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Base-pairing partner modulates alkylguanine alkyltransferase Maureen McKeague, Claudia Otto, Michael H. Räz, Todor Angelov, and Shana J Sturla ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00446 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 28, 2018

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ACS Chemical Biology

Base-pairing partner modulates alkylguanine alkyltransferase Maureen McKeagueǂ, Claudia Ottoǂ, Michael H. Räz, Todor Angelov, Shana J. Sturla* Department of Health Sciences and Technology, ETH Zürich, Schmelzbergstrasse 9, 8092, Zürich, Switzerland

ABSTRACT O6-alkylguanine DNA adducts are repaired by the suicide enzyme alkylguanine alkyltransferase (AGT). AGT facilitates repair by binding DNA in the minor groove, flipping out the damaged base, and transferring the O6-alkyl group to a cysteine residue in the enzyme’s active site. Despite there being significant knowledge concerning the mechanism of AGT repair, there is limited insight regarding how altered interactions of the adduct with its complementary base in the DNA duplex influences its recognition and repair. In this study, the relationship of base pairing interactions and repair by human AGT (hAGT) was tested in the frequently mutated codon 12 of the KRAS gene with complementary sequences containing each canonical DNA base. The rate of O6-MeG repair decreased two-fold when O6-MeG was paired with G, whereas all other canonical bases had no impact on repair rate. We used a combination of biochemical studies, molecular modelling and artificial nucleobases to elucidate the mechanism accounting for the two-fold decrease. Our results suggest that the reduced rate of repair is due to O6-MeG adopting a syn conformation about the glycosidic bond precluding the formation of a repair-active complex. These data provide a novel chemical basis for how direct reversion repair may be impeded through modification of the base pair partner and support the use of artificial nucleobases as tools to probe the biochemistry of damage repair processes. ǂ

These authors contributed equally to this work *Corresponding author E-mail: [email protected] (S. J. Sturla)

Keywords: DNA damage, DNA repair, synthetic nucleobases, alkylguanine alkyltransferase

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Alkylguanine alkyltransferase (AGT) is a DNA repair protein that directly removes alkyl groups from DNA alkylation adducts by extruding the damaged DNA base from the duplex and transferring the alkyl group to an active site cysteine.1-5 AGT-associated functional motifs are conserved in alkyltransferase-like proteins throughout all domains of life.6, 7 AGT acts on O6-alkylguanine adducts, which arise from reactions of DNA with environmental, dietary, and endogenous alkylating agents, as well as drugs. These damage products are cytotoxic and mutagenic, therefore AGT is important for avoiding mutations and is also clinically relevant because it contributes to drug resistance in cancer chemotherapy by protecting tumor cells from the action of DNA alkylating drugs.5, 8-10 Small molecules such as O6–benzylguanine have undergone clinical trials to overcome drug resistance in cancer chemotherapy; however, the therapeutic potential remains unclear. As a result, probing previously unexplored and novel chemical space to modulate AGT function supports our understanding of cancer etiology and strategies for improving cancer therapeutics. There are several factors known to influence AGT repair rates and cellular expression levels: DNA sequence context,11-17 epigenetic silencing by MGMT promoter methylation,18, 19 mutations or single nucleotide polymorphisms,20-23 and the structure of the alkyl group being transferred.24-27 However, there is limited understanding of whether AGT function is influenced by interactions with the base that is paired opposite the alkylguanine. This knowledge gap is important to address because it could lead to completely novel therapeutic approaches for targeting AGT and overcoming its contribution to drug resistance. Moreover, there are several biologically relevant situations where understanding how interactions with the pairing partner modulate AGT function is critical. Examples include the use of nucleoside analogues as antiviral drugs, which are incorporated into DNA,28, 29 or epigenetic mechanisms that involve the presence of non-canonical bases in DNA.30, 31 We were particularly concerned about the biological relevance of DNA mismatches that are known to readily occur upon DNA synthesis opposite O6-MeG.32 For example, due to the altered hydrogen bonding, T is known to be misincorporated opposite O6-MeG at a high frequency.33 Given that O6-MeG blocks synthesis by prokaryotic and eukaryotic processive DNA polymerases, DNA damage tolerance pathways have evolved to rescue DNA replication. Specifically, specialized polymerases allow replication past the blocking DNA adduct in a process called translesion DNA synthesis.34 Several in vitro studies indicate that the translesion synthesis (TLS) polymerases also misincorporate A and G opposite O6-MeG.35 For example, yeast pol η incorporates G and A at an efficiency one order of magnitude lower than C and T nucleotides36 while G is incorporated with a similar efficiency to C opposite O6-MeG by human pol κ.37 Together, these results imply that TLS results in a variety of mismatched canonical bases opposite O6-MeG adducts. Few previous results hint at a potential base pair partner influence on the repair of O6-MeG. In one study, it was shown that changing the base opposite O6-MeG from C to T had essentially no influence on the rates of repair,38 but for the larger adduct O6-pyridyloxobutylguanine, repair by AGT was faster with T vs. C as the pairing partner.27, 39, 40 As another example, 5-methylcytosine paired opposite to O6-MeG reduced AGT repair rate.41 However, the biochemical basis for the altered repair is unknown. Furthermore, there is a lack of data concerning the impact of the other relevant basepairing partners, namely A and G. Therefore, we were interested whether the presence of O6-MeG:A and O6-MeG:G mismatches may reduce AGT repair efficiency. Significant insight concerning how non-canonical and chemically modified bases in DNA influence interactions with enzymes may be gained by using oligonucleotides containing synthetic base analogs as mechanistic probes.42, 43 Such an approach has been used in the study of AGT by using synthetic analogs of O6-MeG as repair substrates to dissect which positions of O6-methylG analogues interact with the enzyme.24 Furthermore, the fluorescent base analogue pyrroloC has been placed opposite O6-MeG and O6-benzylG to compare their flipping rates.44 Adduct-directed nucleobase analogues with variable chemical and physical properties can stabilize duplexes containing O6alkylguanine DNA 2 ACS Paragon Plus Environment

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adducts.45-47 The corresponding synthetic triphosphate analogues can be enzymatically incorporated opposite to DNA adducts thereby promoting extension past an O6-alkylguanine adduct.31, 48, 49 The resulting oligonucleotide products are uniquely suited to test the influences of altered pairing interactions on AGT recognition and repair in a bio-orthogonal manner, which has never been previously reported. To probe AGT repair modulation, we characterized the influence of the base pairing partner in DNA duplexes containing O6-MeG paired opposite all canonical nucleobases. DNA substrates were derived from a biologically relevant AGT substrate KRAS codon 12, which is frequently mutated in human cancers. Common GGTGAT mutations in KRAS are thought to be a consequence of O6-guanine alkylation, and are reduced in frequency with AGT expression.5, 50, 51 Here, we show that the rate of O6-MeG repair is attenuated when O6-MeG is paired with G. To probe the basis of the reduced repair rate we leveraged synthetic nucleobases with known differences in π-stacking, hydrogen bonding, size, and glycosidic bond angles. We compared the thermodynamic properties of the modified duplexes with protein binding, and used molecular modelling to assess the mechanism of reduced repair. The results illustrate that the base paired opposite to O6-MeG can inhibit AGT when it has a high propensity for adopting a syn glycosidic bond and demonstrate that adduct-directed synthetic nucleobases can serve as useful mechanistic probes for assessing DNA repair. RESULTS AND DISCUSSION Base pair partner influences AGT repair of O6-MeG. To measure the impact of base-pairing interactions on O6-MeG repair by hAGT, we determined repair rates as a function of opposing base (either C, T, A, or G) at a mutational hotspot in codon 12 of the KRAS gene. We measured residual O6MeG after reacting 24-mer DNA duplexes with recombinant hAGT (Figure 1). The analysis involved stable isotope dilution mass spectrometry according to a previously described method.40, 52 Apparent second order repair rates robs were calculated by fitting the remaining fraction of O6-MeG versus time (Figure 1C). In the standard case of O6-MeG opposite C, the rate of repair was 1.5 × 106 M-1s-1 (Table 1), similar to the rate previously measured in different sequences.14, 38, 52 When O6-MeG was opposite G rather than C, a 2-fold decrease in the repair rate was observed (0.7 × 106 M-1s-1: Figure 1C, Table 1). In contrast, when O6-MeG was paired with all the other canonical bases A and T, repair rates were similar to O6-MeG opposite C (1.7 and 1.4 × 106 M-1s-1, respectively: Figure 1C, Table 1). The repair rates for O6-MeG:C and O6-MeG:T were similar to previously published data obtained using different sequences;38 whereas rates for O6-MeG:A and O6-MeG:G have not been previously reported.

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Figure 1. AGT repair of O -MeG opposite canonical nucleobases. A) The KRAS codon 12 sequence was synthesized 6 containing O -MeG, indicated as *G. The complementary strand was synthesized containing either A, C, G, or T (indicated 6 by N in green). B) Work flow for measuring hAGT repair of O -MeG (orange) opposite different base pairing partners 6 (green). The sequences were annealed to form a duplex and incubated with AGT to initiate repair of O -MeG. After thermal 2 acid hydrolysis to release the purines, the reaction was purified by solid phase extraction and subjected to LCMS to 6 6 quantify the remaining O -MeG. C) Relative decrease in O -MeG upon hAGT repair of DNA duplexes as a function of the base paired opposite O6-MeG. The curve shown represent best fit of datasets to kt = 1 / (B0 – A0) ln A0 (B0 – Ct ) / B0 (A0 – Ct), 6 where k is the rate of O -MeG repair, A0 is the concentration of AGT protein in the repair assay, B0 is the initial 6 6 concentration of O -MeG containing DNA, and Ct is the concentration of O -MeG repaired at time t. Data represent the mean and standard deviation of three independent experiments.



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O6-MeG:G mismatch does not alter DNA duplex stability or AGT binding. The new observation that the O6-MeG:G mismatch influences AGT repair interested us as a new mechanism to inhibit AGT repair. Therefore, we sought to understand the mechanism of inhibition. While AGT repair is often measured using second order kinetic analysis, a more accurate model involves an initial binding step (Kd) then a first order reaction involving base flipping into the active site, followed by an irreversible transfer of the methyl group to AGT. Furthermore, in our biochemical assay there is an additional step involving the formation of the duplex DNA. Therefore, we evaluated both the thermal stability and thermodynamic parameters of the DNA duplexes, as well as the AGT binding to the duplexes.

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Scheme 1. Key steps in hAGT-mediated repair of O -MeG (orange) opposite different base pairing partners (green). Thermodynamic stability of the duplex is reflected in the equilibrium constant Kduplex. AGT binding and dissociation is 6 represented by the dissociation constant Kd. Experimental repair rates integrate the processes of O -MeG-flipping (kflip) and methyl transfer (ktrans).

Duplex formation was confirmed by native PAGE, and thermodynamic characteristics were determined by variable temperature UV melting analysis for DNA concentrations from 1 to 6 µM.53 When the G at position 11 was changed to O6-MeG, the duplex melting temperature (Tm) decreased from 79.2 to 74.5 °C (Table 1). This decrease is consistent with data from DNA methylation of different DNA sequences.47 When O6-MeG was paired with A, G, or T there were minimal changes in duplex melting (Table 1). Thermodynamic parameters were determined for all the duplexes by variable concentration thermal-denaturation experiments. Surprisingly, the least stable among the duplexes was the one containing O6-MeG paired with T,47, 54 whereas the stability of the duplex with opposing G was similar to C. From these results, we concluded that the reduced repair rate for the O6-MeG:G mismatch was not due to increased duplex stability. To determine whether the base pair partner impacts DNA duplex binding by hAGT and if diminished binding may account for lower repair rate, electrophoretic mobility shift analysis (EMSA) assays were conducted with increasing amounts of catalytically inactive hAGT C145S in protein:DNA ratios from 0 to 5. In this mutant, the active site cysteine (C145) is replaced by serine such that the DNA-binding and base-flipping properties are maintained, but the O6-alkyl transfer reaction cannot occur (Scheme 1).55 AGT exists as a small monomer that binds to DNA with modest affinity. However, solution studies indicate that AGT binding to DNA is cooperative, allowing a high density of binding to DNA (up to 4 bp/protein) which is proposed to facilitate rapid scanning of alkylated bases.55,56 Thus, we calculated AGT binding affinity to each duplex by measuring the ratio of free DNA (bottom band of gel; Figure S7 and S8) to total DNA. The ratio was fit as a function of AGT concentration using a previously described cooperative binding model Kd =[E]n[D]/[EnD], where n molecules of AGT (E) bind to one DNA duplex (D) (GraphPad Prism 7.03).16 AGT binding was strongest when O6-MeG was placed opposite A (Kd= 32 nM, Table 1), however, there was no significant difference between the opposing C, T, and G. These Kd values were similar to previously reported Kd values for duplexes with different sequences (27-76 nM).55 Binding data within the frequently mutated p53 derived sequences (codons 158, 245, and 248; 19-21-mer duplexes), using the alanine AGT mutant (C145A) show Kd values ranging from 0.6-1.3 µM.30 Therefore, we concluded that the binding affinity was also not responsible for the reduced rate of repair observed for the O6-MeG:G mismatch. 4 ACS Paragon Plus Environment

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Table 1. Apparent second order repair rates and dissociation temperatures (Tm) and thermodynamic parameters of duplexes. b Duplex robs robs 6 -1 -1 [x10 M s ] Fold Change G:C ----O6-MeG:C 1.5 ± 0.3 1 6 O -MeG:A 1.7 ± 0.3 1.1 O6-MeG:T 1.4 ± 0.3 0.9 6 O -MeG:G 0.7 ± 0.1 0.5

constants of hAGT and methylated duplexes, melting Kd [nM]

c

--58 ± 6 32 ± 18 77 ± 11 73 ± 14

Tmobs at 6 μM ○ d [ C] 79.2 ± 0.1 74.5 ± 0.1 74.1 ± 0.2 73.1 ± 0.1 74.3 ± 0.2

○ e

ΔG, 16 C [kJ/mol] -151.1 ± 1.3 -141.7 ± 0.4 -139.1 ± 3.8 -121.9 ± 0.2 -137.3 ± 1.3

a Values are mean ± standard deviation from three independent experiments. b Relative rates are with respect to O6-MeG:C. c Dissociation constants were derived from electrophoresis mobility shift assays. Gel images and binding isotherms available in the Supporting Information. d Tm was analyzed at different duplex concentrations (1, 2, 3, 4, 6 µM); values shown for 6 µM duplex concentrations. Representative plots available in the Supporting Information. e Free energy values ∆G were determined at AGT repair reaction conditions (16 °C). ΔH and ΔS available in Table S1.

On the basis of these results and the processes illustrated in Scheme 1, the reduced rate of alkyl repair observed with O6-MeG:G must be related to the rates of base flipping and the alkyl transfer. In a previous study, Zang and coworkers measured the impact of alkyl substituents on repair rates using O6-MeG vs. O6-BenzylG, and found that for O6-MeG, the alkyl transfer step was the slowest forward process.44 However, in our present study, the chemical identity of the transferred alkyl group is unchanged, therefore a significant difference in the kinetics of the alkyl transfer step is unlikely. This situation therefore points toward a reduction in base flipping rates as a basis of repair inhibition. Unfortunately, base flipping is measured by placing a pyrroloC opposite the adduct being flipped, and thus cannot be translated to our study comparing various opposing base pair partners. We hypothesized that a reduced capacity for AGT to flip O6-MeG out of the duplex could be related to non-canonical conformations of one of the nucleobases resulting from the mismatch (either the substrate O6-MeG, or its base pairing partner G). Indeed, NMR studies indicated that when A, C, or T are paired opposite O6-MeG, both nucleobases remain in the anti conformation. Conversely, for O6MeG:G, the O6-MeG adopts a syn conformation (Figure 2A).57 This altered conformation could indeed impact the rate of base flipping. To further test this hypothesis, we employed a combination of modelling and synthetic nucleobases to test the impact of conformational changes on repair efficiency. Synthetic nucleobases as conformational probes. Unnatural nucleobases have been used to detect conformational changes at modification sites58 as well as to probe local folding dynamics.59 Here, we tested the use of synthetic nucleobases with altered π-stacking, hydrogen bonding, and size to probe the impact of conformational changes on the repair capacity of AGT. A recently described suite of synthetic nucleobases containing hydrophobic base analogues and hydrogen bonding capacities have been synthesized and reported to preferentially pair with O6-alkylguanine adducts.46, 47, 60 These probes provided insight into the influence of hydrogen bonding, base pair size, and nucleobase shape on DNA synthesis past O6-MeG adducts. A crystal structure of the perimidinone-derived synthetic nucleobase Per revealed that it adopts a syn conformation when paired to O6-BnG, indicating its potential utility as a conformational probe within the O6-MeG containing base pair.61 We conducted molecular mechanics simulations to characterize the Per probe and a smaller variant, Benzi (Figure 2B,C) opposite O6-MeG (Molecular Operating Environment, MOE). Molecular mechanics is a well-established computational approach, which can provide insights of biological interest not accessible by experimental methods. Computational modeling has been effective for elucidating DNA-protein interactions ranging from methyltransferase-mediated base flipping,62 specific recognition of a DNA adducts by 8-oxoguanine-glycosylase,63 as well as opening-64 and closing-65 dynamics of DNA polymerase I in DNA replication. In addition, in silico modeling of nucleic acids has been applied to understand conformational features of Watson-Crick-66 and synthetic base pairs,67-69 or artificial DNA backbone topologies.70 Hydrogen bonding patterns between canonical and synthetic bases, related to the probe used in this study, in the active site of polymerase enzymes,31, 71, 72 were 5 ACS Paragon Plus Environment


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predicted by the same modeling approach used herein, and further supported by x-ray crystallography.73 We used the two most relevant and available experimental structures as starting points: the solution structure of a Dickerson-Drew dodecamer duplex (pdb file 2M1161, Figure 2C) and the solid state structure of hAGT C145S in a DNA-bound base-extruded complex with O6-MeG opposite C (pdb file 1T384, Figure S11, Table S3). By replacing the O6-BnG with O6-MeG in the Dickerson-Drew dodecamer duplex, we calculated the potential energies (Epot), number of putative hydrogen bonds, and minor groove widths with both G, C, Per, and Benzi opposite O6-MeG in either a syn or anti conformation. The results of the modelling indicated the lowest potential energies when O6-MeG is in a syn conformation (Figure 2D), thus supporting Per as a nucleobase analogue to probe the impact of repair of O6-MeG in a syn conformation. The predicted structure with the lowest potential energy was O6-MeG(syn):Per(syn), which precludes hydrogen bonding (Figure 2B,C). This observation is consistent with a previous study indicating that π-stacking interactions of these nucleobase analogs promote duplex stability.74However, the next-lowest energy structure was with Per in the anti conformation (Figure S11), thus restoring hydrogen bonding. Thus, whether Per is in the syn or anti conformation, for both lowest energy conformations, O6-MeG is predicted to be in a syn conformation. In contrast, there is a smaller difference between the minimized potential energies of O6-MeG in syn vs anti conformation when paired with Benzi compared to when they are paired with Per (Figure 2D), potentially due to the smaller size of Benzi, which may facilitate a greater ease of rotational isomerism (Figure 2, Table S2, Table S3).75

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Figure 2. Base pairing of O -MeG with synthetic nucleobase probes Per and Benzi. A) Structure and hydrogen bonding 6 6 6 6 pattern of O -MeG:C and O -MeG:G mismatch B) Structure of hypothetical of O -MeG(anti):Benzi and O -MeG(syn):Per 6 based on the crystal structure of O -BnG:Per (pdb file 2M11s) and computational modelling. C) Computational modelling 6 (molecular mechanics) of the modified Dickerson-Drew dodecamer at the site of methylation. The O -MeG adduct is modeled in anti (I-IV) or syn (V-VIII) conformation and paired with C, G, or PER in anti- (I-III and V-VII) or PER in syn conformation (IV and VIII) as base pairing partners. Predicted hydrogen bonds between the base pairs are shown as red dashed lines. D) Minimized potential energies calculated using molecular modelling of the modified Dickerson-Drew dodecamer duplex structure (pdb file 2M11s).


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With an indication that the synthetic probes may allow us to interrogate the impact of O6-MeG nucleoside configuration on its repair, we evaluated the stability the KRAS DNA duplex and binding affinity of AGT when Per and Benzi were present. When Per or Benzi were paired opposite O6-MeG, we observed relatively little change in hAGT binding affinity (Kd = 118 and 77 nM, Table 2) and the duplex stability (Tm = 75.4 and 74.1 °C, Table 2) compared to duplexes containing the canonical nucleobases. These data are consistent with the results from O6-MeG:G and is also consistent with previous results of mechanistic studies of AGT that indicate the alkyl transfer, not AGT binding, is the rate limiting step.44 Furthermore, AGT has a high affinity for unmodified DNA, binding to and flipping out even unmodified guanine bases.5 Thus, despite the large errors in Km, previous work and the EMSA data support the indication that base-pairing partner does not significantly impact AGT binding. As a result, we focused our further analysis on the potential impact of altered adduct conformation on repair rate. Altered adduct conformation may reduce repair rate. To further interrogate differences in repair proficiency resulting from altered adduct conformation, we measured the apparent second order repair rates with the synthetic nucleobase probes opposite O6-MeG in the KRAS duplex. Benzi served as a probe of the typical anti conformation of O6-MeG (as observed with canonical bases A, C, and T), and when it was opposite O6-MeG, there was minimal change from the rate measured for O6-MeG:C (1.2 × 106 M-1s-1 vs 1.5 × 106 M-1s-1, respectively, Table 2). In contrast, the repair rate was reduced 5fold, to 0.3 × 106 M-1s-1 (Table 2) when O6-MeG was paired opposite Per, a probe of the potential influence of O6-MeG in a syn conformation. The suggestion that O6-MeG adopts a syn conformation opposite Per is based on previous crystallographic data for a related adduct,61 together with the results of modelling studies described above, however it cannot be excluded that the large size of Per may reduce the repair rate by some other direct interaction with the protein. However, results for repair of O6-MeG opposite an abasic site, where there are no predicted steric effects, indicate similar binding and rates of repair (Table S2) to those of O6-MeG paired to A, C, T, and Benzi (Table 1). Furthermore, modelling results of Per in the AGT complex (pdb file 1T38) do not predict any steric clash and, importantly, Per is away from the active site (Figure S11). These observations suggest that probe sterics influence conformational changes to the DNA, but direct steric interactions of the probe with the protein alone does not have a significant influence on the observed reduction in repair rates. Therefore, taken together, the results of all of these experiments point toward the reduced repair rate observed for O6-MeG paired to G could be explained by the adduct experiencing a conformational change such adopting a syn conformation about the glycosidic bond. While approximations of relative AGT repair rates when O6-MeG was paired opposite C, G, Benzi, or Per were readily obtained from fitting data as a second order repair process (Table 2), the process is more complex due to the high affinity of AGT for DNA. Thus, for more detailed insight concerning AGT repair, the process is more accurately modeled on the basis of a reversible protein:DNA binding step, followed by a first-order chemical reaction (Scheme 1).17, 44 Therefore, we repeated our evaluation of the AGT repair, this time using a more rapid sampling method combined with a range of increasing AGT concentrations. We plotted the first-order rate constants against the initial concentration of AGT. The Km and kinact (where the kinact of the reaction represent a combination of the kflip and ktrans constants) were then derived as previously described.4, 44. The results demonstrate that O6-MeG opposite G or Per cause a 2-3 fold decrease in the first order rate constants as compared to C and Benzi (Table 2). Together with the conformational preferences of these pairing interactions, these results are consistent with conformational changes such as O6-MeG adopting a syn glycosidic bond formation reduce AGT repair efficiency. This finding is of biological relevance given that G can be inserted opposite O6-MeG by TLS polymerases.36, 37



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Table 2. Thermodynamic and kinetic parameters for the reaction of hAGT and duplexes containing synthetic nucleobases. a b c d ○ f g h h Duplex robs Kd [nM] Tmobs at 6 kinact ΔG, 16 C Km kinact robs 6 -1 -1 ○ e -1 [x10 M s ] Fold μM [ C] [nM] [s ] Fold [kJ/mol] Change Change O6-MeG:C 1.5 ± 0.3 1 58 ± 6 74.5 ± 0.1 -141.7 ± 0.4 560 ± 400 0.22 ± 0.06 1 6 O -MeG:Benzi 1.2 ± 0.1 0.8 77 ± 17 74.1 ± 0.1 -128.3 ± 1.5 250 ± 70 0.18 ± 0.02 0.8 O6-MeG:G 0.7 ± 0.1 0.5 73 ± 14 74.3 ± 0.2 -137.3 ± 1.3 170 ± 50 0.12 ± 0.01 0.5 6 O -MeG:Per 0.3 ± 0.1 0.2 118 ± 45 75.4 ± 0.2 -132.3 ± 0.5 660 ± 500 0.06 ± 0.02 0.3 a

Values of robs, Kd, Tmobs and ΔG for O6-MeG:C and O6-MeG:G are from Table 1 for comparison purposes. b Apparent second order reaction rates. c Relative rates are with respect to O6-MeG:C. d Dissociation constants were derived from electrophoresis mobility shift assays with the inactive hAGT C145S. Gel images and binding isotherms available in the Supporting Information. e Tm was analyzed at different duplex concentrations (1, 2, 3, 4, 6 µM); values shown for 6 µM duplex concentrations. Representative plots available in the Supporting Information. f Free energy values ∆G were determined at AGT repair reaction conditions (16 °C). ΔH and ΔS available in Table S1. g Km represents the binding affinity with active hAGT calculated by plotting the first-order rate constants against the initial concentration of AGT (Figure S12 and S13). h Kinact represents the rate of methyl transfer; value calculated by plotting the first-order rate constants against the initial concentration of AGT (Figure S12 and S13). i Relative rates of kinact are with respect to O6-MeG:C.

In summary, results obtained in this study demonstrate for the first time a chemical basis of how AGT repair of O6-MeG depends on the structure of the base it is paired with. It provides new insight regarding how altering base-pairing interactions could serve as a strategy for AGT inhibition. Furthermore, elucidation of fundamental aspects of AGT repair with regards to peripheral modifications in its DNA substrate has yielded new mechanistic insight relevant in the context of situations involving DNA alterations, such as base mismatches, nucleoside drugs, non-canonical base biotechnologies, or epigenetic processes.

METHODS A description of all chemicals, enzymes, instrumentation, apparatus, and synthesis procedures is available in the Supporting Information. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website and includes experimental procedures and methods, supporting figures and table.

ACKNOWLEDGEMENTS We thank S. Kanugula, Pennsylvania State University, for providing expression plasmids and purified human AGT and AGT C145S proteins, and P. Röthlisberger and C. Leumann, University of Bern, for providing phosphoramidite reagents that enabled preliminary experiments. We are grateful to H. Gahlon, ETH Zurich, M. Egli, Vanderbilt University, and C. Oostenbrink, BOKU, Vienna, for valuable discussion. This work was supported by the European Research Council (260341), the Swiss National Science Foundation (156280), and the ETH research commission (ETH-43 14-1).

REFERENCES 1. Demple, B., Jacobsson, A., Olsson, M., Robins, P., and Lindahl, T. (1982) Repair of alkylated DNA in Escherichia coli. Physical properties of O6-methylguanine-DNA methyltransferase, J. Biol. Chem. 257, 13776-13780. 2. Pegg, A. E., Dolan, M. E., Scicchitano, D., and Morimoto, K. (1985) Studies of the repair of O6alkylguanine and O4-alkylthymine in DNA by alkyltransferases from mammalian cells and bacteria, Environ. Health Perspect. 62, 109-114. 8 ACS Paragon Plus Environment

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3. Pegg, A. E., Dolan, M. E., and Moschel, R. C. (1995) Structure, function, and inhibition of O6alkylguanine-DNA alkyltransferase, Prog. Nucleic Acid Res. Mol. Biol. 51, 167-223. 4. Daniels, D. S., Woo, T. T., Luu, K. X., Noll, D. M., Clarke, N. D., Pegg, A. E., and Tainer, J. A. (2004) DNA binding and nucleotide flipping by the human DNA repair protein AGT, Nat. Struct. Mol. Biol. 11, 714-720. 5. Pegg, A. E. (2011) Multifaceted Roles of Alkyltransferase and Related Proteins in DNA Repair, DNA Damage, Resistance to Chemotherapy, and Research Tools, Chem. Res. Toxicol. 24, 618-639. 6. Cline, S. D., and Hanawalt, P. C. (2003) Who's on first in the cellular response to DNA damage?, Nat. Rev. Mol. Cell Biol. 4, 361-372. 7. Tubbs, J. L., Latypov, V., Kanugula, S., Butt, A., Melikishvili, M., Kraehenbuehl, R., Fleck, O., Marriott, A., Watson, A. J., Verbeek, B., McGown, G., Thorncroft, M., Santibanez-Koref, M. F., Millington, C., Arvai, A. S., Kroeger, M. D., Peterson, L. A., Williams, D. M., Fried, M. G., Margison, G. P., Pegg, A. E., and Tainer, J. A. (2009) Flipping of alkylated DNA damage bridges base and nucleotide excision repair, Nature 459, 808-813. 8. Gerson, S. L. (2002) Clinical relevance of MGMT in the treatment of cancer, J. Clin. Oncol. 20, 23882399. 9. Margison, G. P., and Santibanez-Koref, M. F. (2002) O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy, BioEssays : news and reviews in molecular, cellular and developmental biology 24, 255-266. 10. Kaina, B., Margison, G. P., and Christmann, M. (2010) Targeting O(6)-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy, Cell. Mol. Life Sci. 67, 3663-3681. 11. Scicchitano, D., Jones, R. A., Kuzmich, S., Gaffney, B., Lasko, D. D., Essigmann, J. M., and Pegg, A. E. (1986) Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanineDNA-alkyltransferase, Carcinogenesis 7, 1383-1386. 12. Dolan, M. E., Oplinger, M., and Pegg, A. E. (1988) Sequence specificity of guanine alkylation and repair, Carcinogenesis 9, 2139-2143. 13. Georgiadis, P., Smith, C. A., and Swann, P. F. (1991) Nitrosamine-induced cancer: selective repair and conformational differences between O6-methylguanine residues in different positions in and around codon 12 of rat H-ras, Cancer Res. 51, 5843-5850. 14. Bender, K., Federwisch, M., Loggen, U., Nehls, P., and Rajewsky, M. F. (1996) Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context, Nucleic Acids Res. 24, 2087-2094. 15. Delaney, J. C., and Essigmann, J. M. (1999) Context-dependent mutagenesis by DNA lesions, Chem. Biol. 6, 743-753. 16. Meyer, A. S., McCain, M. D., Fang, Q., Pegg, A. E., and Spratt, T. E. (2003) O6-alkylguanine-DNA alkyltransferases repair O6-methylguanine in DNA with Michaelis-Menten-like kinetics, Chem. Res. Toxicol. 16, 1405-1409. 17. Coulter, R., Blandino, M., Tomlinson, J. M., Pauly, G. T., Krajewska, M., Moschel, R. C., Peterson, L. A., Pegg, A. E., and Spratt, T. E. (2007) Differences in the rate of repair of O6-alkylguanines in different sequence contexts by O6-alkylguanine-DNA alkyltransferase, Chem. Res. Toxicol. 20, 1966-1971. 18. von Wronski, M. A., Harris, L. C., Tano, K., Mitra, S., Bigner, D. D., and Brent, T. P. (1992) Cytosine methylation and suppression of O6-methylguanine-DNA methyltransferase expression in human rhabdomyosarcoma cell lines and xenografts, Oncol. Res. 4, 167-174. 19. von Wronski, M. A., and Brent, T. P. (1994) Effect of 5-azacytidine on expression of the human DNA repair enzyme O6-methylguanine-DNA methyltransferase, Carcinogenesis 15, 577-582. 20. Margison, G. P., Heighway, J., Pearson, S., McGown, G., Thorncroft, M. R., Watson, A. J., Harrison, K. L., Lewis, S. J., Rohde, K., Barber, P. V., O'Donnell, P., Povey, A. C., and Santibanez-Koref, M. F. (2005) Quantitative trait locus analysis reveals two intragenic sites that influence O69 ACS Paragon Plus Environment


Page 10 of 14

alkylguanine-DNA alkyltransferase activity in peripheral blood mononuclear cells, Carcinogenesis 26, 1473-1480. 21. Candiloro, I. L., and Dobrovic, A. (2009) Detection of MGMT promoter methylation in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter single nucleotide polymorphism, Cancer Prev. Res. 2, 862-867. 22. Zhong, Y., Huang, Y., Huang, Y., Zhang, T., Ma, C., Zhang, S., Fan, W., Chen, H., Qian, J., and Lu, D. (2010) Effects of O6-methylguanine-DNA methyltransferase (MGMT) polymorphisms on cancer: a meta-analysis, Mutagenesis 25, 83-95. 23. Leng, S., Bernauer, A. M., Hong, C., Do, K. C., Yingling, C. M., Flores, K. G., Tessema, M., Tellez, C. S., Willink, R. P., Burki, E. A., Picchi, M. A., Stidley, C. A., Prados, M. D., Costello, J. F., Gilliland, F. D., Crowell, R. E., and Belinsky, S. A. (2011) The A/G allele of rs16906252 predicts for MGMT methylation and is selectively silenced in premalignant lesions from smokers and in lung adenocarcinomas, Clin. Cancer Res. 17, 2014-2023. 24. Spratt, T. E., Wu, J. D., Levy, D. E., Kanugula, S., and Pegg, A. E. (1999) Reaction and binding of oligodeoxynucleotides containing analogues of O6-methylguanine with wild-type and mutant human O6-alkylguanine-DNA alkyltransferase, Biochemistry 38, 6801-6806. 25. Peterson, L. A., Vu, C., Hingerty, B. E., Broyde, S., and Cosman, M. (2003) Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex, Biochemistry 42, 13134-13144. 26. Mijal, R. S., Thomson, N. M., Fleischer, N. L., Pauly, G. T., Moschel, R. C., Kanugula, S., Fang, Q., Pegg, A. E., and Peterson, L. A. (2004) The repair of the tobacco specific nitrosamine derived adduct O6-[4-Oxo-4-(3-pyridyl)butyl]guanine by O6-alkylguanine-DNA alkyltransferase variants, Chem. Res. Toxicol. 17, 424-434. 27. Mijal, R. S., Kanugula, S., Vu, C. C., Fang, Q., Pegg, A. E., and Peterson, L. A. (2006) DNA sequence context affects repair of the tobacco-specific adduct O(6)-[4-Oxo-4-(3-pyridyl)butyl]guanine by human O(6)-alkylguanine-DNA alkyltransferases, Cancer Res. 66, 4968-4974. 28. Triemer, T., Messikommer, A., Glasauer, S. M. K., Alzeer, J., Paulisch, M. H., and Luedtke, N. W. (2018) Superresolution imaging of individual replication forks reveals unexpected prodrug resistance mechanism, Proc. Natl. Acad. Sci. 115, E1366-E1373. 29. Poirier, M. C., Olivero, O. A., Walker, D. M., and Walker, V. E. (2004) Perinatal genotoxicity and carcinogenicity of anti-retroviral nucleoside analog drugs, Toxicol. Appl. Pharmacol. 199, 151161. 30. Guza, R., Ma, L., Fang, Q., Pegg, A. E., and Tretyakova, N. (2009) Cytosine methylation effects on the repair of O6-methylguanines within CG dinucleotides, J. Biol. Chem. 284, 22601-22610. 31. Nilforoushan, A., Furrer, A., Wyss, L. A., van Loon, B., and Sturla, S. J. (2015) Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase eta by reducing synthesis in the presence of the major cisplatin DNA adduct, J. Am. Chem. Soc. 137, 47284734. 32. Fu, D., Calvo, J. A., and Samson, L. D. (2012) Balancing repair and tolerance of DNA damage caused by alkylating agents, Nat. Rev. Cancer 12, 104-120. 33. Singh, J., Su, L., and Snow, E. T. (1996) Replication across O6-methylguanine by human DNA polymerase beta in vitro. Insights into the futile cytotoxic repair and mutagenesis of O6methylguanine, J. Biol. Chem. 271, 28391-28398. 34. Raz, M. H., Dexter, H. R., Millington, C. L., van Loon, B., Williams, D. M., and Sturla, S. J. (2016) Bypass of Mutagenic O(6)-Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases, Chem. Res. Toxicol. 29, 1493-1503. 35. Choi, J. Y., Chowdhury, G., Zang, H., Angel, K. C., Vu, C. C., Peterson, L. A., and Guengerich, F. P. (2006) Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases, J. Biol. Chem. 281, 38244-38256. 36. Haracska, L., Prakash, S., and Prakash, L. (2000) Replication past O(6)-methylguanine by yeast and human DNA polymerase eta, Mol. Cell. Biol. 20, 8001-8007. 10 ACS Paragon Plus Environment

Page 11 of 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37. Haracska, L., Prakash, L., and Prakash, S. (2002) Role of human DNA polymerase kappa as an extender in translesion synthesis, Proc. Natl. Acad. Sci. 99, 16000-16005. 38. Lips, J., and Kaina, B. (2001) Repair of O(6)-methylguanine is not affected by thymine base pairing and the presence of MMR proteins, Mutat. Res. 487, 59-66. 39. Kotandeniya, D., Murphy, D., Seneviratne, U., Guza, R., Pegg, A., Kanugula, S., and Tretyakova, N. (2011) Mass spectrometry based approach to study the kinetics of O6-alkylguanine DNA alkyltransferase-mediated repair of O6-pyridyloxobutyl-2'-deoxyguanosine adducts in DNA, Chem. Res. Toxicol. 24, 1966-1975. 40. Kotandeniya, D., Murphy, D., Yan, S., Park, S., Seneviratne, U., Koopmeiners, J. S., Pegg, A., Kanugula, S., Kassie, F., and Tretyakova, N. (2013) Kinetics of O(6)-pyridyloxobutyl-2'deoxyguanosine repair by human O(6)-alkylguanine DNA alkyltransferase, Biochemistry 52, 4075-4088. 41. Bentivegna, S. S., and Bresnick, E. (1994) Inhibition of human O6-methylguanine-DNA methyltransferase by 5-methylcytosine, Cancer. Res. 54, 327-329. 42. Dziuba, D., Jurkiewicz, P., Cebecauer, M., Hof, M., and Hocek, M. (2016) A Rotational BODIPY Nucleotide: An Environment-Sensitive Fluorescence-Lifetime Probe for DNA Interactions and Applications in Live-Cell Microscopy, Angew. Chem. Int. Ed. Engl. 55, 174-178. 43. Cahova, H., Panattoni, A., Kielkowski, P., Fanfrlik, J., and Hocek, M. (2016) 5-Substituted Pyrimidine and 7-Substituted 7-Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs, ACS Chem. Biol. 11, 31653171. 44. Zang, H., Fang, Q., Pegg, A. E., and Guengerich, F. P. (2005) Kinetic analysis of steps in the repair of damaged DNA by human O6-alkylguanine-DNA alkyltransferase, J. Biol. Chem. 280, 3087330881. 45. Dahlmann, H. A., Vaidyanathan, V. G., and Sturla, S. J. (2009) Investigating the biochemical impact of DNA damage with structure-based probes: abasic sites, photodimers, alkylation adducts, and oxidative lesions, Biochemistry 48, 9347-9359. 46. Gong, J., and Sturla, S. J. (2007) A synthetic nucleoside probe that discerns a DNA adduct from unmodified DNA, J. Am. Chem. Soc. 129, 4882-4883. 47. Gahlon, H. L., and Sturla, S. J. (2013) Hydrogen bonding or stacking interactions in differentiating duplex stability in oligonucleotides containing synthetic nucleoside probes for alkylated DNA, Chemistry 19, 11062-11067. 48. Gahlon, H. L., Schweizer, W. B., and Sturla, S. J. (2013) Tolerance of base pair size and shape in postlesion DNA synthesis, J. Am. Chem. Soc. 135, 6384-6387. 49. Wyss, L. A., Nilforoushan, A., Eichenseher, F., Suter, U., Blatter, N., Marx, A., and Sturla, S. J. (2015) Specific incorporation of an artificial nucleotide opposite a mutagenic DNA adduct by a DNA polymerase, J. Am. Chem. Soc. 137, 30-33. 50. Swann, P. F. (1990) Why do O6-alkylguanine and O4-alkylthymine miscode? The relationship between the structure of DNA containing O6-alkylguanine and O4-alkylthymine and the mutagenic properties of these bases, Mutat. Res. 233, 81-94. 51. Ronai, Z. A., Gradia, S., Peterson, L. A., and Hecht, S. S. (1993) G to A transitions and G to T transversions in codon 12 of the Ki-ras oncogene isolated from mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related DNA methylating and pyridyloxobutylating agents, Carcinogenesis 14, 2419-2422. 52. Guza, R., Rajesh, M., Fang, Q., Pegg, A. E., and Tretyakova, N. (2006) Kinetics of O(6)-methyl-2'deoxyguanosine repair by O(6)-alkylguanine DNA alkyltransferase within K-ras gene-derived DNA sequences, Chem. Res. Toxicol. 19, 531-538. 53. Angelov, T., Dahlmann, H. A., and Sturla, S. J. (2013) Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O(6)methyl-G in DNA duplexes, Bioorg. Med. Chem. 21, 6212-6216.



Page 12 of 14

54. Gaffney, B. L., and Jones, R. A. (1989) Thermodynamic comparison of the base pairs formed by the carcinogenic lesion O6-methylguanine with reference both to Watson-Crick pairs and to mismatched pairs, Biochemistry 28, 5881-5889. 55. Melikishvili, M., and Fried, M. G. (2012) Lesion-specific DNA-binding and repair activities of human O(6)-alkylguanine DNA alkyltransferase, Nucleic Acids Res. 40, 9060-9072. 56. Fried, M., and Crothers, D. M. (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis, Nucleic Acids Res. 9, 6505-6525. 57. Patel, D. J., Shapiro, L., Kozlowski, S. A., Gaffney, B. L., Kuzmich, S., and Jones, R. A. (1985) Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes, Biochimie 67, 861-886. 58. Berger, F. D., Sturla, S. J., Kung, R. W., Montina, T., Wetmore, S. D., and Manderville, R. A. (2018) Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl-Guanine Lesion in the NarI Mutational Hotspot: Evidence for Enhanced Syn Adduct Formation, Chem. Res. Toxicol. 31, 37-47. 59. Mata, G., and Luedtke, N. W. (2015) Fluorescent probe for proton-coupled DNA folding revealing slow exchange of i-motif and duplex structures, J. Am. Chem. Soc. 137, 699-707. 60. Trantakis, I. A., Nilforoushan, A., Dahlmann, H. A., Stauble, C. K., and Sturla, S. J. (2016) In-Gene Quantification of O(6)-Methylguanine with Elongated Nucleoside Analogues on Gold Nanoprobes, J. Am. Chem. Soc. 138, 8497-8504. 61. Kowal, E. A., Lad, R. R., Pallan, P. S., Dhummakupt, E., Wawrzak, Z., Egli, M., Sturla, S. J., and Stone, M. P. (2013) Recognition of O6-benzyl-2'-deoxyguanosine by a perimidinone-derived synthetic nucleoside: a DNA interstrand stacking interaction, Nucleic Acids Res. 41, 75667576. 62. Huang, N., Banavali, N. K., and MacKerell, A. D., Jr. (2003) Protein-facilitated base flipping in DNA by cytosine-5-methyltransferase, Proc. Natl. Acad. Sci. 100, 68-73. 63. Banerjee, A., Yang, W., Karplus, M., and Verdine, G. L. (2005) Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA, Nature 434, 612-618. 64. Miller, B. R., 3rd, Parish, C. A., and Wu, E. Y. (2014) Molecular dynamics study of the opening mechanism for DNA polymerase I, PLoS Comput. Biol. 10, e1003961. 65. Miller, B. R., 3rd, Beese, L. S., Parish, C. A., and Wu, E. Y. (2015) The Closing Mechanism of DNA Polymerase I at Atomic Resolution, Structure 23, 1609-1620. 66. Poltev, V. I., Anisimov, V. M., Sanchez, C., Deriabina, A., Gonzalez, E., Garcia, D., Rivas, F., and Polteva, N. A. (2016) Analysis of the conformational features of Watson–Crick duplex fragments by molecular mechanics and quantum mechanics methods, Biophysics 61, 217226. 67. Cosman, M., Hingerty, B. E., Luneva, N., Amin, S., Geacintov, N. E., Broyde, S., and Patel, D. J. (1996) Solution conformation of the (-)-cis-anti-Benzo[a]pyrenyl-dG adduct opposite dC in a DNA duplex: Intercalation of the covalently attached BP ring into the helix with base displacement of the modified deoxyguanosine into the major groove, Biochemistry 35, 98509863. 68. Xie, X. M., Geacintov, N. E., and Broyde, S. (1999) Origins of conformational differences between cis and trans DNA adducts derived from enantiomeric anti-benzo[a]pyrene diol epoxides, Chem. Res. Toxicol. 12, 597-609. 69. Roethlisberger, P., Istrate, A., Marcaida Lopez, M. J., Visini, R., Stocker, A., Reymond, J. L., and Leumann, C. J. (2016) X-ray structure of a lectin-bound DNA duplex containing an unnatural phenanthrenyl pair, Chem. Commun. (Cambridge, U. K.) 52, 4749-4752. 70. Evequoz, D., and Leumann, C. J. (2017) Probing the Backbone Topology of DNA: Synthesis and Properties of 7',5'-Bicyclo-DNA, Chemistry 23, 7953-7968. 71. Gahlon, H. L., Boby, M. L., and Sturla, S. J. (2014) O6-alkylguanine postlesion DNA synthesis is correct with the right complement of hydrogen bonding, ACS Chem. Biol. 9, 2807-2814.


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72. Wyss, L. A., Nilforoushan, A., Williams, D. M., Marx, A., and Sturla, S. J. (2016) The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts, Nucleic Acids Res. 44, 6564-6573. 73. Betz, K., Nilforoushan, A., Wyss, L. A., Diederichs, K., Sturla, S. J., and Marx, A. (2017) Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase, Chem. Commun. (Cambridge, U. K.) 53, 12704-12707. 74. Dahlmann, H. A., Berger, F. D., Kung, R. W., Wyss, L. A., Gubler, I., McKeague, M., Wetmore, S. D., and Sturla, S. J. (2018) Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O6-Alkylguanine Adducts, Helv. Chim. Acta 101, e1800066. 75. Hritz, J., and Oostenbrink, C. (2009) Efficient free energy calculations for compounds with multiple stable conformations separated by high energy barriers, J. Phys. Chem. B 113, 12711-12720.



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