Genetic Control of Replication through N1-methyladenine in Human Cells*

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.

N1-methyladenine (1-MeA) 3 is formed in DNA by reaction with S n 2 methylating agents such as methyl methanesulfonate and naturally occurring methyl halides (1)(2)(3). The S n 2 methyl halides are among the most abundant environmental methylating agents released from biomass burning and from decaying vegetation. Exposure to alkylating agents could also occur from food, occupational hazards, and chemotherapeutic treatments (1).
1-MeA is highly cytotoxic because the N1 atom is engaged in Watson-Crick (W-C) base pairing and its modification by a methyl group impairs W-C base pairing and blocks normal DNA replication. In Escherichia coli, AlkB repairs 1-MeA by oxidative demethylation, which liberates formaldehyde from the methylated base and results in complete reversal of the damage (4,5). In humans, there are nine potential AlkB homologs, two of which, ABH2 and ABH3, can repair the same spectrum of DNA lesions as AlkB (6,7); ABH2, however, is the primary housekeeping enzyme in humans for repairing 1-MeA (8). Mouse embryonic fibroblast lines derived from ABH2 null mice are highly defective in repairing 1-MeA residues generated in response to methyl methanesulfonate treatment. Because in the absence of any exposure to alkylating agents, 1-MeA residues accumulate over time in the genomic DNA of livers from ABH2 null mice, endogenous DNA methylation contributes to their generation (8).
Previously, we reported on the genetic control of translesion synthesis (TLS) opposite UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts and opposite thymine glycol (Tg), which is the most common oxidation product of thymine (9 -12). Of the two UV lesions, cyclobutane pyrimidine dimer does not significantly affect the ability of two pyrimidines to form a correct W-C base pair with the purine bases, and it has only a modest effect on DNA structure (13); by contrast, a (6-4) pyrimidine-pyrimidone photoproduct induces a large structural distortion in DNA. It confers a 44º bend in the DNA helix and the 3ЈT is oriented perpendicular to the 5ЈT in the (6-4) TT photoproduct (14 -16). The Tg lesion also has no significant effect on the ability of oxidized T to form a correct base pair with an A; however, because of the addition of hydroxyl groups at C5 and C6 on Tg, the damaged base becomes non-planar and that prevents the base 5Ј to Tg from stacking above it (17)(18)(19)(20). Consequently, Tg presents a strong block to extension of synthesis from the Tg:A base pair. Despite the fact that these DNA lesions differ vastly in their effects on DNA structure and on base pairing, they generate only ϳ2% mutagenic TLS products in human cells (10 -12). This is rather surprising in view of the fact that the various TLS DNA polymerases (Pols) synthesize DNA with a low fidelity (21).
Here we identify the TLS Pols that promote replication through the 1-MeA lesion in human cells and show that TLS opposite this lesion is mediated by three independent pathways, involving Pols and in one pathway and Pols and , respectively, in the other two pathways. The observation that similar to that opposite UV and Tg lesions, TLS opposite this W-C blocking lesion also occurs in a highly error-free manner reinforces the notion that the fidelity of TLS Pols is actively regulated in human cells.

Experimental Procedures
Construction of Plasmid Vectors Containing 1-MeA-The 16-mer oligonucleotides containing an N1-methyl deoxyadenosine (Fig. 1A) were purchased from Trilink Biotechnologies (Santa Cruz, CA). The methods used by this company for the synthesis of 1-MeA-derived phosphoramidite and for its incorporation into oligonucleotide employ N 6 -chloroacetyl protection and controlled anhydrous deprotection conditions to prevent the formation of N 6 -methyl-2Ј-deoxyadenosine via a Dimroth rearrangement (22). The in-frame target sequence of lacZЈ gene containing 1-MeA is shown in Fig. 1A. In the lacZЈ gene, the 1-MeA-containing DNA strand harbors an MfeI restriction site, and it encodes functional ␤-galactosidase (␤-gal), whereas the opposite strand harbors an SpeI site containing a ϩ1 frameshift, making it non-functional for ␤-gal. The 1-MeA containing strand carries the kanamycin gene (Kan ϩ ), whereas the other DNA strand has the kan Ϫ gene (Fig. 1B). The detailed method for construction of 1-MeA-containing plasmids was as described previously (11).
Translesion Synthesis Assays and Mutation Analyses of TLS Products in Human Cells-Normal (MRC5) and XPV (XP30RO) human fibroblast cells were grown in DMEM media (GenDEPOT) with 10% FBS (GenDEPOT) and plated in 6-well plates at 70% confluence (approximately 3 ϫ 10 5 cells per well). Cells were transfected with 100 pmol of siRNAs with Lipofectamine 2000 (Invitrogen). For the simultaneous siRNA FIGURE 1. Strategy for examining TLS opposite 1-MeA. A, the target 16-mer sequence containing 1-MeA. The sequence of the N-terminal part of the lacZЈ gene in pBS vector (leading strand) including the 1-MeA site, indicated by mA, is shown. B, selection strategy for TLS assays. The top DNA strand containing the 1-MeA lesion carries a wild type kanamycin gene (Kan ϩ ) so that TLS through the 1-MeA lesion will produce a blue colony on LB/Kan plates with isopropyl-1thio-␤-D-galactopyranoside and X-Gal. The other DNA strand harbors an Spe1 sequence opposite 1-MeA and carries the kan Ϫ gene. Because the Spe1 sequence puts the lacZ sequence out of frame (see Fig. 1A), replication of this strand will produce kan Ϫ colonies. C, assays for TLS and mutation analysis opposite 1-MeA in human cells. The purified cDNA and siRNAs are cotransfected into siRNA-treated human cells. The target sites of 1-MeA and for kanamycin gene selection are shown in the vector. After 30 h of incubation, the rescued plasmid DNA is treated with DpnI to remove any unreplicated plasmid and then transformed into XL-1 blue E. coli cells. TLS frequency is determined by phenotypic selection of transformed bacterial cells grown on LB/kan/X-Gal plates. The mutations induced by TLS opposite 1-MeA are analyzed by colony PCR, MfeI digestion, and by sequencing of the target site. HF, human fibroblast. knockdown of two genes, 100 pmol of siRNAs for each gene were mixed and transfected. After 48 h of incubation, the heteroduplex target vector DNA (1 g) and 50 pmol of siRNA (second transfection) were cotransfected with Lipofectamine 2000 (Invitrogen) (Fig. 1C). After 30 h of incubation, plasmid DNA was rescued from cells by the alkaline lysis method and digested with DpnI to remove unreplicated plasmid DNA. The plasmid DNA was then transformed into E. coli XL1-Blue super competent cells (Stratagene). Transformed bacterial cells were diluted in 1 ml of SOC media (Thermo Fisher Scientific) and plated on LB/kan (25 g/ml kanamycin, Sigma) plates containing 1 M isopropyl-1-thio-␤-D-galactopyranoside (Gen-DEPOT) and 100 g/ml X-Gal (GenDEPOT). After 16 h of incubation at 37°C, blue and white colonies were counted from kanamycin plates (Fig. 1C). The actual TLS frequency was determined from the number of blue colonies out of the total colonies growing on LB/kan plates. Plasmid DNA obtained from blue colonies was analyzed to determine the mutation frequency, and the mutational changes were incorporated during TLS. For details of these methods, see Yoon et al. (11).
DNA Polymerase Assays-DNA substrates consisted of a radiolabeled oligonucleotide primer annealed to a 75-nucleotide (nt) oligonucleotide DNA template by heating a mixture of primer/template at a 1:1.5 molar ratio to 95°C and allowing it to cool to room temperature for several hours. The template 75-mer oligonucleotide contained the sequence 5Ј-AGC AAG TCA CCA ATG TCT AAG AGT TCG TAT AAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3Ј, and it harbored an undamaged A or a 1-MeA at the underlined position. For examining the incorporation of dATP, dTTP, dCTP, or dGTP nucleotides individually or of all 4 dNTPs, a 44-mer primer 5Ј-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3Ј was annealed to the above-mentioned 75-mer template.
The standard DNA polymerase reaction (5 l) contained 25 mM Tris⅐HCl (pH 7.5), 5 mM MgCl 2, 1 mM dithiothreitol, 100 g/ml BSA, 10% glycerol, 10 nM DNA substrate, and 0.2 nM Pol or 0.25 nM Pol. For nucleotide incorporation assays with Pol, 50 M dATP, dTTP, dCTP, or dGTP (Roche Applied Science) was used, and for examining synthesis through the 1-MeA lesion all 4 dNTPs (50 M each) were used. For nucleotide incorporation assays with Pol, 25 M dATP, dTTP, dCTP, or dGTP (Roche Applied Science) was used, and for examining synthesis through the 1-MeA lesion by Pol all 4 dNTPs (25 M each) were used. Reactions were carried out for 10 min at 37°C.
For extension studies, a 75-mer oligonucleotide template 5Ј-AGC AAG TCA CCA ATG TCT AAG AGT TCG TAT AAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3Ј containing an undamaged A or a 1-MeA at the underlined position was annealed to a 23-mer primer 5Ј TCC GGT ACT CCA GTG TAG GCA TX-3Ј, which contained a T at the position indicated by X.
Primer extension by human Pol (1 nM) or yeast Pol (1 nM) was assayed in the presence of 10 M each dATP, dGTP, dTTP, and dCTP (Roche Applied Science) on DNA containing an A/T or 1-MeA/T primer terminal base pair; the standard DNA polymerase reaction (5 l) contained 25 mM Tris⅐HCl (pH 7.5), 5 mM MgCl 2, 1 mM dithiothreitol, 100 g/ml BSA, 10% glycerol, and 10 nM DNA substrate. Reactions containing human Pol were carried out at 37°C and yeast Pol at 30°C for 10 min.

TLS Pols Required for Replicating through the 1-MeA
Lesion-To identify the TLS Pols required for replicating through the 1-MeA lesion, we determined the effects of siRNA depletions of TLS Pols individually and in combinations on TLS frequency opposite this lesion carried on the leading strand template of the SV40-based plasmid. To ascertain that the observed defects result from the deficiency of the particular Pol and not from any off-target effects, we confirmed that the defect in TLS engendered by the siRNA depletion of a Pol was complemented by the respective wild type Pol.
TLS in normal human fibroblasts treated with control (NC) siRNA occurred with a frequency of ϳ65% (Table 1). In Poldepleted cells, TLS frequency was reduced to ϳ52%. TLS frequency was reduced to ϳ45% in Pol-depleted cells, and a similar reduction in TLS frequency was observed in cells depleted for the Rev3 or the Rev7 subunits of Pol or for Pol. To determine whether these Pols function independently of one another or whether some of them function together in replicating through the 1-MeA lesion, we examined the effects of simultaneous depletion of two Pols on TLS frequencies. Our observation that simultaneous depletion of Pol and Pol confers no further reduction in TLS frequency than that seen upon their individual depletion indicates that Pols and function together in conducting TLS opposite 1-MeA. By contrast, the observation that simultaneous depletion of Pol with Pol or of Pol with Pol results in a greater reduction in TLS frequency, to ϳ30%, than that observed upon their individual depletion implies that Pol functions in TLS opposite 1-MeA independently of Pols and . And, because the simultaneous depletion of Pol with the Rev3 or Rev7 subunit of Pol reduces TLS frequency to a greater extent (ϳ30%) than that observed upon their individual depletion (ϳ45-52%), Pols and act independently of one another. Additionally, because the simultaneous depletion of Pol with Pol confers a greater reduction in TLS frequency than that observed upon their individual depletion, these Pols function in TLS independently of one another.
As deduced from these genetic observations, TLS opposite 1-MeA occurs via three independent pathways mediated by the  (Table 2), and as expected from the involvement of Pols and in the same TLS pathway, the simultaneous depletion of Pol and Pol causes no further reduction in TLS frequency than that seen upon their individual depletion. Because only the Pol/Poland Pol-dependent TLS pathways would remain functional in XPV cells, we expect the simultaneous depletion of Pol and Pol or of Pol and Pol will confer a drastic reduction in TLS frequency. In accord with this, we find that simultaneous depletion of Pol and Rev3 or of Pol and Rev3 reduces TLS frequency to ϳ4 -5% (Table 2). These results provide confirmatory evidence that TLS opposite 1-MeA occurs via three independent pathways that require Pols and in one pathway and Pol and Pol, respectively, in the other two pathways.
Mutagenicity of TLS Opposite 1-MeA-As determined by sequence analyses of TLS products, TLS opposite the 1-MeA lesion occurs in a predominantly error-free fashion, as in NC siRNA-treated cells only ϳ1% of TLS products harbor mutations. Among the ϳ400 TLS products that were analyzed, we observed mutations in only four of the TLS products in which an A or G was inserted opposite 1-MeA instead of the correct nucleotide, T (Table 3). Even though the overall TLS-mediated via three different pathways opposite 1-MeA generates only ϳ1% mutations, among the TLS Pols involved in 1-MeA bypass some may function in an error-free manner whereas others may promote a more mutagenic mode of TLS. Thus, for example, even though the overall TLS opposite a Tg lesion generates only ϳ2% mutations, the Pol/Pol-dependent pathway mediates a predominantly error-free mode of TLS, whereas the Pol-dependent pathway functions in a more error-prone manner (9,10). For this reason we examined the frequency and types of mutations generated by the action of different TLS Pols opposite 1-MeA. Interestingly, among the ϳ200 -300 TLS products analyzed from cells depleted for Pol, Pol, Pol, or Pol, we observed mutation frequencies ranging from 0% in Pol-de-pleted cells to ϳ1.5% in Pol-depleted cells (Table 3). These differences are not statistically significant. We conclude from these results that opposite 1-MeA, all three pathways function in a predominantly error-free manner, generating only ϳ1% mutagenic TLS products where an A, or a G, is inserted opposite 1-MeA rather than a T.
Biochemical Studies for Roles of TLS Pols in DNA Synthesis Opposite 1-MeA-TLS through a DNA lesion can occur by the action of one Pol, wherein the TLS Pol inserts one nt opposite the DNA lesion and then extends synthesis from the inserted nt (21). Alternatively, TLS through a DNA lesion may occur by the sequential action of two Pols wherein one Pol inserts the nt opposite the lesion and then another Pol extends synthesis (21,23). To determine the potential of TLS Pols for DNA synthesis opposite 1-MeA, we examined their ability to insert dATP, dTTP, dGTP, or dCTP opposite 1-MeA and to synthesize DNA through the 1-MeA lesion in the presence of all 4 dNTPs. As shown in Fig. 2, Pol inserts dTTP opposite undamaged A, and in the presence of all 4 dNTPs, it extends synthesis by incorporating 1-2 additional nts. Opposite 1-MeA, Pol inserts dTTP, but it also inserts a dATP or dCTP, and in the presence of 4 dNTPs, Pol inserts one nt opposite 1-MeA, but it fails to extend synthesis any further. Thus, Pol is more error-prone opposite 1-MeA than opposite an undamaged A, and because of its failure to extend synthesis from the nt opposite 1-MeA, replication through the lesion would require the action of another TLS Pol. The inability of Pol to extend synthesis opposite from 1-MeA and the requirement of Pol for Pol-dependent TLS indicated from genetic studies suggested that Pol would extend synthesis from the nt inserted by Pol opposite 1-MeA. Hence, we examined if in the presence of four dNTPs, Pol could extend synthesis from a T inserted opposite 1-MeA. As shown in Fig. 3, Pol exhibits the same propensity for extending synthesis from the T nt opposite 1-MeA as from opposite undamaged A. These biochemical observations support the inference that in the Pol/Pol pathway, TLS through the 1-MeA lesion would occur by the sequential action of Pol and Pol, wherein after nt insertion by Pol opposite 1-MeA, Pol performs the subsequent extension step of TLS.
Biochemical studies show that opposite 1-MeA, Pol incorporated dTTP better than the incorrect nts, and the pattern of correct and incorrect nt incorporation opposite 1-MeA resembled that opposite undamaged A (Fig. 4). Thus, in addition to dTTP, Pol inserted the other three nts dATP, dGTP, or dCTP almost equally well opposite 1-MeA and undamaged A. In the presence of four dNTPs, Pol synthesized full-length products   DECEMBER 11, 2015 • VOLUME 290 • NUMBER 50 on 1-MeA containing DNA, but its ability to extend synthesis from the nt inserted opposite 1-MeA was reduced, as indicated by the presence of a prominent stall site opposite 1-MeA but not opposite undamaged A. Pol is very inefficient in inserting nts opposite DNA lesions but it is highly proficient at extending synthesis from nts opposite from a diverse array of DNA lesions (21,(23)(24)(25)(26). To verify that Pol plays a similar role opposite 1-MeA, we examined the proficiency of Pol for extending from the 1-MeA:T base pair. As shown in Fig. 3, in the presence of 4 dNTPs, Pol extends synthesis from the 1-MeA:T base pair as well as from the undamaged A:T base pair.

Discussion
The important findings of this study are as follows. 1) TLS provides a major means for replication of 1-MeA containing DNA. 2) Multiple pathways promote replication through the 1-MeA lesion. 3) TLS opposite 1-MeA occurs in a predominantly error-free manner. We consider the implications of these observations for the prominent role of TLS in the replication of 1-MeA-damaged DNA, for the roles of TLS Pols in 1-MeA bypass as related to their structural and biochemical features, and for the fact that in human cells, TLS opposite 1-MeA occurs in a highly error-free fashion.
A Major Role of TLS in the Replicative Bypass of 1-MeA-Previously, we have determined the extent, genetic control, and mutagenicity of TLS opposite three DNA lesions, cis-syn TT dimer, (6-4) TT photoproduct, and thymine glycol. These studies indicated that in nucleotide excision repair-defective XPA human fibroblasts, TLS opposite a cis-syn TT dimer or a (6-4) TT photoproduct occurs with a frequency of ϳ40%, whereas TLS opposite these lesions in nucleotide excision repair-proficient fibroblasts occurs with a frequency of ϳ20%. Thus, a very substantial fraction of these UV photoproducts are excised from plasmid DNA before its replication. Similar to that for UV photoproducts, in wild type fibroblasts, TLS opposite the thymine glycol lesion occurs with a frequency of ϳ20 -25%. In striking contrast to the TLS frequency of ϳ20 -25% opposite UV photoproducts or thymine glycol in wild type fibroblasts, we find that opposite 1-MeA, TLS contributes to ϳ65% of lesion bypass. Because the 1-MeA lesion carried on the duplex plasmid will be subject to removal by base excision repair or nucleotide excision repair, we presume that TLS provides a predominant, if not the sole means for replicating through this lesion.
Multiple Pathways for Replication of 1-MeA-damaged DNA-From siRNA depletion of TLS Pols, we have inferred that replication through the 1-MeA lesion occurs via three different pathways that, respectively, require Pol/Pol, Pol, or Pol (Fig. 5). Because biochemical studies have indicated that Pol can insert a nt opposite 1-MeA but lacks the ability to extend synthesis from the inserted nt whereas Pol can carry out efficient extension of synthesis from the nt opposite 1-MeA, we suggest that Pol/Pol-dependent TLS would occur by the sequential action of Pol and Pol at the insertion and extension  step of TLS, respectively (Fig. 5). In the Pol-dependent pathway, Pol alone would carry out both the insertion and extension steps of TLS, and because of the proficient ability of Pol to extend synthesis from the nt opposite 1-MeA, we expect that in the Pol-dependent TLS pathway, an as yet unidentified TLS Pol would insert a nt opposite 1-MeA from which Pol would extend (Fig. 5). We are now checking whether any of the Pols not yet examined for their role in TLS opposite 1-MeA, such as Pol, Pol, or Pol, could function as an inserter in this pathway.
Roles of TLS Pols in the Replication of 1-MeA-damaged DNA: Structural and Biochemical Considerations-In the Pol/Poldependent pathway, the ability of Pol to push 1-MeA into a syn conformation and to form a Hoogsteen base pair with the incoming T residue would allow it to carry out an nt insertion opposite 1-MeA from which Pol would extend (26,27). Previously, we have shown that Pol can replicate through the Tg lesion by both inserting an A opposite Tg and then by extending from there (9). The ability of Pol to extend synthesis on DNA containing a Tg lesion that blocks the base 5Ј to Tg from stacking above it, and the potential ability of Pol to extend from the 1-MeA:T Hoogsteen base pair handed over to it by Pol would suggest that Pol, an A-family Pol, can extend synthesis opposite from a diverse array of DNA lesions which distort the DNA helix or which inhibit the W-C base pairing.
Our genetic and biochemical observations support a role for Pol in replicating through the 1-MeA lesion by inserting a nt opposite 1-MeA and then by extending synthesis. Such a role for Pol is intriguing in view of the fact that biochemical studies have indicated that Pol uses W-C base pairing for DNA synthesis. For example, synthesis by Pol is severely impaired by a difluorotoluene base, which is virtually identical in shape, size, and conformation to thymine but lacks the ability to form W-C hydrogen bonds with adenine (28). And opposite the UV-induced cis-syn TT dimer, Pol forms W-C base pairs with an A opposite both the 3Ј-T and 5Ј-T residues of the dimer (29 -31). Curiously, although biochemical studies with yeast Pol have indicated that the incorporation of 1-MeA opposite the 3ЈT or the 5ЈT of a TT dimer or opposite the undamaged T residue is reduced by ϳ100 -200-fold compared with the incorporation of an A opposite the undamaged or UV-damaged T residue (30), human Pol inserts a T opposite 1-MeA almost as well as it inserts a T opposite undamaged A. It remains to be seen how in the absence of W-C base pairing, Pol can manage to insert the correct T nt opposite 1-MeA and then extend synthesis from the 1-MeA:T base pair.
In the Pol-dependent pathway, the proficient ability of Pol to extend synthesis from the 1-MeA:T base pair is in accord with the role of this Pol in extending synthesis opposite from a wide variety of DNA lesions that distort the DNA helix or which impair W-C base pairing. It may turn out that the TLS Pol found to be involved in inserting the nt opposite 1-MeA requires W-C hydrogen bonding for synthesizing DNA. The ability of TLS Pols such as Pol and others to bypass the requirement of W-C base-pairing opposite 1-MeA and presumably opposite other such lesions would suggest that at these lesion sites, TLS Pols can stabilize the nascent base pair in their active site by using means other than W-C hydrogen bonding.
Predominantly Error-free TLS Opposite 1-MeA-Rather surprisingly, in human cells, despite the poor fidelity of Pols and for nt insertion opposite 1-MeA, TLS opposite this lesion occurs with a high fidelity, and only ϳ1% of TLS products harbor mutations. The high fidelity with which TLS Pols manage to replicate 1-MeA damaged DNA recapitulates the relatively high fidelity TLS that occurs opposite the cis-syn TT dimer,  (6-4) TT photoproduct, and thymine glycol, where despite the low fidelity of DNA synthesis by the TLS Pols involved in their replicative bypass, only ϳ2% of TLS products harbor mutations (10 -12). Thus, opposite these different types of DNA lesions, which confer structural distortions or affect W-C base pairing, human cells have adapted TLS Pols to function in a much more error-free manner than would be predicted from their fidelity for nt incorporation. It remains enigmatic how human cells manage to achieve such a high fidelity for replicating through DNA lesions. A likely possibility is that protein-protein interactions and post-translational modifications regulate the fidelity of TLS Pols in human cells.