Active Site Mutations in Mammalian DNA Polymerase δ Alter Accuracy and Replication Fork Progression

DNA polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging DNA strand and also functions in DNA repair. In previous work, we demonstrated that heterozygous expression of the pol δ L604G variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, L604K, is associated with shortened life span and accelerated carcinogenesis. Here, we report in vitro analysis of the homologous mutations at position Leu-606 in human pol δ. Four-subunit human pol δ variants that harbor or lack 3′ → 5′-exonucleolytic proofreading activity were purified from Escherichia coli. The pol δ L606G and L606K holoenzymes retain catalytic activity and processivity similar to that of wild type pol δ. pol δ L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis, whereas pol δ L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild type pol δ. However, pol δ L606K is impaired in the bypass of DNA adducts, and the homologous variant in mouse embryonic fibroblasts results in a decreased rate of replication fork progression in vivo. These results indicate that different substitutions at a single active site residue in a eukaryotic polymerase can either increase or decrease the accuracy of synthesis relative to wild type and suggest that enhanced fidelity of base selection by a polymerase active site can result in impaired lesion bypass and delayed replication fork progression.     

Lyase activities intrinsic to Escherichia coli polymerases IV and V

Escherichia coli DNA polymerase IV and V (pol IV and pol V) are error-prone DNA polymerases that are induced as part of the SOS regulon in response to DNA damage. Both are members of the Y-family of DNA polymerases. Their principal biological roles appear to involve translesion synthesis (TLS) and the generation of mutational diversity to cope with stress. Although neither enzyme is known to be involved in base excision repair (BER), we have nevertheless observed apurinic/apyrimidinic 5'-deoxyribose phosphate (AP/5'-dRP) lyase activities intrinsic to each polymerase. Pols IV and V catalyze cleavage of the phosphodiester backbone at the 3'-side of an apurinic/apyrimidinic (AP) site as well as the removal of a 5'-deoxyribose phosphate (dRP) at a preincised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase beta, which is a key enzyme used in short patch BER. Pol IV forms a covalent Schiff's base intermediate with substrate DNA that is trapped by sodium borohydride, as proscribed by a beta-elimination mechanism. In contrast, a NaBH(4) trapped intermediate is not observed for pol V, even though the lyase specific activity of pol V is slightly higher than that of pol IV. Incubation of pol V (UmuD'(2)C) with a molar excess of UmuD drives an exchange of subunits to form UmuD'D+insoluble UmuC causing inactivation of polymerase and lyase activities. The concomitant loss of both activities is strong evidence that pol V contains a bona fide lyase activity.     

A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′₂C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol V-catalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.     

A RecA Protein Surface Required for Activation of DNA Polymerase V

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3''-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3''-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA''₂B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis.     

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.     

The E295K Cancer Variant of Human Polymerase β Favors the Mismatch Conformational Pathway during Nucleotide Selection

DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damaged DNA as part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (∼30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for binding dCTP opposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.     

The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis

Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.     

DNA polymerase V and RecA protein, a minimal mutasome

A hallmark of the Escherichia coli SOS response is the large increase in mutations caused by translesion synthesis (TLS). TLS requires DNA polymerase V (UmuD'2C) and RecA. Here, we show that pol V and RecA interact by two distinct mechanisms. First, pol V binds to RecA in the absence of DNA and ATP and second, through its UmuD' subunit, requiring DNA and ATP without ATP hydrolysis. TLS occurs in the absence of a RecA nucleoprotein filament but is inhibited in its presence. Therefore, a RecA nucleoprotein filament is unlikely to be required for SOS mutagenesis. Pol V activity is severely diminished in the absence of RecA or in the presence of RecA1730, a mutant defective for pol V mutagenesis in vivo. Pol V activity is strongly enhanced with RecA mutants constitutive for mutagenesis in vivo, suggesting that RecA is an obligate accessory factor that activates pol V for SOS mutagenesis.     

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 β (pol β) using ¹⁹F 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 β. The addition of a non-hydrolysable 2′-deoxycytosine-5′-[(α,β)-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 β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with ¹⁹F NMR data. Surface plasmon resonance binding kinetics revealed that pol β 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.     

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction.     

The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.     

DNA polymerase V allows bypass of toxic guanine oxidation products in vivo

Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.     

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.     

Role of RNase H enzymes in maintaining genome stability in Escherichia coli expressing a steric-gate mutant of pol VICE391

pol V_ICE391 (RumAʹ₂B) is a low-fidelity polymerase that promotes considerably higher levels of spontaneous “SOS-induced” mutagenesis than the related E. coli pol V (UmuDʹ₂C). The molecular basis for the enhanced mutagenesis was previously unknown. Using single molecule fluorescence microscopy to visualize pol V enzymes, we discovered that the elevated levels of mutagenesis are likely due, in part, to prolonged binding of RumB to genomic DNA leading to increased levels of DNA synthesis compared to UmuC.We have generated a steric gate pol V_ICE391 variant (pol V_ICE391_Y13A) that readily misincorporates ribonucleotides into the E. coli genome and have used the enzyme to investigate the molecular mechanisms of Ribonucleotide Excision Repair (RER) under conditions of increased ribonucleotide-induced stress. To do so, we compared the extent of spontaneous mutagenesis promoted by pol V and pol V_ICE391 to that of their respective steric gate variants. Levels of mutagenesis promoted by the steric gate variants that are lower than that of the wild-type enzyme are indicative of active RER that removes misincorporated ribonucleotides, but also misincorporated deoxyribonucleotides from the genome.Using such an approach, we confirmed that RNase HII plays a pivotal role in RER. In the absence of RNase HII, Nucleotide Excision Repair (NER) proteins help remove misincorporated ribonucleotides. However, significant RER occurs in the absence of RNase HII and NER. Most of the RNase HII and NER-independent RER occurs on the lagging strand during genome duplication. We suggest that this is most likely due to efficient RNase HI-dependent RER which recognizes the polyribonucleotide tracts generated by pol V_ICE391_Y13A. These activities are critical for the maintenance of genomic integrity when RNase HII is overwhelmed, or inactivated, as ΔrnhB or ΔrnhB ΔuvrA strains expressing pol V_ICE391_Y13A exhibit genome and plasmid instability in the absence of RNase HI.     

A novel variant of DNA polymerase ζ, Rev3ΔC,highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches.     

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.     

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.