Adduct Fluorescence as a Tool to Decipher Sequence Impact on

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Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion. Florence Daniela Berger, Richard A. Manderville, and Shana J Sturla Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00016 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on March 9, 2019

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Chemical Research in Toxicology

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Florence D. Berger, † Richard A. Manderville*,‡ and Shana J. Sturla,*, †

†Department

‡

of Health Sciences and Technology, ETH Zurich, 8092 Zurich, Switzerland

Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada N1G

2W1

*Corresponding Authors: Richard A. Manderville: Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada, N1G 2W1 Tel: 519-824-4120, x53963 E-mail: [email protected] Shana J. Sturla: Department of Health Sciences and Technology ETH Zürich, 8092 Zürich, Switzerland Tel:+41 44 632 91 75 E-mail: [email protected] 1

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ABSTRACT: Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2′-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CGdinucleotide repeat sequences, such as NarI (5′-GGCXCC-3′, where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts and hence chemistry-based strategies are in need for probing relationships between nucleic acid sequence and structure with replication errors. In the

NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) 4


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bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (−2 frameshift mutation).

In the current study, incorporating the fluorescent C-linked 4-

fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CGdinucleotide repeats (NarI: 3′-CXCGGC-5′ and CG3: 3′-CXCGCG-5′, X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo− (Kf−) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf− extended the primer past FBP-dG within a CG3 template to afford full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes.

This 5



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fluorescence sensitivity provides a rationale for sequence impact on −2 frameshift mutations mediated by the C-linked FBP-dG lesion.

INTRODUCTION DNA adducts (addition products) can be an initiating event in mutagenesis and ultimately carcinogenesis.1,2 Covalent attachment of aromatic moieties to the C8-site of 2′-deoxyguanosine (C8-dG) is a common adduct type.3 The aryl ring may be directly attached to the C8-site of dG to afford C-linked varieties that are produced by the phenolic food toxin ochratoxin A (OTA),4-6 the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene7 or carcinogenic aryl hydrazines.8,9 Alternatively, the aryl group and dG nucleobase may be separated by a flexible N- or O-linked tether. The N-linked C8-dG adducts are produced by arylamine and heterocyclic aromatic amine carcinogens,10-13 while the O-linked C8-dG adducts are produced by chlorophenolic toxins, such as pentachlorophenol.14-16 Amongst the multiple factors that dictate the relationship between adduct formation and mutagenesis, a combination of adduct structure and sequence context are a critical chemical basis of how enzymes involved in the DNA damage response and replication cope with these changes. The N-Linked C8-dG adduct derived from N-acetylaminofluorene (AAF) has served as a model lesion to study the mechanism of frameshift mutagenesis, especially in the recognition sequence of the NarI restriction enzyme (5′-G1G2CG3CC-3′).17,18 The G3 position is within the reiterated CG-dinucleotide repeat; a hotspot for frameshift mutations19,20 that are likely important contributors to human cancers.21 A single AAF-dG adduct at the reiterated G3 position induces −2 6


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frameshift mutations at very high frequency (> 107-fold over background in single-stranded vectors under SOS-induced conditions) in Escherichia coli.22 The AAF-dG adduct at G3 can trigger the formation of a misaligned primer-template intermediate, referred to as a slipped mutagenic intermediate (SMI), that contains AAF-dG and the 3′-flanking C within a two nucleotide bulge in the template strand.23,24 The AAF-dG adduct strongly stabilizes the SMI compared to an unmodified two-nucleotide bulge by adopting a syn conformation that permits favorable stacking interactions between the AAF residue and base-pairs flanking the bulge.25 The mutagenicity of AAF-dG has also been observed during DNA synthesis over adducted templates with a variety of purified polymerases including E. coli DNA polymerase I Klenow fragment exo− (Kf−).26-28 In non-repeat sequences, AAF-dG is a strong block to DNA synthesis by Kf−, with the least efficient extension occurring at the n + 1 site (i.e. following base insertion opposite AAFdG), although the effect of extension 5 bases from the lesion is also greatly reduced.26 The base found opposite AAF-dG is C or A, with only a 3-fold preference for C.27 However, when the AAF-dG adduct is positioned at G3 within the NarI sequence context, efficient bypass synthesis by Kf− occurs to afford the GC dinucleotide deletion as the major product and small amounts of the full-length oligonucleotide.28 Our interest in C8-dG adducts has focused on the C- and O-linked analogs29,30 in which a variety of C- and O-linked C8-dG adducts have been inserted into the G3 position of NarI to determine the impact of linkage type on adduct conformation and in vitro mutagenicity using primer extension assays.16,31-33 Bulky O- and C-linked C8-dG adducts strongly stall primer extension by Kf− at the n + 1 site. The correct base C is mainly inserted opposite the lesion, along with small amounts of A.16,32,33 In most instances, full-length extension products are not observed and the GC dinucleotide deletion product has never been detected. These observations appear to 7



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mimic the results of synthesis by Kf− over AAF-dG at noniterated positions.26 In this regard, it is noteworthy that none of the O-linked C8-dG adducts studied to date stabilize the SMI compared to an unmodified two-nucleotide bulge.16,34

For the C-linked C8-dG adducts, fused-ring

derivatives with a linear extended aromatic ring system can stabilize the SMI (increase in thermal melting temperature (Tm) = 3−6 °C compared to the unmodified two-nucleotide bulge),33 but not nearly to the extent of the AAF-dG lesion (Tm ~ 15 °C).35,36 However, despite the tendency of C-linked C8-dG adducts to strongly stall primer extension, they were deemed as potential adduct bioprobes due to their fluorescence that can be utilized to decipher adduct conformation (syn vs. anti) within duplex structures.32 Recently, we developed a C-linked C8-biphenyl-dG adduct analog bearing a fluorine atom (FBP-dG) with a turn-on emission response suited for monitoring the formation of SMI structures mediated by polymerase processing of adducted templates.37 The adduct strongly favors the syn conformation in the fully paired (FP) duplex and stabilizes the SMI compared to an unmodified two-nucleotide bulge by 6-7 ºC; the largest Tm for the SMI containing a C-linked C8-dG adduct that we have observed to date. Furthermore, the FBP-dG lesion displayed fluorescence sensitivity to SMI formation with a 4-fold increase in emission intensity within the SMI compared to the FP duplex. We now report on the ability of Kf− to carry out extension past FBP-dG within NarI versus a three GC-repeat (CG3) sequence, which is also a hotspot for −2 frameshift mutations.38 Extension past FBP-dG within NarI correlates with previous extension data for NarI containing bulky C-linked C8-dG adducts,32,33 resulting in partially extended primers due to strong blockage by the lesion. However, Kf− extended the primer past FBP-dG within the CG3 template to afford 8


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full-length product and the GC dinucleotide deletion, which represents the first instance that primer extension past a C-linked C8-dG adduct has afforded a two-base deletion product. Using a simulated translesion synthesis approach reported previously by Liang and Cho to model primer extension past a bulky N-linked fluoroaminofluorene adduct (FAF-dG),39 FBP-dG-adducted oligonucleotide duplex constructs were generated to model FP versus SM pathways that were characterized by UV thermal denaturation and fluorescence spectroscopy. The resulting thermal and emission measurements provide valuable insight for explaining the origins of the observed differences in primer extension bypass mediated by Kf− on template strands containing the FBPdG lesion within hotspot sequences for −2 frameshift mutations.

EXPERIMENTAL PROCEDURES DNA Synthesis. All unmodified primers and template oligonucleotides were purchased from Sigma-Aldrich Ltd. (Oakville, ON). The 22-mer adducted oligonucleotide sequences (NarI: 5'-ATCGGCXCCATCCCTTACGAGC-3' and CG3: 5'-ATGCGCXCCATCCCTTACGAGC-3', X = FBP-dG) were prepared on a BioAutomation MerMade 12 automatic DNA synthesizer using standard and the FBP-dG modified phosphoramidite. Full synthetic details of the FBP-dG phosphoramidite have been described previously.37 Following synthesis, oligonucleotides were cleaved from the solid support and deprotected using 2 mL of 30 % ammonium hydroxide solution at 55 °C for 12 h and purified by RP-HPLC. Oligonucleotides were analyzed by electrospray ionization mass spectrometry (ESI-MS), as previously outlined.16,31-34 Modified or unmodified oligonucleotides were annealed with primer strands at a final concentration of 3 µM in 50 mM Na2HPO4 buffer and 100 mM NaCl (pH 7). The strands were annealed by heating to 95 °C and 9



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then slowly cooling (1 °C/min) to 10 °C. The samples were used for UV thermal melting analysis and fluorescence measurements. UV Thermal Melting. Melting temperatures (Tm) of the duplexes (3 M) were determined by variable temperature UV analysis using a Cary 300-Bio UV-vis spectrophotometer equipped with a 6x6 Multicell Peltier block-heater using Hellma 114-QS 10 mm light path cells. Tm values were determined as previously outlined.37 All measurements were performed three times and the mean Tm values are reported. Fluorescence. All fluorescence spectra were recorded on a Cary Eclipse Fluorescence spectrophotometer equipped with a 1 x 4 multicell block Peltier stirrer and temperature controller, using excitation and emission slit widths of 2.5 nm. Once inserted into oligonucleotides the FBPdG lesion undergoes excitation at 315 nm, with emission at ~ 420 nm. Primer elongation experiments. T4 polynucleotide kinase and [γ-32P]ATP were used to label the 15-mer primer strand at the 5’-end according to the manufacturers protocol. Primers and templates were annealed by heating the sample to 95 °C for 5 min in a heating block and cooling over the course of 16 h. Reaction mixtures contained 10-100 nM polymerase (Kf−), 100 nM 5′[32P]primer:template DNA and 25 µM dNTPs in 10 µL of reaction buffer (1x Buffer: 50 mM NaCl, 10mM Tris-HCl (pH7.9), 10 mM MgCl2, 1 mM dithiothreitol (DTT)). Reaction mixtures were incubated at 37 °C for 60 min, then quenched by adding 20 µL PAGE gel loading buffer (80% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanole FF). The resulting mixtures (4 µL) were analyzed on 15% polyacrylamide/7M urea denaturing gels. Radioactive bands were visualized by autoradiography (Bio-Rad, Hercules, CA) and quantified using the Bio-Rad Quantity One software.

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RESULTS Primer Extension. To determine the miscoding potential of FBP-dG, single nucleotide insertion assays were carried out with the adducted NarI and CG3 22-mer templates annealed to a 15-mer primer (100 nM duplex) with Kf− (10 nM) and 100 M individual dNTP (Figure 1). For the adducted NarI template, we observed 47% of the product containing two C’s relative to 28% of the single C insertion product. Misincorporation of G (17 %) and A (36 %) were also observed (Figure 1b). The incorporation of two C’s with a C-linked C8-dG adduct at the G3-position of NarI has been observed previously32 and implies two-base slippage with CC pairing with GG one position removed from the adduct site (positions 3 and 4 in the NarI template, Figure 1a).40 For the adducted CG3 template that lacks a GG site only one C was incorporated

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and the levels of misincorporation (G (12 %) and A (22 %)) were diminished compared to the adducted NarI template. Having established the propensity for one or two C’s to be preferentially inserted amongst individual nucleotides, we evaluated the outcome of adding all four dNTPs to the reaction. It was clear that primer extension one nucleotide past the FBP-dG adduct (n + 1) was strongly hindered for the NarI template using 10 nM Kf− (Figure 1c), consistent with observations made previously for other bulky C-linked C8-dG adducts32,33 and for the N-linked AAF-dG adduct within non-repeat sequences.26 Increasing the Kf− concentration (50 and 100 nM) led to further primer extension, but was halted after the forth incorporation, to generating a new blockage site at n + 4. This site of blockage has been observed previously for the adducted 22-mer NarI containing the C-linked C8benzo[b]thienyl-dG adduct,33 and to a small extent with the C8-phenyl-dG lesion.32 For extension past FBP-dG within the CG3 template, three sites of blockage (n + 1, n + 2 and n + 4) were observed using 10 nM Kf−. However, in this instance increasing the Kf− concentration (50 and 100 nM) led to new bands for full-length product (n + 7/F) and the 12


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GC dinucleotide deletion product (n + 5/T) that comigrated with authentic samples in the M2 lane. The gel also exhibited an n + 8 band above the n + 7/F product that was ascribed to blunt end extension (Figure 1c).32

Thus, the propensity for overcoming stalls in

synthesis strongly depended on the sequence, which can influence adduct conformation and lesion bypass efficiency.41 Model Translesion Synthesis. To gain further insight on the chemical basis of the sequence-dependant differences in overcoming the stalling propensity of FBP-dG, DNADNA interactions in the translesion synthesis process were modeled by annealing the adducted 22-mer templates to primers with increasing lengths corresponding to the FP versus SM pathways (n, n + 1, n + 2, n + 3, n + 5/T and n + 7/F, where n + 1 = FBP-dG site, Figure 2). For the NarI sequence only one of the chosen primers (n + 1) can be in equilibrium between the FP and SM duplex (indicated by green triangle in Figure 2), providing a total of eight distinct template:primer duplexes. For the CG3 template the number of primers in equilibrium is increased to three (n + 1, n + 2 and n + 3), which reduces the total number of distinct template:primer duplexes to six.

The UV thermal 13



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melting parameters (Tm values) for the various duplexes are provided in Table 1; values in parentheses are from unmodified control duplexes. Evaluating Tm values between the starting 22-mer template:15-mer primer (n) duplex and the other duplex revealed the step-wise increases in duplex stability during translesion synthesis (Table 1, Figure 3). For the FP pathway the C-linked FBP-dG lesion strongly destabilized duplex stability during translesion synthesis (Figure 3a). For the control 22-mer templates, extending the 15-mer (n) primer to n + 1 increased the Tm by 5 °C, while for the adducted templates the minor increase was not significant.

Both

adducted templates also exhibited a small increase for n + 2 (Tm = 3 °C) and this trend continued for the adducted NarI template for n + 3 (Tm also 3 °C, solid blue trace, Figure 3a). However, the CG3 template displayed a significant increase in duplex stability for n + 3 (Tm = 6.4 °C), which clearly distinguishes it from the corresponding NarI duplex for the FP pathway (solid traces, Figure 3a). For the SM pathway the changes in duplex stability for the two adducted templates were very similar, although the CG3 template exhibited slightly greater Tm values for the n + 2, n + 3 and n + 5/T duplexes (Figure 3b). 14


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In the SM pathway both adducted templates displayed greater Tm values for template:primer (n + 3) duplex versus the NarI control duplex and produced duplexes with the truncated primer (n + 5/T) that were more stable than the controls by 3.1 (CG3) and 2.0 °C (NarI). To gain insight into adduct conformation the fluorescence intensity of the FBP-dG lesion in the various adducted template:primer duplexes were also recorded and the changes in emission intensity relative to the starting template:15-mer primer (n) duplex were evaluated (Figure 4). For FBP-dG within NarI (solid blue trace, Figure 4), the emission intensity increased upon extension to n + 1. The emission then exhibited a slight decrease in intensity upon extension to n + 2 for the FP pathway (solid blue trace) that was followed by a sharp decrease in intensity for extension to n + 3. The emission intensity of the adduct within the template:primer (n + 3) duplex was very similar to its intensity within the full-length template:primer (n + 7/F) duplex, suggesting that template and primer were standardly paired, which caused the sharp decrease in the adduct emission intensity. For the SM pathway (dashed blue trace, Figure 4) the emission 15



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intensity of the lesion increased slightly upon extension from n + 1 to n + 2 and remained fairly constant in the n + 3 and n + 5/T duplexes. For the adducted CG3 template, the lesion emission intensity increased only slightly upon extension to n + 1, and then reached the level noted within the NarI template upon extension to n + 2 (red trace, Figure 4). In contrast to the NarI trend, only a slight decrease in emission intensity was observed upon extension to n + 3. From this point, the emission intensity either increased significantly upon formation of the SMI by annealing the adducted template to n + 5/T (dashed red trace, Figure 4), or decreased sharply upon formation of the FP duplex.

DISCUSSION

The main objective of the present study was to determine the basis of how sequence context influences the efficiency and fidelity of Kf− mediated primer extension of oligonucleotide templates containing the C-linked FBP-dG lesion within CGdinucleotide repeat sequences, which are hotspots for −2 frameshift mutations.19,20 We

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previously demonstrated the capacity of FBP-dG to stabilize the SMI within the NarI sequence and exhibit emission sensitivity to SMI formation.37

Specifically, FBP-dG

displayed a 4-fold increase in emission intensity at 420 nm following excitation at 315 nm within the SMI versus the FP duplex. Therefore it was reasoned that the C-linked FBPdG lesion could serve as a model fluorescent adduct to distinguish FP from SM template:primer duplexes and permit additional insight into translesion synthesis of C8dG adducts. For the present experiments, FBP-dG was inserted into the reiterated G3 position of a 22-mer NarI template and the corresponding position within a 22-mer CG3 template. These sequences differ at positions 4 and 5, which are three and four bases 5′-removed from the lesion site (1) that in flanked by two C residues (see Figure 1a for sequence numbering). Given the similarity of the two sequences, it was somewhat surprising to observe drastic differences in polymerase bypass efficiency mediated by Kf− (Figure 1c).

However, given that polymerase blockage can be governed by lesion

conformation,41 it was predicted that a simulated translesion synthesis model approach (Figure 2) would display differences in UV thermal denaturation (Table 1, Figure 3) and 17



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adduct fluorescence (Figure 4) to help explain the origins of the observed differences in primer extension bypass. The key findings from the thermal and fluorescence analysis of the template:primer duplexes were the observed differences in thermal stability of the adducted template:primer (n + 3) duplexes (Figure 3a) and differences in adduct emission intensity with the adducted templates annealed to the n + 1, n + 3 and n + 5/T primers (Figure 4). For both adducted templates the first incorporation (n + 1) is the correct base C that can be paired with the adduct in the FP pathway or matched with an unmodified G located 5′ from the adduct to generate the two-base bulge in the SM pathway (see Figure 2). At this point in the synthesis the resulting SMI can rapidly interchange with the non-slipped FP configuration for both adducted templates,42 which is indicated by the green triangle in Figure 2. This incorporation generates essentially no increase in thermal stability of the template:primer duplex compared to the starting 22-mer template:15-mer primer (n) duplex (Figure 3). As pointed out by Petruska and coworkers,43 this lack of efficient annealing should disfavor polymerization and consequently the n + 1 site presents a 18


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strong block to Kf− elongation, especially for the NarI adducted template. However, even at 10 nM Kf−, the primer was extended past the n + 1 site with FBP-dG within the CG3 template, but was blocked at the n + 2 and n + 4 sites. The relative emission of the FBPdG lesion within the template:primer (n + 1) duplex (Figure 4) indicates greater emission intensity for the adduct in the NarI duplex versus the CG3 duplex. This observation suggests that the equilibrium between the SM and FP duplexes favors more partnered adduct in the CG3 template.37 In the FP NarI duplex our previous structural studies revealed a strong syn preference for FBP-dG, producing an intercalated structure (80%) with the opposing dC partially displaced from the helix.37 The minor structure (B-type, 20%) maintains Watson-Crick hydrogen bonds between FBP-dG and the opposing C with the adduct in the anti conformation and the biphenyl moiety located in the major groove. The decreased emission of the adduct in the CG3:primer (n + 1) duplex raises the possibility for a greater percentage of B-type structure with correct FBP-dG pairing with C. This conformation would provide a rationale for the increased ability of Kf− to extend the primer past the n + 1 site with the adducted CG3 template. 19



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For the NarI template the next step in the synthesis involves insertion of G (FP pathway) versus C (SM pathway) for primer extension to n + 2. It is interesting that Kf− was strongly blocked at n + 1 in the full-length extension assay, because the single nucleotide assay demonstrated incorporation of two C’s by Kf− indicating two-base slippage (Figure 1b). This observation suggested that Kf− is capable of extending past the n + 1 position if only provided dCTP. In this scenario the terminal C of the n + 1 primer is paired with unmodified G at position 3 of NarI, which produces the two-nucleotide bulge that contains FBP-dG and the 3′-flanking C. The dinucleotide bulge must be formed upstream of the polymerase active site to permit the second C insertion. However, in the full-length extension assays the polymerase is also provided with dGTP. The melting data for the adducted NarI template:primer (n + 2) duplex indicates that the FP and SM duplexes have identical Tm values (62.2 vs. 62.3 °C, Table 1), suggesting no thermal advantage for either pathway. Consequently, the SMI structure is in rapid equilibrium with the FP duplex.

Molecular dynamics (MD) simulations of the NarI SMI duplex

containing FBP-dG indicates conformational heterogeneity with little energy difference 20


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between the anti and syn adduct conformations.37 The anti adduct conformation would be expected to undergo rapid realignment to the FP duplex,32,33 in which the anti adduct conformation is favored.

Structural studies on an AAF-dG-modified template:primer

duplex bound to T7 DNA polymerase demonstrate that the syn adduct conformation facilitates intercalation of the AAF moiety into a hydrophobic pocket on the surface of the fingers domain, partially blocking the nucleotide binding site to strongly stall polymerization.44 Thus, it is likely that Kf− cannot extend the n + 1 primer in the FP pathway for NarI due to the strong syn preference of the lesion, 37 which strongly inhibits G insertion to cause the n + 1 blockage site. In the GC3 context, the same conformational factors are relevant in promoting bulge formation; however, there is a possibility for sequence realignment that is not accessible in the NarI context. Thus, the subtle sequence change leads to different polymerase bypass efficiency. Annealing the adducted templates with the n + 3 FP primer caused a sharp decrease in the emission intensity of FBP-dG within the NarI template (Figure 4), indicating that the duplex is FP at this stage in the synthesis. However, the thermal 21



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stability of the duplex is virtually the same as the duplex with the template annealed to the n + 2 primer (See Table 1 and Figure 3a). Thus, for the adducted NarI template extending the starting n primer by three bases in the FP pathway leads to little increase in thermal stability. This lack of effective annealing may promote dissociation of the polymerase from the primer termini to generate partially replicated primer.43 This sequence of events may provide a rationale for the n + 4 block site (Figure 1c). In contrast, annealing the n + 3 primer to the adducted CG3 template produces a FP duplex that is still in equilibrium with the SM duplex (see Figure 2) and exhibits a substantial increase in thermal stability compared to the starting template:primer (n) duplex (Tm = 6.4 °C, Table 1, Figure 3a). This observation suggests favorable polymerization of the adducted CG3 template once the third base is added to the primer. The FBP-dG lesion within the CG3 template:primer (n + 5/T) duplex displayed a substantial increase in emission intensity (Figure 4), while in the corresponding NarI duplex the adduct emission intensity remained roughly constant for the SM pathway (dashed blue trace, Figure 4). The constant emission intensity of the FBP-dG adduct 22


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during the SM pathway for NarI is suggestive of complex equilibria between duplex structures and adduct conformations. The adducted CG3 template is also predicted to be in equilibrium between FP and SM duplexes when paired with the n +1, n + 2 and n + 3 primers. In these duplexes the adduct emission intensity is similar to the corresponding emission intensity during the SM pathway for NarI (Figure 4). However, when paired with the n + 5/T and n + 7/F primers the duplex can lock into a single structure. The heightened emission intensity of the FBP-dG adduct within the SMI for CG3 suggests production of a unique adduct conformation, most likely syn given the stability of the SMI compared to an unmodified two-nucleotide bulge. A greater propensity for a syn conformation in the SMI would be expected to favor production of the CG dinucleotide deletion product, as observed for the AAF-dG lesion.28 The inability of Kf− to efficiently extend a primer past the n + 1 and n + 4 sites relative to FBP-dG within a NarI sequence template appears be related to the instability of the template:primer duplexes for the first three extension sites (n + 1, n + 2 and n + 3) in the FP pathway. This instability may cause the polymerase to dissociate from the 23



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primer termini to generate partially replicated extension products (Figure 1c). Furthermore, the emission intensity of FBP-dG within NarI suggests that the terminal C within the n + 1 primer does not base-pair effectively with the lesion, leaving it unpartnered. This lack of base-pairing hinders polymerase extension one nucleotide past FBP-dG in the FP structure, which is required to generate full-length product. In contrast, the emission intensity of FBP-dG within the CG3 template suggests adduct base-pairing with the terminal C of the n + 1 primer, which would facilitate primer extension one nucleotide past the adduct to produce the n + 2 primer (Figure 1c).

The CG3

template:primer (n + 3) duplex also exhibits a substantial increase in stability compared to the starting template:primer (n) duplex, which will further promote extension. This study highlights the importance of sequence context for frameshift mutations mediated by a Clinked C8-dG adduct and demonstrates adduct fluorescence as a tool to gain a better understanding of the molecular mechanisms governing frameshift mutations.

24


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Supporting Information Available. ESI-MS analysis and spectra of modified oligonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org.

Funding Sources Support for this research was provided by the Swiss National Science Foundation (156280) and the Natural Sciences and Engineering Research Council (NSERC) of Canada (Discovery grant to RAM (04621-2018)).

Abbreviations: dG, 2′-deoxyguanosine; OTA, ochratoxin A; PAH, polycyclic aromatic hydrocarbon, AAF, N-acetylaminofluorene; FP, fully paired; SM, slipped mutagenic; SMI, slipped mutagenic intermediate; FBP-dG, C8-fluoro-biphenyl-dG; Tm, thermal melting temperature.

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Table 1. Thermal Melting Parameters FP Duplex

Tm (°C)a

Tm (°C)b

SM Duplex

Tm (°C)

Tm (°C)b

CG3, n

59.4 (59.6)

0.0 (0.0)

CG3, n

59.4 (59.6)

0.0 (0.0)

n+1

60.5 (65.1)

1.1 (5.5)

n+1

60.5 (65.1)

1.1 (5.5)

n+2

62.8 (66.6)

3.4 (7.0)

n+2

62.8 (66.6)

3.4 (7.0)

n+3

65.8 (71.0)

6.4 (11.4)

n+3

65.8 (71.0)

6.4 (11.4)

n + 7/F

66.6 (74.6)

7.2 (15.0)

n + 5/T

68.1 (65.0)

8.8 (5.4)

NarI, n

59.3 (60.7)

0.0 (0.0)

NarI, n

59.3 (60.7)

0.0 (0.0)

n+1

60.4 (65.7)

1.1 (5.0)

n+1

60.4 (65.7)

1.1 (5.0)

n+2

62.2 (68.3)

2.9 (7.6)

n+2

62.3 (65.3)

3.0 (5.0)

n +3

62.6 (72.0)

3.3 (11.3)

n +3

65.0 (65.3)

5.7 (4.6)

n + 7/F

68.1 (77.6)

8.8 (16.9)

n + 5/T

67.7 (65.7)

8.4 (5.0)

Mean Tm values of duplexes (3 M) measured in 50 mM sodium phosphate buffer, pH 7, with 0.1 M NaCl, a heating rate of 1 °C/min and are reproducible within 3%. Values in parentheses are from unmodified control duplexes. b Tm = Tm (22-mer:primer extension duplex) – Tm (22-mer:15-mer (n) duplex). a

30


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Figure 1. a) Adducted template (X = FBP-dG) and primer sequences used in this study. b) Single nucleotide extension with Kf− (100 nM template:primer duplex, 100 μM dNTPs, 10 nM Kf−, incubation for 15 min at 37 °C). c) Full length extension with Kf− (100 nM template:primer duplex, 100 μM dNTPs, 10, 50, 100 nM Kf−, incubation for 30 min at 37 °C). M1 = 15-mer primer, M2 = mixture of n+1 primer, n+2 primer, n+3 primer, truncated sequence (n+5/T) and full length complementary sequence (n+7/F).

31



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Figure 2. Model duplexes for full length extension versus the slipped mutagenic pathway for adducted NarI (shown in blue) versus CG3 (shown in red) with the primer in black and the FBP-dG lesion represented by the green ball attached to G. Fully paired and slipped mutagenic duplexes that may be in equilibrium are marked with the green triangle.

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Figure 3. Plots of change in thermal melting temperatures (Tm) for 22-mer:primer extension duplexes versus Tm for the 22-mer:15-mer (n) primer duplexes for (a) FP pathway (n + 7/F product) and (b) SM pathway to afford GC dinucleotide deletion (n + 5/T product). Plots for CG3 are in red, NarI are in blue, dashed lines are for the control duplexes (X = dG), solid lines are for the adducted duplexes (X = FBP-dG).

33



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Figure 4. Plots of relative emission intensity for FBP-dG (ex = 315 nm, em = 420 nm) at various primer extension positions versus its emission intensity within the starting 22mer:15-mer primer (n position) duplex. Plots for CG3 are in red, NarI are in blue, dashed lines for the SM pathway, solid lines for the FP pathway.

34


Adduct Fluorescence as a Tool to Decipher Sequence Impact on

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