural change and genome instability. However, it is unclear whether the rNTP incorporation into DNA follows any specific sequence patterns. We analyzed multiple datasets of ribonucleoside monophosphates (rNMPs) embedded in DNA, generated from three rNMP-sequencing techniques. These rNMP libraries were obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late-firing autonomously replicating sequences (ARSs) of the yeast genome, where leading and lagging DNA synthesis starts bidirectionally. We found the preference of rNTP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNTP incorporation changes dramatically within the first 1,000 nucleotides from ARSs, highlighting the Pol δ - Pol ϵ handoff during early leading-strand synthesis. Furthermore, the pattern of rNTP incorporation is markedly distinct between the leading and lagging strands not only in mutant but also in wild-type polymerase cells. Such specific signatures of Pol δ and Pol ϵ provide a new approach to track the labor of these polymerases.     

Phosphorylation of human DNA polymerase lambda by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen

DNA polymerase (Pol) λ is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol λ has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol λ has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol λ. Pol λ is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol λ was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol λ was decreased by its interaction with PCNA. Finally, Pol λ is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycle.     

A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Polμ and Polλ

Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3′ single-stranded overhangs and trans-polymerization using one 3′ end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence (‘brooch’), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr¹⁴¹, Ala¹⁴², Cys¹⁴³, Gln¹⁴⁴ and Arg¹⁴⁵ in Polµ, and by Trp²³⁹, Val²⁴⁰, Cys²⁴¹, Ala²⁴² and Gln²⁴³ in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.     

Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

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

Restricted 5′-End Gap Repair of HIV-1 Integration Due to Limited Cellular dNTP Concentrations in Human Primary Macrophages

HIV-1 proviral DNA integration into host chromosomal DNA is only partially completed by the viral integrase, leaving two single-stranded DNA gaps with 5′-end mismatched viral DNA flaps. It has been inferred that these gaps are repaired by the cellular DNA repair machinery. Here, we investigated the efficiency of gap repair at integration sites in different HIV-1 target cell types. First, we found that the general gap repair machinery in macrophages was attenuated compared with that in dividing CD4⁺ T cells. In fact, the repair in macrophages was heavily reliant upon host DNA polymerase β (Pol β). Second, we tested whether the poor dNTP availability found in macrophages is responsible for the delayed HIV-1 proviral DNA integration in this cell type because the K_m value of Pol β is much higher than the dNTP concentrations found in macrophages. Indeed, with the use of a modified quantitative AluI PCR assay, we demonstrated that the elevation of cellular dNTP concentrations accelerated DNA gap repair in macrophages at HIV-1 proviral DNA integration sites. Finally, we found that human monocytes, which are resistant to HIV-1 infection, exhibited severely restricted gap repair capacity due not only to the very low levels of dNTPs detected but also to the significantly reduced expression of Pol β. Taken together, these results suggest that the low dNTP concentrations found in macrophages and monocytes can restrict the repair steps necessary for HIV-1 integration.     

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol ζ) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol ζ alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol ζ. Pol ζ is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol ζ is substantially lower than that of homologous B family pols α, δ and . Pol ζ is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol ζ in vitro is consistent with Pol ζ-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol ζ contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.     

NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5'-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans

8-oxo-dGTP is generated in the nucleotide pool by direct oxidation of dGTP or phosphorylation of 8-oxo-dGDP. It can be incorporated into DNA during replication, which would result in mutagenic consequences. The frequency of spontaneous mutations remains low in cells owing to the action of enzymes degrading such mutagenic substrates. Escherichia coli MutT and human MTH1 hydrolyze 8-oxo-dGTP to 8-oxo-dGMP. Human NUDT5 as well as human MTH1 hydrolyze 8-oxo-dGDP to 8-oxo-dGMP. These enzymes prevent mutations caused by misincorporation of 8-oxo-dGTP into DNA. In this study, we identified a novel MutT homolog (NDX-1) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. NDX-1 did not hydrolyze 8-oxo-dGTP, 2-hydroxy-dATP or 2-hydroxy-dADP. Expression of NDX-1 significantly reduced spontaneous A:T to C:G transversions and mitigated the sensitivity to a superoxide-generating agent, methyl viologen, in an E. coli mutT mutant. In C. elegans, RNAi of ndx-1 did not affect the lifespan of the worm. However, the sensitivity to methyl viologen and menadione bisulfite of the ndx-1-RNAi worms was enhanced compared with that of the control worms. These facts indicate that NDX-1 is involved in sanitization of 8-oxo-dGDP and plays a critical role in defense against oxidative stress in C. elegans.     

Regulation of B family DNA polymerase fidelity by a conserved active site residue: characterization of M644W, M644L and M644F mutants of yeast DNA polymerase epsilon

To better understand the functions and fidelity of DNA polymerase ε (Pol ε), we report here on the fidelity of yeast Pol ε mutants with leucine, tryptophan or phenylalanine replacing Met644. The Met644 side chain interacts with an invariant tyrosine that contacts the sugar of the incoming dNTP. M644W and M644L Pol ε synthesize DNA with high fidelity, but M644F Pol ε has reduced fidelity resulting from strongly increased misinsertion rates. When Msh6-dependent repair of replication errors is defective, the mutation rate of a pol2-M644F strain is 16-fold higher than that of a strain with wild-type Pol ε. In conjunction with earlier studies of low-fidelity mutants with replacements for the homologous amino acid in yeast Pol α (L868M/F) and Pol δ (L612M), these data indicate that the active site location occupied by Met644 in Pol ε is a key determinant of replication fidelity by all three B family replicative polymerases. Interestingly, error specificity of M644F Pol ε is distinct from that of L868M/F Pol α or L612M Pol δ, implying that each polymerase has different active site geometry, and suggesting that these polymerase alleles may generate distinctive mutational signatures for probing functions in vivo.