STRUCTURAL AND CONFORMATIONAL INSIGHTS INTO BULKY ARYLAMINE-INDUCED MUTAGENESIS

Cancer is the second deadliest disease in the United States. Over 100 different types of cancers exist, among which lung, breast and prostate cancers are those most frequently diagnosed. Genetic factors are important. However, exposures to tobacco smoke and environmental pollutants are considered to be responsible for 75%–80% of cancer. About 6% of cancer deaths every year in the US are reportedly to be directly linked to known carcinogen exposures. Therefore, it is important to study the mechanisms of how the environmental carcinogens trigger cancer initiation. Most chemical carcinogens are metabolized into reactive species in vivo to interact with DNA, consequently producing covalent DNA adducts. These harmful lesions can be removed by various repair systems including base excision and nucleotide excision repair machinery in the cell. However, unrepaired lesions can enter into cell’s DNA replication cycle and generate various point and frameshift mutations. In particular, the latter represents a gain or loss of base pairs, which alters the genome information. As an example, mutations on the specific genes such as the tumor suppressor p53 may trigger cancer initiation. Arylamine is known as an important group of environmental chemical carcinogens. Some members of this group, such as 4-aminobiphenyl (ABP), benzidine and 2-naphthylamine, are classified as human bladder carcinogens. These chemicals are found commonly in cigarette smoke, incomplete diesel exhausts, and hair dye products. 2-Aminofluorene is a prototype animal carcinogen that undergoes metabolic activation by liver enzymes to form electrophilic nitrenium ion to form two major C8 substituted DNA-adducts: N-(2-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(2deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF). Similarly, the human carcinogen ABP produces N-(2-deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). Encountering these lesions in a replicative or a bypass polymerase will result in different types of biological outcomes, such as error-free, error-prone, or frameshifts. Manuscript I (published in Chemical Research in Toxicology, 2012) is a rapid report. In this communication, we used a real-time, label-free chip-based technique named surface plasmon resonance (SPR) to determine the binding interaction between the DNA replicative polymerase exonuclease-free Klenow fragment and three arylamine DNA lesions (FAF/FAAF/FABP). We designed biotin labeled DNA hairpin construct with modified lesions and immobilized the DNA on the streptavidin coated chip. The analyte Kf-exo was added over the DNA surface in the presence or absence of dNTP. The results showed a tight binding between the enzyme and unmodified DNA with great dNTP selectivity. In contrast, the dNTP selectivity was minimal in adduct modified DNA. Moreover, lesion included DNA tended to have better and stronger binding than unmodified DNA. Manuscript II (published in Chemical Research in Toxicology, 2014) contains the full details of Manuscript I. The full paper involves two 5’-flanking sequence (CG*A and TG*A), two adducts (FAAF and FABP), and two different polymerases (E. coli replicative polymerase Kf-exo and human repair polymerase ). We employed the same SPR methodology to study the binding interaction and complementary 19 F NMR and primer steady-state kinetics. Results showed significant substrate specificity for Kfexo and polymerase , which are double-stranded/single-stranded junction and a doublestranded DNA with a nucleotide gap structure, respectively. Tight binding with native DNA was observed, as well as the high nucleotide selectivity. However, Kf-exo binds tightly to lesion DNA, but not for polymerase . A minimal nucleotide selectivity for modified was observed with both enzymes. Moreover, the dynamic 19 F NMR and primer steady-state kinetics results indicated the importance of lesion-induced conformational heterogeneity in polymerase binding. In Manuscript III (to be submitted to Journal of Molecular Biology), we conducted a series of systematic studies to probe the conformational mechanisms of arylamine-induced -2 base deletion mutations frequently observed in the NarI mutational hot sequence (5’---TCGGCG*CN---3’; N= dC and dT) of E. coli during translesion synthesis (TLS). We employed two well-characterized fluorinated bulky DNA lesions FAAF and FABP that were derived from the environmental carcinogens 2-aminofluorene and 4-aminbiphenyl. Our work focused primarily on elucidating the effects of lesion size, bulkiness, and overall topology and the 3’-next flanking base N in producing the bulge structure responsible for -2 frameshift mutations. Two chemical simulated TLS models were examined, in which the FAAF/FABP lesion is positioned at G3 position of two 16mer NarI sequences, which were annealed systematically with increasing primer lengths in the full length and -2 deletion pathways. Their thermodynamic, conformational, and binding profiles at each elongation step were measured by various biophysical techniques including spectroscopic (dynamic 19 F NMR/CD), thermodynamic (UV-melting/DSC), and affinity binding (SPR). Results showed two different -2 bulge formations, which are triggered by the conformational stability of the G3*: C base pair at the replication fork, as well as the nature of base sequences surrounding the lesion site. Each bulge structure exists in a mixture of “external solvent exposed” B-type (B-SMI) and “inserted solvent protected “stacked” S-type (S-SMI), and their conformational rigidity increases as a function of primer lengths. The results indicate the importance of conformational stability, heterogeneity, and flexibility in the mechanisms of bulky arylamine-induced frameshift mutagenesis.

DNA is under constant assault by various endogenous and exogenous pathways, which result in different types of DNA damage. When a polymerase encounters a lesion, it can bypass by replicative polymerase, either inserting the correct base (error-free) or incorrect base (error-prone) 1 . The environmental arylamine carcinogens are known to form C8substituted dG adducts in vivo. We have shown that these lesions exist in a mixture of the base-displaced stacked, major groove B-type, and wedge conformers, with each leading to potentially unique sequence-dependent mutation and nucleotide excision repair It is important to understand the nature of interactions between polymerase and DNA lesions. Crystal structure and kinetic analyses have been used to elucidate details of polymerase action at an atomic resolution 1 . However, similar structural details of bulky DNA lesions have been challenging due to difficulties with obtaining crystals 3,4 .
Consequently, various alternative techniques such as fluorescence, circular dichroism spectroscopy, gel mobility shift assays, and footprinting assays have been used 5,6 However, these techniques are either qualitative or semi-quantitative, non-compatible with fast dissociation rates, and require labeling of at least one of the components of interest. Although gel-based assay is relatively simple and robust, samples of interest will not be in chemical equilibrium and the system's components are not amenable to testing across temperatures or salt concentrations 6 . Surface plasmon resonance (SPR) is a chipbased, label free solution technique that allows real-time monitoring of binding interactions between DNA and proteins [5][6][7][8] .
In this report, a SPR study was conducted to examine polymerase interactions of DNA lesions derived from three fluorinated prototype arylamine carcinogens: 2-aminofluorene (FAF), 2-acetylaminofluorene (FAAF), and 4-aminobiphenyl (FABP) (Fig. 1c). We employed exonuclease-free E. coli DNA polymerase I Klenow fragment (Kf-exo -) as it avoids complication of proofreading activity. The features of fluorinated arylamines as effective conformational probes are well documented 2,9 . The present study takes advantage of the sensitivity of Biacore T200 to conduct SPR analysis of the binary and ternary polymerase complexes of bulky carcinogen-DNA adducts. Figure 1a and S1a show the construction scheme for a biotinylated hairpin-based template-primer strand on a gold sensor chip. The hairpin-DNA was used to improve stability of oligonucleotides during performance of kinetics experiments. Arylaminemodified 31-mer oligonucleotides were purified by HPLC and characterized by mass spectrometry (Fig. S2). The biotin-hairpin-template/primer strands were annealed, ligated, and purified by denaturing polyacrylamide gel (Fig. S1b). The incorporation of dideoxythymidine (ddT) was carried out using Kf-exoand the 3 terminal ddT allowed capture of the ternary polymerase/template-primer/ dNTP complex without primer extension.
The kinetic assays were optimized with respect to regeneration buffer, surface density, and surface testing, as described elsewhere 10 (Fig. S3). The binding kinetics analysis was performed by injecting varying amounts of Kf-exoto cover the hairpin template-primer DNA (Fig. 1b) coated on streptavidin surface in the absence (binary) and presence (ternary) of dNTPs (100 M). The injections were repeated three times for each concentration in random, and the resulting data were fitted to the Langmuir model (1:1) ( Fig. 2). From the fitting, binding constants (k on , k off and K D ) were calculated (Table 1 and S1) using Biacore's BIAsimulation software. The Chi-squared values for the 1:1 fitting were less than 1% of R max (0.002-0.003 for all experiments with R max in the range of 0.7-3.5RU) (Figs. S4 and S5). The K D values for ternary systems were determined using affinity analysis as the association rate (k on ) reaches the near-diffusion limit. This procedure allowed the monitoring of interactions between unmodified or adducted DNA with different polymerases on a single chip. Furthermore, DNA over the chip surface was found to be stable for at least 7-10 days, without loss in binding activity under buffered reaction conditions.
The results from the binding assay (Fig. S6) are summarized in Table 1. The Kf-exobound tightly to unmodified DNA in the presence of a correct incoming dCTP opposite the templating dG. However, relative to dCTP binding, binding tightness was reduced by 30-, 60-, 34-, and 264-fold in binary, dATP, dTTP, and dGTP, respectively ( Fig. 2b and Table 1). The discrimination ability of correct versus incorrect nucleotides was significant, as the Watson-Crick base pair dCTP bound tightly and dGTP does not bind significantly. In contrast, the discrimination effect on Kf-exobinding was weaker for binding to FAF than for binding to unmodified DNA. The specificity of binding between the correct dCTP and incorrect nucleotides, as well as for the binary system, differed by only 2-to 16-fold. The tightness of Kf-exobinding in the presence of dCTP was reduced by 4-fold, as compared to that of the unmodified control.
Moreover, the difference in binding affinity between dCTP and dATP was less for FAF-dG (10-fold), as compared to that of unmodified DNA (60-fold) (Fig. 2b). The Kf-exobound more tightly to FAAF (k off = 0.02s -1 ) and FABP-dG (k off = 0.01s -1 ) lesion sites than to the unmodified control (k off = 0.13s -1 ) while k on values are similar. However, discrimination between correct and incorrect nucleotides was not maintained with FAAF and FABP-dG, for which binding affinities differed by only 1-to 3-fold (Fig. 2b).
Highly specific binding of Kf-exoto unmodified DNA in the presence of dCTP opposite a dG templating base is in line with the polymerase undergoing conformational change from an open to a closed system to form Watson-Crick base pairs 11 . However, Kf-exodoes bind weakly with incorrect nucleotides, probably retaining the open polymerase conformation. In particular, the binding of dGTP is very poor compared to other nucleotides.
To further confirm that the binding of polymerase to DNA is 1:1, theoretical R max values were calculated and compared with experimental values. The data presented here are consistent with data from sedimentation studies in which polymerase was shown to bind template-primer junction in a 1:1 ratio 12 . Interestingly, the K D value for Kf-exobinding to FAF adducts was higher in the presence of dCTP than with unmodified DNA (Table 1), indicating that the lesion prevents the nucleotide-induced, catalytically-favored closed conformation. Previous studies have shown that the carcinogenic aminofluorene orients into the energetically favorable solvent-exposed major groove, which causes less disruption at the replication fork, but may perturb the groove structures and the geometry in the active site of the polymerase 3 .
The aforementioned crystal structure of AF on T7 DNA polymerase showed fuzzy electron densities around the carcinogenic aminofluorene moiety in line with sequencedependent conformational heterogeneity in solution 4 . The present kinetics data also fit with previously published findings from a single nucleotide insertion assay study in which dATP was the next preferred nucleotide after dCTP 13 .
The higher binding affinity of Kf-exoto the bulky N-acetylated FAAF lesion, compared to unmodified DNA, could be due to the adduct perturbing the template-primer junction while maintaining some specific interactions with amino acids on the active site of the polymerase. It has been shown that the AAF lesion has two hydrogen bond interactions between the N 2 -amino group of the modified guanine and Asp-534, as well as between the N 7 -guanine and Arg-566 4 . In addition, the lesion adopts a syn-glycosidic conformation wherein the fluorene moiety is inserted between the hydrophobic pocket of the O-helix finger subdomain. These changes also keep the polymerase in the open and maintain a distorted conformation of the subdomain fingers, causing the Tyr-530 residue to occupy the binding region of the nucleotide and preventing interaction between the incoming nucleotide and polymerase 4 . The present data are also in agreement with previous results from tryptic digestion studies, in which the polymerase was shown to bind very tightly to unmodified DNA in the presence of the correct nucleotide and to be insensitive to digestion; FAAF did not exhibit any additional stability in relation to the incoming nucleotide 14 . FAF adducts are known to exist in a sequence-dependent equilibrium of B and S conformers 2,9 . FABP is similarly N-deacetylated; however, its biphenyl moiety is not as coplanar as fluorene, thereby resulting in a lesser basedisplaced stacked conformer population 15 . Consequently, FABP may behave similar to FAAF at the replication fork in the active site of a polymerase.
In summary, tight binding of Kf-exowas observed with unmodified dG in the presence of a correct dCTP in this study. Nucleotide selectivity was pronounced with K D values in the order of dCTP << dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates: Kf-exobound tightly to FAAF-dG and FABP-dG lesions as compared to FAF-dG. The SPR results for FAF and FAAF agreed with those obtained from gel-based assays, 16               .

INTRODUCTION
Polymerases are critical to the replication and repair of DNA. 1 While replication of DNA is an essential first step for cell division, repair of DNA is needed when insults such as UV rays, environmental toxins, and some drugs chemically modify DNA. 2 These modifications can yield a diverse array of mutations. 3 To understand the mechanisms of DNA replication and repair, it is crucial to understand how a polymerase processes DNA lesions. 4,5 As part of ongoing carcinogenesis research, and to understand the mechanisms of DNA mutation and repair, we have been studying how the bulky and mutagenic arylamine-DNA lesions ( Figure 1a) interact with a polymerase or a repair protein. [6][7][8][9][10][11] Using 19 F NMR, microcalorimetric and other biophyisical methods, we have shown that It has been shown that most replicative polymerases easily bypass the planar and Ndeacetylated aminofluorene (AF) adducts after a brief stall at the lesion site. On the other hand, the bulkier N-(2-deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF) analogs cannot be readily bypassed, and thus stall DNA synthesis. 16 In vitro studies with Xfamily polymerase β, AAF adducts lead to -2 base deletion mutations, while AF extends full length primers. 17 A recent study via single-molecule fluorescence spectroscopy showed that high-fidelity polymerases cannot extend a primer whose terminus occurs across from AAF. 18 In E. coli, AAF adducts results mostly in frameshift mutations, while both AF and AAF adducts cause point mutations . 19 In mammals, both adducts afford point mutations. 17 This difference in mutagenic profiles has been attributed to the presence of a bulky acetyl group on the central nitrogen, which causes the AAF adduct to adopt a syn conformation. 20 In contrast, the AF adduct adopts an anti-/syn-conformation, while the N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl (FABP) adduct adopts exclusively an anti-conformation. 9 Other factors influencing adduct-induced mutations include topology, insertion of the nucleotide opposite the lesion site, and the characteristics of the polymerase. 21,22 Numerous crystal structure and kinetic analysis studies are available and provide information on actions of native 23 SPR relies on changes in the refractive index that are due to changes in mass, and can thus measure a small difference in binding (K D ) at sub-nanomolar level. SPR is thus ideal for probing interactions of binary and ternary polymerase-DNA interaction. We have recently communicated our initial SPR work on the binding affinities of Kf-exoto arylamine DNA lesions. 6,49 Subsequently, a similar study was conducted to elucidate how FAF lesions affect the active site conformation of the human repair enzyme pol β, and how the structure and sequence of the DNA affects its ability to be repaired. 7 In the present study, we are providing a complete set of SPR data on the binding of Kf-exoor pol β to FAAF and FABP lesions in two different sequences (CG*A and TG*A). To complement the SPR binding results, we also conducted dynamic 19 F NMR as well as steady-state nucleotide insertion kinetics. The results are discussed in terms of adduct-induced conformational heterogeneity, the effect of the 5-flanking base sequence, substrate specificity, and the nature of a polymerase. The purpose of the present paper is two-fold: 1) to give the full details of our previous SPR work ("Rapid Report") 6 and 2) to introduce SPR to the chemical toxicology community as a powerful alternative to existing techniques for investigating protein-DNA interactions. As a result, the choice of polymerases used in the present study was based largely on the experimental systems in our previous work. 7,8,50 Obviously, future SPR studies should be expanded to a range of Y-family bypass polymerases, which is more likely to be involved in replication of bulky DNA lesions.

F NMR
Approximately 70 µM of a FAAF-or FABP-dG modified 16-mer template was annealed with a 9-mer primer in a 1:1 molar ratio to produce ds/ss junction containing duplexes ( Figure 3). The samples were lyophilized and dissolved in 300 µL of typical pH 7.0 NMR buffer containing 10% D 2 O/90% H 2 O with 100 mM NaCl, 10 mM sodium phosphate, and 100 µM EDTA. All 19 F NMR spectra were recorded using a dedicated 5 mm 19 F/ 1 H dual probe on a Varian 500 MHz spectrometer operating at 476.5 MHz, using acquisition parameters described previously. 11,51,52 The spectra were acquired in the 1 Hdecoupled mode and referenced relative to that of CFCl 3 by assigning external C 6 F 6 in C 6 D 6 at -164.9 ppm. 19 F NMR spectra were measured at two different temperatures, 5 and 25 C.

Standing start experiments
Single nucleotide/full length extension experiments for both FABP-and FAAF-dG adducts in Kf-exowere performed as described previously. 8 Briefly, the 9-mer primer was 5-radiolabeled using [γ-32 P] ATP and T4 polynucleotide kinase (T4 PNK) following the manufacturer's protocol. The 32 P-labeled primer (50 pmol) was annealed to either an unmodified or adducted template oligonucleotide (60 pmol) by heating to 95 °C for 5 min and then slowly cooling to room temperature in 3 h. For pol  assays, 1 nt-gap was generated by adding downstream 9-mer primer with 5-phosphate group while annealing with radiolabeled primer (9-mer) and template (19-mer). 7 The ds/ss primer-template sequence (20 nM) was incubated with Kf-exo − (0.5 or 1.0 nM) for 5 min to form a binary complex in Tris buffer (Tris, 50 mM pH 7.4; BSA, 50 g/mL; 5% (v/v) glycerol). The reaction was initiated by adding a dNTP (100 µM)/MgCl 2 (5 mM) solution to a binary mixture and incubated at 22°C for 10 min. The reaction was arrested with gel loading buffer (containing 50 mM EDTA (pH 8.0)/95% formamide solution). The quenched sample was heated to 95 °C for 5 min and immediately cooled on ice. The products were resolved with a denaturing polyacrylamide gel (20% polyacryamide (w/v)/7 M urea) electrophoresed at 2500 V for 4 h. The gel was exposed on a Kodak phosphor imaging screen overnight and scanned with a Typhoon 9410 variable mode imager.

Steady-state kinetics analysis
To determine the efficiency of dCTP insertion opposite the adducted site, steady-state kinetic parameters for incorporation of the nucleotide opposite the unmodified and FABP-modified templates were determined by using the reported literature procedures. 7,8 The reactions were performed with pol  (0.5 nM) and oligonucleotide (20 nM) at 22°C.
For the unmodified sequence, reactions were performed in shorter time period of 0.5-10 min for nucleotide incorporation and up to 30 min in the case of modified templates. The band intensities were quantitated using ImageQuantTL from GE Healthcare. The percentage of primer extended in kinetic assays was determined by taking the ratio of extended primer to the total amount of primer (unextended + extended primer). The kinetic parameters k cat and K m were determined as described earlier. 7,8

Arylamine-modified hairpin template/primer constructs
The modification of 5-biotin CGA/TGA sequences (31-mer) was carried out using the previously reported procedures ( Figure 2) 6, 7 and the modified products were purified by by RP-HPLC and characterized by MALDI-TOF mass spectrometer. Biotinylated unmodified (20 M) or modified 31-mer (20 M) was annealed with 20 M of 52-mer hairpin by heating to 95C for 5 min and cooling down to room temperature ( Figure 2).
The annealed mixture was ligated by using 4000 U T4 DNA ligase in 1× ligase buffer for 16 h at room temperature. The ligated 83-mer oligonucleotide was purified by 10% denaturing polyacrylamide gel ( Figure S1) and extracted using crush and soak method.

Characterization of oligonucleotides by MALDI-TOF
Either biotinylated 31-mer, 83-mer or 84-mer DNA sequences (100 pmol) was mixed with 2 L matrix containing 1 L of 3-hydroxy picolinic acid (3-HPA) (50 mg/mL dissolved in acetonitrile/water 50% v/v) and 1 L of diammonium hydrogen citrate (DAHC) (50 mg/mL dissolved in acetonitrile/water 50% v/v). MALDI-TOF experiments were performed using Axima Performance from Shimadzu Biotech. The mass spectrometric measurement of 31-mer oligonucleotides was carried out in a reflectron positive mode. The calibration of the instrument in reflectron positive mode was performed using low molecular weight oligonucleotide or peptide standard calibration kit.
For high molecular weight oligonucleotides (>10,000 Da), calibration was done in a linear negative mode using 52-, 80-, 90-, 100-mer standards with laser power 120 in order to enhance the signal intensity. The spectral data was processed by using Shimadzu Biotech MALDI-MS software with processing parameters as follows: smoothing filter width as 20 channels; baseline filter width as 80 channels and double threshold.

DNA coating on biosensor chip
SPR measurements were conducted with Biacore T200 (GE Healthcare). A carboxymethylated dextran coated CM5 chip supplied by GE Healthcare was used to immobilize streptavidin (SA) via the amine coupling kit on flow cells by following the previously reported literature. 6,7,49 The EDC/NHS mixture was injected over the surface mM MgCl 2 was injected over the surface for 5 min followed by 0.05% SDS to remove the polymerase. For pol  experiments, 1 nt-gap was created by using the same DNA coating approach and in addition corresponding downstream complementary sequence (21-mer) containing 5-phosphate group (2 nM) was injected over the surface for 5 min.

Real-time kinetic analysis
Kf-exowas injected with or without dNTPs (100 M) over the DNA surface in random order (neither ascending nor descending concentrations). Each concentration was repeated twice. Langmuir model.

Model hairpin template/primer constructs
An overall scheme for the construction of the biotinylated hairpin-based templateprimer strands is depicted in Figure 2a. FABP-or FAAF-modified biotin-31-mer oligonucleotides were prepared according to published procedures. 7, 10, 12 The 52-mer hairpin-DNA was annealed and ligated to the biotinylated 31-mer (Figure 2b, c). ddTTP was incorporated at the 3 primer terminus using Kf-exo -. 25,53 The hairpin structure was created to improve the thermal stability of the oligonucleotide constructs on a gold chip during kinetics experiments. As a result, the same oligonucleotide constructs could be used multiple times with different polymerases and buffer conditions. Finally, the lesion was positioned at the 22 nd base, with 21 bases on the 5'-side and 28 bases on the 3'-side, in order to avoid close contact between the polymerase and the chip surface. The resulting template/primer strands, containing the biotinylated 84-mer hairpin, were purified by denaturing polyacrylamide gel ( Figure S1) and used for further study.

F NMR
To examine lesion-induced conformational heterogeneity, we measured 19 F NMR spectra of modified 16/9-mer template/primer duplexes. As shown in Figure  conformer. 11,13 Hence, the present FAAF-induced heterogeneity could be a variation of the B/S/W heterogeneity. In contrast to the aforementioned study, however, the 19 F signals in the present study are derived from the lesions at the ds/ss junction, not fully paired double helical duplexes. 8,12 The relative shielding of 19 F signals and the narrow the narrow chemical shift range (~ 2 ppm) in the present work are probably due to the flexible lesions at the ds/ss junction. As a result, we could not unequivocally assign the signals to the B-, S-, or W-conformer.

Primer extension assay
Single nucleotide incorporation was carried out using the E. coli exonuclease-deficient Klenow fragment (Kf-exo -) and the human base excision repair polymerase  (pol ) We observed preferential dCTP incorporation opposite the lesion.
As for FAAF, no nucleotide insertion was observed with either Kf-exoor pol , even at high enzyme concentrations or longer incubation period (data not shown) because the lesion had completely blocked elongation.

Steady-state kinetics
We conducted steady-state experiments to investigate the impact of conformational heterogeneity on nucleotide insertion kinetics. The results for Kf-exoand pol  are summarized in Tables 1 and 2, respectively. To examine the influence of lesions, we used the relative insertion efficiency f ins , which was defined as (k cat /K m ) modified or mismatched /(k cat /K m ) unmodified . With Kf-exo -, the f ins of dCTP opposite -CG[FABP]A-was 500-fold lower than that of the unmodified control (Table 1). This is contrasted with -TG[FABP]A-which was reduced only 33-fold. In the pol  assay (Table 2), the f ins of dCTP opposite FABP in the CGA sequence was 142-fold lower than that of the control, while in the TGA sequence the f ins was 59-fold lower than that of the control. These results indicate that the nucleotide insertion efficiency is consistently greater in the TGA sequence compared to the CGA sequence, regardless of the polymerase structure. We were unable to perform similar steady-state kinetics experiments for FAAF because this lesion caused a major blockage at the replication fork.

DNA coating and mass transport limitation studies
After activation with streptavidin (SA), flow cells 1 and 3 were retained as blank references, and DNA was coated on the SA surface of flow cells 2 and 4. Surface testing, regeneration buffer scouting, and the mass transport limitation test were performed before the kinetics experiments as described previously. 6 DNA coating at 0.7 resonance units (RU) did not show any influence of mass transport; an increase in flow rate of the analyte did not alter the association rate. However, at 10 RU, mass transport became a limiting factor, as the association rate deviated with the flow rate of the analyte (data not shown). Based on this study of mass transport limitation, all the experiments were carried out in the DNA coating range between 0.7 and 3.5 RU.

Kf-exo -
The sensorgrams for the binary binding between Kf-exoand the unmodified TGA controls or the modified TG*A oligonucleotide constructs are shown in Figure 5a. We performed steady-state affinity analysis of the binary and ternary complexes in the presence of four dNTPs ( Figure 6). A similar set of results for the CGA sequence have been reported previously 6 and the results on the binding affinity of Kf-exoto both TGA and CGA sequences are summarized in Table 3.
As for the unmodified controls, Kf-exobinds tightly in both sequences in the presence of the correct dCTP. The affinity of binding for the CGA sequence was reduced by 30-, 62-, 264-, and 34-fold in binary, dATP, dGTP and dTTP, respectively, compared to the correct dCTP binding (Table 3). Similar results were obtained for TGA, where the binding affinity was reduced by 15-, 39-, 180-, and 40-fold in binary, dATP, dGTP and dTTP, respectively (Table 3). These results are consistent with those of the nucleotide insertion assay, which showed preferential insertion of the correct dCTP.
Kf-exobound strongly to the modified TG*A templates. In the TG*A sequence, the K D value for FABP was 4.9-fold greater than the control, and the K D value for FAAF was 8.8-fold greater than the control. Similar changes were observed in the CG*A sequence, where the K D for FABP was 10.8-fold larger than for the control, and the K D for FAAF was 7.2-fold larger than for the control. These differences are primarily due to the much slower dissociation rates observed for the modified template/primer for both the CG*A sequence (FAAF, k d : 0.02 s -1 ; FABP k d : 0.01 s -1 ) and the TG*A sequence (FAAF, k d : 0.01 s -1 , FABP, k d : 0.01 s -1 ). The net stabilization energies were positive and ranged from 1.10 to1.47 kcal/mol (Table S2).
Nucleotide selectivity was low in the modified ternary complexes. Pol  For pol , binding assays were performed on two distinct substrates: nongapped ds/ds and 1 nt-gap. The results for the binary and ternary systems on both CGA and TGA sequences are summarized in Table 4. Weak binding was observed for the nongapped DNA, with K D values of ~0.8 M (data not shown). In contrast, the binding affinity of pol  increased 1,000 fold with the 1 nt-gap.
As for the unmodified controls, pol  binds to the correct dCTP more tightly. The binding affinity for the dCTP is 2.7-fold higher in the TGA sequence, and 4.5-fold higher in the CGA sequence (Table 4). In contrast to Kf-exo -, the binding in the binary complex between the modified template and pol β is less tight than that in the complex containing the unmodified template, where the differences in binding are approximately 3-fold for  (Table S2). Figure S9 show the sensorgrams for the ternary complexes between pol  and the FAAF-and FABP-modified CG*A constructs. We have recently reported a similar set of binding results for the N-deacetylated FAF. 7 With the correct nucleotide dCTP, the pol  binds 2.7-fold more tightly in the ternary complex than in the binary complex and ~3,000-fold more tightly than to the non-gapped DNA. The binding affinity to the incorrect nucleotide was 4 to 5-fold lower than to the correct dCTP. The lesion in the 1 nt-gap reduced the binding affinity of pol  by 6-fold for FAAF and 3-fold for FABP, virtually eliminating the nucleotide selectivity of pol  at the lesion site. The affinity for pol  binding decreased in the order dG > FABP > FAAF.

DISCUSSION
In the present study, we have employed SPR to investigate the binary and ternary binding interactions of Kf-exoand pol β to two prototype arylamine-DNA lesions (FABP and FAAF) in the context of two different sequences (CG*A and TG*A). Kfexois a 68-kDa high fidelity replicative A-family bacterial DNA polymerase, 56 which carries a polymerase and 3'-5'-exonuclease activities and has been used extensively as a model enzyme for studying adduct-induced DNA synthesis. Pol β is the smallest (39 kDa) eukaryotic polymerase, belonging to the X-family of base-excision repair DNA polymerases, and has been characterized extensively. 57 With pol β, primer extension past AAF adduct was blocked, but full length products were shown to contain exclusively -2 deletion mutations. 17 Although its role is limited in base excision repair, pol β has been additionally implicated in the replication of various DNA damage. For example, deregulation of pol β may enhance the genetic instability induced by bulky lesions such as cis-platin 32 and UV radiation. 33 Pol β can also bypass abasic site 58 and bulky polyaromatic hydrocarbons adducts. 34 FABP and FAAF are C8-substituted dG adducts adducts which contain structurally unique arylamine structures, i.e., Nacetylated/coplanar-fluorene and N-deacetylated/twisted-biphenyl, respectively ( Figure   1a). Finally, the two sequences (CG*A vs. TG*A) were selected because of their marked difference in the S/B population ratios observed with the N-deacetylated FAF. 11 The SPR results, along with data from 19 F NMR and steady-states primer kinetics, elucidate how lesion-induced conformational heterogeneity alters the binding capacity of a polymerase and thus its nucleotide insertion efficiency.

Model hairpin oligonucleotide constructs for SPR binding assays
We constructed the 84-mer hairpin-based oligonucleotides for SPR ( Figure 2) based on the following considerations. First, the incorporation of ddT at the 3-end of the primer prevents the usual nucleophilic attack of the 3'-hydroxyl to the incoming dNTP, and thus blocks the formation of a phosphodiester bond. 25,59 This ensures the stability of the ternary complex polymerase/template-primer/dNTP for SPR measurements. Previous assays using gel electrophoresis, single-molecule FRET, or crystallography have consistently shown that the absence of 3-OH at the primer terminus does not affect the affinity with which polymerases bind to binary and ternary complexes of DNA. 25,59 Second, while Kf-exorequires a minimum of 11 bases, because it covers approximately 5 bases downstream from the primer/template junction and 6-7 bases upstream to the 3primer terminus, 60 pol β can operate on any length of DNA containing a 1 nt-gap.

Binary and ternary binding affinities with unmodified control DNA
We observed very tight binding of Kf-exowith native unmodified dG, in the presence of the correct incoming nucleotide dCTP. This system exhibited high nucleotide selectivity, with K D values increasing in the order dCTP << dTTP ~ dATP << dGTP ( The stoichiometry, however, is highly concentration dependent. As shown in Figure S11, comparison between theoretical and experimental Rmax for pol β and Kf-exoare in good match, indicating a 1:1 complex.
Initially, we carried out a SPR binding assay of pol  using the non-gapped ds/ss junction replication fork. The binding was very weak, with K D values in the range 7 However, upon introduction of the 1 nt-gap (Figure 2c 61 The results are also in agreement with gel assays, which had previously shown that addition of the correct dCTP opposite unmodified DNA enhances the binding affinity of polymerase compared to other nucleotides, by an induced-fit model adopted by pol . 61

Lesion and sequence effects on binary binding affinities with modified DNA
An unusually greater binding of Kf-exowas observed for modified dG, where the K D of this interaction was 5 -11-fold higher than the K D for interaction with the unmodified native DNA substrate. The binary binding affinity decreased in the order FABP > FAAF > dG for the CG*A sequence, and FAAF> FABP > dG for the TG*A sequence (Table 3).
Previous studies have also shown tighter binary binding of Kf-exowith the AAF adduct. 62 Using gel-retardation assays, Dzantiev and Romano 62 showed that the bulky and hydrophobic AAF interacts with nearby hydrophobic amino acid residues, strengthening its binding to the active site of Kf-exo -. The authors suggested that such lesion-induced conformational adjustment may block the conformational change required to properly accommodate an incoming nucleotide. 27 It is well established that the N-deacetylated fluorinated analog FAF adducts ( Figure 1) adopt sequence-dependent equilibrium between B-and S-conformers. FABP is similarly N-deacetylated, but lacks a methylene bridge, resulting in a bulky twisted biphenyl moiety. 21 In other words, FABP may behave like FAAF at the replication fork of the template in the active site of a polymerase. In contrast to the unmodified control, modified adducts displayed a significant decrease (7-to 13-fold) in dissociation rate, with positive net stabilization energy (Table S2). The markedly slower off-rates are consistent with single-molecule FRET studies as well as gel shift assay in which the presence of the bulky DNA adduct stabilizes the binary complex and does not induce dissociation before the nucleotide incorporation. 16,61 In contrast to Kf-exo -, pol β exhibited significantly lesser binary binding affinity to the modified templates. Furthermore, the modified sequences exhibited significantly faster dissociation rates and more negative net stabilization energies. As in the ds/ss situation discussed above, it is likely that FAAF promote conformational heterogeneity in a sequence containing a 1-nt gap. Such heterogeneity may hinder the interaction of that sequence with key amino acids in the polymerase, thus preventing the polymerase from undergoing conformational change that is necessary for strong binding.

Lesion and sequence effects on ternary binding affinities with modified DNA
Nucleotide selectivity was low in the ternary complexes with Kf-exo -, where the K D values indicate poor discrimination between the correct (K D 0.19 -0.30 nM) and incorrect (K D 0.28 -0.67 nM) nucleotides. Variance in these values ranged from 1.5-to 3.5-fold (Table 3) The low selectivity for incoming nucleotides could also arise from the high stability of binary complex, which may hinder the polymerase's ability to recognize the incoming nucleotide. No crystal structures or high-resolution NMR structures are currently available for complexes between any DNA polymerase and ABP or the fluorinated FABP.
In the present study, FABP in both sequences exhibited a single 19 F signal possibly for a B-or a B/S-conformational mix owing to the presumed conformational flexibility at the ds/ss junction. These NMR data, albeit in the absence of a polymerase, are in agreement with the gel based kinetics data, which reveal a preference towards inserting the correct nucleotide over other nucleotides (Table 1).
In the case of Kf-exo -, TG*A sequence favored the insertion of dCTP more efficiently than the CG*A sequence. The relative insertion efficiency f ins of dCTP opposite FABP was significantly lower in the CG*A (500-fold) and TG*A (33-fold) sequences compared to the unmodified controls (Table 1). This 15-fold difference in f ins is puzzling because FABP at ds/ss junction exhibited a single 19 F signal in both sequences ( Figure 3).
However, we have shown previously that FAF in the duplex setting displayed a greater Sconformer in the CG*A duplex (50%) relative to the TG*A (38%). As mentioned above, it is likely that the absence of co-planarity in FABP would embrace intermediate structures between FAAF and FAF, as observed from 19 F NMR, gel and SPR assays.
The SPR results with pol β (Table 4) indicated that a modified templating base weakens the polymerase binding affinity and the nucleotide selectivity ( Figure S9, Table   4). The reduced binding affinity of pol β to the modified template DNA could be related to the lesion-induced conformational heterogeneity in the active site of the polymerase.
In the closed conformation, key amino acids such as Lys 234 and Tyr 271 interact with the minor groove of the primer strand, while Arg 283 interacts with the template strand of DNA. As mentioned above, it is possible that the FAAF at the 1 nt-gap may hinder the active site geometry, and thus prevent the conformational change necessary to form the catalytic ternary complex. We previously observed similar conformational heterogeneity caused by FAF bound to 1 nt-gap DNA in both the absence and presence of pol β. 7 The .

SPR as a powerful tool for probing polymerase action
In the present study we have taken advantage of the sensitivity of SPR, which allowed us to probe the delicate interaction between polymerases and DNA strands containing arylamine-DNA lesions at the binary and ternary complex levels. We were able to measure a sub-nanomolar difference in binding affinity among dNTPs. We found that 0.7 -3.5 RU of DNA coating was sufficient, with no significant interference from mass transport limitation.
The binding specificity ratios (K D of the control binary complex over the K D of a ternary complex) in the presence of dNTPs, for the unmodified (dG) and FAAF-and FABP-modified lesions are plotted as in Figure 7. The dNTPs are color-coded in the plot.
We observed highly specific binding between Kf-exoand the native DNA substrates in the presence of the correct dCTP (green) opposite a dG templating base (Figure 7a,b).   (Table S1) and kinetics details of sequence binding with Kf-exoand pol  in binary system (1:1 binding) (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

Introduction
Arylamine is an important group of 'bulky' environmental pollutants that has been implicated in various sporadic human cancers such as the bladder, breast, and liver cancer.
[1] 2-Aminofluorene and its derivatives have been most extensively studied as model bulky carcinogens. In vivo, these chemicals are reduced to N-hydroxylamine and subsequently activated to the acetyl or sulfate derivatives by the action of ubiquitous Nacetyltransferase or sulfotransferase enzymes. [2] Consequently, these pro-carcinogenic esters produce highly reactive electrophilic nitrenium ions, which are known to interact directly with cellular DNA to form DNA adducts. [2] In vivo, 2-aminoflurene produces two major C8-subsituted dG adducts, N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-

C8-AF, simply designated as AF here on), and N-acetyl-(2'-deoxyguanosin-8-yl)-2-
aminofluorene (dG-C8-AAF, simply designated as AAF here on). [3] The related arylamine 4-aminobiphenyl is a known human bladder carcinogen that also binds to dG at C8 to form N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP, designated as ABP here on) as a major adduct. The structures of AF and AAF differ only in that the latter contains a bulky acetyl group on the central linking nitrogen (Figure 1). Despite the structural similarity, they produce different mutational and repair outcomes. In E. coli, AF produces both point and frameshift mutations, whereas AAF results in mostly frameshift mutations. [4][5][6][7] However, both lesions produce primarily G to T point mutations in the COS-7 mammalian cells replication. [8,9] The N-deacetylated AF adduct in fully paired duplexes adopts an equilibrium between syn-glycosidic stacked (S) and anti-glycosidic major groove (B)-type conformations. mutations. This process for frameshift mutagenesis is known as "Streisinger Slippage Model", which is proposed by Streisinger and colleagues decades ago. [21,22] Compared to AF, the bulkier N-acetylated AAF has shown much greater propensity to induce frameshift mutation in the NarI sequence. AAF is a strong blocker in highly replicative polymerase, and is bypassed by the low fidelity polymerase, ultimately producing various deletion mutations. [6,7,23,24] Clearly, the nature of polymerases can also contribute to the efficiency of deletion mutations. Gill  Using primer extension assays coupled with MALDI-TOF mass spectrometry, Schorr and Carell have shown that frameshift mutation is triggered by the unstable molecular association of the AAF-dG lesion with the correct incoming nucleotide dC. [26] Such configurations have been observed in both replicative and bypass polymerases and are likely to promote the lesion-containing dG and flanking bases to slip to form bulge structures. Hence, the stability of bulged-out structures and subsequent elongation will determine the propensity for frameshift mutagenesis. To that end, we recently performed systematic structure and conformational studies of FAAF-modified NarI-sequence based −1, −2, and −3 deletion duplexes. [27] FAAF is the 19 F analog of AAF. These SMIs existed in a mixture of the so-called external "solvent exposed" B-type (B-SMI) and inserted "solvent protected" "stacked" S (S-SMI) conformers, with the population of the S conformer and thermodynamic stability in the order of −1 > −2 > −3 deletion duplexes.
The results showed greater thermal and thermodynamic stabilities of S-SMI over the flexible B-SMI, which supports the aforementioned Carell's hypothesis. We also studied

NarI-based -2 deletion [(5'-CTCGGCG*CNATC-3') (5'-GATNGCCGAG-3'), N = dC
or dT] duplexes, in which G* was FAF, the 19 F analog of AF. These sequences mimic a SMI for -2 deletion mutations. The results indicated that the NarI-dC/-2 deletion duplex adopts mostly a S-SMI conformer, whereas the NarI-dT/-2 deletion duplex exists as a mixture of S-SMI and various 'exposed" B-SMI ( Figure 1). [28] In the present study, we hypothesize that the NarI-induced frameshift mutagenesis is stimulated by the conformational stability of SMI formed during TLS. The conformational, thermodynamics, and binding affinity details of the two progressive TLS models were examined, in which the FAAF/FABP lesion is positioned at G 3 position of 16-mer NarI sequence (5'-CTCTCG 1 G 2 CG 3 * CNATCAC-3', N=C: NarI-dC Series; N=T: NarI-dT Series). These templates were both annealed systematically with increasing primer lengths (full length extended or -2 deletion), and their thermodynamic, conformational, and binding profiles at each elongation step were investigated and analyzed. We have utilized a powerful array of biophysical techniques such as differential scanning calorimetry (DSC), surface plasmon resonance (SPR), as well as circular dichroism (CD) and dynamic 19 F and imino NMR spectroscopy. The results are discussed the critical role of conformational stability, heterogeneity and flexibility in the mechanisms of bulky arylamine-induced frameshift mutagenesis.

Materials and Methods
Caution: Aminofluorene and aminobipheyl are animal, human carcinogens respectively therefore caution is required when handling. and filtered with 0.2 μm filter paper. The worked out reaction mixture was injected to reverse phase preparative HPLC (Figure 5a) and appropriate peaks were purified up to 99% purity. In theory there should be a total of seven FAAF adducts due to the presence of three guanines in the model sequence: three mono-, three di-and one tri-adduct. As such, a stringent HPLC condition is required. Our HPLC mobile systems entails a gradient of 3-9% acetonitrile for 5min followed by 9-30% acetonitrile for 20-min, in pH 7.0 100 mM of ammonium acetate buffer with a flow rate of 2.0 mL/min. The modified DNA was collected and purified up to 99% purity by repeating the mixture injections following the method involving a gradient system of 7.5-12.2% acetonitrile in 100 mM of ammonium acetate buffer with a flow rate of 2.0 mL/min for 30min, followed by 12.2-40% for 5min and then 40%-7.5% for 5min.
All seven modified adducts were isolated and three mono-adducts were characterized by MALDI-TOF using 3'→5' or 5'→3' exonuclease enzyme digestion method. The isolated G 3 -FAAF/FABP modified 16-mer sequences were each annealed with appropriate primers with different length to form the various ds/ss duplexes starting from n-1, n, n+1, n+2, n+3, n+6 to full duplex ( Figure 2) for structural studies. A similar set of unmodified templates with appropriate primers was also prepared as controls.

Translesion synthesis (TLS) model Systems:
Two TLS models were designed, in which FAAF or FABP lesion is at G 3 position of a 16-mer NarI sequence (5'-CTCTCG 1 G 2 CG 3 * CNATCAC-3', N = C: NarI-dC Series; N = T: NarI-dT Series). FAAF and FABP are fluorine-tagged AAF and ABP lesions, which are intended for obtaining dynamic 19 F NMR spectra. The underline 12-mer portion of this 16-mer NarI sequence is identical to that used in our previous study, in which the sequence effect of the FAF was investigated in the context of -2 deletion mutation. [28] In that study, the NarI-dC/-2 deletion duplex was found to adopt the S-SMI conformer exclusively, whereas the NarI-dT/-2 deletion duplex showed multiple conformers, presumably consisting of S-and B-SMI conformers among others. Initially, we tried to use the same TLS sequences; however, the initial 12/5-mer template/primer (e.g., n-1) was too short to form proper duplexes to give meaningful thermo-melting and thermodynamic parameters. As such, two more bases were included on both sides (CT on the 5' and AC on the 3') to make a 16-mer, whereas the inner core was kept exactly the same.
Sequence Issues: Figure 3b shows two different -2 SMI models assumed for each lesion, i.e., G 3 *C and CG 3 * bulges for FAAF and FABP respectively, based on the previous high resolution 1 H NMR and fluorescence results that are described below. Using the simple Streisinger model depicted in Figure 3a, insertion of the correct cytosine opposite the lesion at G 3 * is the first step. [34] The potentially unstable G 3 *: C pair causes a polymerase to pause at the replication fork, triggering a slippage of the nascent strand and leaves two bases bulge out in the template. However, there are two slippage possibilities, either a G 3 *C or a CG 3 * bulge out. As detailed in Figure 4, the G 3 *C bulge out involves a slippage of two terminal bases ("CG" slip) in the primer hydrogen bonded with the downstream complementary 5'-G 2 C-3' dinucleotide. Alternatively, CG 3 * bulge out can be formed by a single base "C" slippage. Regardless, continued replication of either scenario will lead to a chemically identical daughter strand that is two bases shorter than the parent strand. Figure 4 shows that each of the two pathways (two bases "CG" or one base "C" slippage) is expected to produce a conformational mixture of S-SMI and B-SMI. Both the G 3 *C or CG 3 * bulge out scenarios will lead to the same -2 deletion mutation. The biological outcome of the two models is identical, however, it is important to understand the structural and sequence aspects of the SMI involved in the different lesions. Evidence indicates the importance of the thermodynamic stabilities of the initial base pairing of G 3 *: C at the replication fork. The delicate conformational structures of bulged-out SMI may determine the propensity for frameshift mutagenesis.
There are conflicting reports as to which SMI structure is responsible for the NarI-based -2 deletion. Mao et al [35] conducted NMR/molecular modeling studies on a 12/10-mer -2 deletion duplex [(5'-CTCG 1 G 2 CG 3 *CCATC-3') (5'-GATGGCCGAG-3')], in which G 3 is modified with AF. Their NMR results showed the exclusive presence of the CG 3 * bulge out S-SMI (underlined above), in which the AF-modified guanine in the syn conformation and 5'-C reside in the major groove and the aminofluorene moiety is fully inserted into the bulge. This result is consistent with the results from our 19  The structures of AF and AAF are essentially identical except that AAF possesses a bulky N-acetyl group on the central nitrogen of adduct, thereby exhibiting unique conformational features and different mutational and repair outcomes. The term "Nacetyl factor" was previously coined to describe their repair differences. Schorr and Carell [26] showed that AAF-induced -2 frameshift mutation on NarI sequence by the bypass polymerase pol  indeed follow the Milhe's [36] G 3 *C bulge out model ( Figure  3b). We have utilized fluorescence spectroscopy (unpublished) to investigate the two SMI pathways by using sequences, which include the fluorescent tag pyrrolodeoxycytidine ( P C) in either 5'-or 3'-side of the lesion. The fluorescence results indicated that AAF and AF induce G 3 *C and CG 3 * slipped mutagenic structures, respectively, supporting the NMR results discussed above. Therefore, the conformational stability and flexibility of the G 3 *: C base pairing at the replication fork dictates the types of a slippage, i.e., the conformationally flexible N-deacetylated AF promotes one base (C) slip, whereas the bulky and rigid N-acetylated AAF induces two base (CG) slippage.
Evidently, the nature of the adduct structure (N-acetyl, bulkiness, coplanarity, overall topology) and base sequence contexts surrounding the lesion are important factors for determining the types of -2 frameshifts. FABP is considered as an analog of FAF because both are N-deacetylated, thus susceptible for conformational heterogeneity; however, FABP lacks a bridging methylene group, therefore less coplanar than the FAF. Hence, the G 3 *C bulge model was selected for FAAF and the CG 3 * bulge model for FABP.

Preparation and characterization of modified template sequences
The 16-mer NarI template sequence (5'-CTCTCG 1 G 2 CG 3 CNATCAC-3', N = C or T for dC and dT series, respectively) was treated with either an activated FAAF or FABP, according to the biomimetic procedures published previously. [4,28,29] In principle, there should be at least seven adducts because of the three guanines in the NarI sequence; such as three mono-, three di-and one tri-adduct. The guanines in the sequence maintain similar chemical reactivity, and consequently it is possible to regulate the relative ratios of mono-, di-and tri-adducts by adjusting reaction time. The complexity of the adduct profiles called for development of an efficient HPLC separation method.
Previously, the separation of this complex mixture took 90 min to collect all seven modified peaks (Figure 6b). [11] In the present study, an efficient HPLC method was developed to purify the same reaction mixture in a much shorter time frame (Figure 6a) (see Material and Methods for details). Figure 6 compares the two HPLC chromatograms. di-and tri-adducts, respectively (Figure 5b). The structural identities of the FAAFadducts were characterized by exonuclease enzyme digestions-MALDI-TOF mass spectrometry as described below. The results showed that peak 1was G 1 , peak 2 was G 3 , and peak 3 was G 2 in both the NarI dC and dT sequences (Figures 8-13).
Similarly, the treatment of the same 16-mer NarI dC/dT sequence with FABP (5'-CTCTCG 1 G 2 CG 3 CNATCAC-3', N = C or T for dC and dT series) gave a reaction mixture that showed all three group of adducts in less than 45 min, such as the three mono-adducts at 19-24 min, three di-adducts at 35-38 min and the tri-adduct at 42 min ( Figure 7a). The UV shoulder absorbance in the range of 300-320 nm indicated the number of FABP adducts (mono di, and tri). The three mono-FABP adducts were collected with repeated HPLC injections and were characterized by MALDI-TOF as detailed below. The results indicated that peak 1was G 1 , peak 2 was G 3 , and peak 3 was G 2 in both the NarI dC and dT sequences (Figures 14-19). The order of elution was same as the FAAF case above.

NarI-FAAF-16-mer dC sequence:
The FAAF modified NarI 16-mer dC sequence (5'-CTCTCG 1 G 2 CG 3 *CCATCAC-3') was characterized previously using ESI-QTOF-MS. [11] The overall HPLC elution patterns were similar. All three mono-FAAF adducts have been characterized by the analysis of MALDI-TOF spectra (Figures 8-10). Here details of the characterization of peak 2 as G 3 modification is presented, which is relevant to the present study.

NarI-FAAF-16-mer dT sequence:
The FAAF modified dT sequence has not been characterized previously. Three mono-adduct peaks were characterized by MALDI using both 3' and 5' enzyme digestions. Figure 11   one or no base extra to the 5' side of the lesion, respectively. These data confirmed that peak 3 was G 2 . Hence, the results identified the HPLC peaks 1, 2, and 3 as the FAAF at G 1 , G 3 , and G 2 , respectively.

NarI-FABP-16-mer dT sequence:
The HPLC order of elution should be noted to be identical with FAAF/FABP modified NarI 16-mer sequences, i.e., peak 1, 2, and 3 were G 1 , G 3 , and G 2 , respectively, regardless of lesion and next flanking base sequences.
UV melting: All TLS model duplexes showed mostly monophasic sigmoidal curves on UV melting (Figures 20 and 21). A correlation (R 2 > 0.9) between lnC t and Tm -1 was observed, confirming typical helix-coil melting transitions. Tables 1-4 summarize the thermal and thermodynamic parameters calculated from UV melting curves.

UV melting Curves:
FAAF series: Figure 20 shows UV-melting curves of FAAF-modified full (NarI-FAAF-Full-dC and NarI-FAAF-Full-dT) and -2 deletion duplexes (NarI-FAAF-SMI-2-dC and NarI-FAAF-SMI-2-dT) in the dC and dT series along with corresponding unmodified control models. The unmodified n-1 duplex (16/7-mer) in the dC series ( Figure 20a, dotted black), in which the primer is elongated to the one base before the lesion, was not clearly defined presumably because of a short primer. However, the duplex melting gradually improved to produce well-behaved sigmoidal curves, i.e., increase of Tm as function of temperature (n to n+8). In contrast with the FAAF modified model, the Tm from n to n+3 barely increased. The results indicated a lesion-induced destabilization. By contrast, the corresponding -2 SMI (FAAF-SMI-2-dC) duplex exhibited well-behaved melting curves of all duplexes including the n-1, with generally higher melting (for n to n+2). However, for the unmodified -2 SMI model, the Tm did not change between n and n+3. A higher melting of -2 SMI over the full duplex at n and n+1 indicated lesion-induced duplex stabilization. The opposite result was observed in the dT series, in which the Tm -2 SMI at n and n+1 was lower than that of the full duplex.
This finding indicated the direct effect of the next flanking base N (e.g., T over C) on the bulge stability of FAAF at G 3 ( Figure 20).
FABP series: Figure 21 shows the UV-melting curves of FABP-modified full and -2 SMI duplexes in both dC and dT series along with the unmodified controls. As in FAAF, FABP stabilized the duplex at n-1 in both the dC and dT series. FABP modified -2 SMI models showed a gradual increase of Tm, suggesting FABP-induced stabilization in the -2 bulge structure.

UV melting thermodynamics:
FAAF-dC series: Figures 22a and 22b show plots of UV-based thermal-melting (Tm) and thermodynamics (ΔG) for the FAAF modified full (NarI-FAAF-Full-dC; left) and -2 SMI (NarI-FAAF-SMI-2-dC; right) duplexes of dC Series with increasing length of primers (n+8 and n+6 for full duplex and -2 SMI, respectively). FAAF-modified duplexes (red, empty circles) are compared with unmodified ones (blue, filled circles). In the unmodified full duplex model, values increased consistently as expected from standard primer elongation (blue lines). However, for the unmodified -2 SMI models (blue lines), the thermal and thermodynamic values slightly changed from n to n+3. In both full and -2 SMI cases, the lesion effects were minimal at and prior to the lesion site (n-1 to n+1), but became significant between n+2 and full (n+6 and n+8 for -2 SMI and Full, respectively). In the -2 SMI models, the modified (NarI-FAAF-SMI-2-dC) showed greater stability (higher T m and lower ΔG) than the unmodified controls (ΔTm, 2.10 °C to 13.06 °C, ΔΔG, -0.27 to -4.10 kcal/mol) for the n+2 to n+6 positions. By contrast, the FAAF modified full-length duplexes (NarI-FAAF-Full-dC) showed lower thermal and thermodynamic stabilities (lower ΔTm -6.20 °C to -15.61°C) and higher ΔΔG (1.77 to 4.98 kcal/mol) values compared with those of the unmodified controls (Table 1).
These results indicate that the lesion effect at n-1 to n+1 is minimal in both -2 SMI and full TLS models. Remarkably, no SMI is expected to form up to this point although some discernible differences appear at n+1. The lesion effect was quite consistent between n+2 and n+6/n+8. The thermal and thermodynamic stability of the -2 SMI model over the control SMI was clearly due to the formation of a stable -2 SMI structure in which FAAF is stacked in the solvent protected bulge environment. By contrast, the negative thermodynamic effect on the full-length duplex models is contributed to the FAAF-induced S/B/W-conformational heterogeneity at both replication fork and duplex settings.
FAAF-dT series: Similar trend was observed in the dT series. Figure 23 shows the Tm and ΔG comparison between Full (NarI-FAAF-Full-dT; left) and -2 SMI (NarI-FAAF-SMI-2-dT; right) models for the FAAF dT series as a function of increasing length of primers. In the fully extended model, FAAF-modified duplex is thermally and thermodynamically less stable than the unmodified control (lower ΔTm -9.61 °C to -  Figure 24 shows the Tm and ΔG comparison between FABPmodified full and -2 SMI models with increasing length of primers for the dC series. For FAAF, the fully extended FABP duplexes are thermally and thermodynamically less stable than the unmodified controls in the dC (NarI-FABP-Full-dC) (ΔTm -7.17 °C to -14.47 °C) series. However, in the -2 SMI models, thermal and thermodynamic stability significantly increased from n+2 to n+6 (ΔTm 0.48 °C to 8.17 °C and ΔΔG -0.1 kcal/mol to -2.18 kcal/mol) ( Table 3). Figure 25 shows the Tm and ΔG comparison between FABPmodified fully extended and -2 SMI models with increasing length of primers for the dT series. In the fully extended model, the FABP modified duplex destabilized the structure by ΔTm -7.32 °C to -15.25 °C and higher ΔΔG 2.16 kcal/mol to 4.27 kcal/mol ( Table 4).

DSC
We also conducted DSC experiments on FAAF-modified and unmodified control -2 SMI TLS models in the dC and dT series. Figure 26 shows the overlays of plots of heat capacity change with increasing temperatures. The maximum point of the Gaussian bell curves in the DSC thermograms represents duplex melting (Tm), and the areas under the curve denote transition enthalpy values (ΔH). The DSC results are independent of concentration and thus provide reliable thermal and thermodynamic parameters compared with those of UV melting. Figure 26a shows an overlay of the unmodified dC series (NarI-SMI-2-dC) from n-1 to n+6 as controls. The n-1 curve (cyan), which represents a 16/7ds/ss duplex, shows a broad curve with Tm of 35.1 °C and ΔH of -45.0 kcal/mol ( Table   5). The curve shapes up nicely with one additional base (n) with Tm of 48.5 °C and ΔH of -48.5 kcal/mol. Both Tm and ΔH have mostly stalled between n+1 and n+3. However, a significant increase existed at n+6 in Tm (57.3 °C) and ΔH (-120.9 kcal/mol). This DSC profile is inconsistent with the regular full-paired TLS cases, which generally show an incremental Tm/ΔH increases with increasing primer elongation. [37] Therefore, these results reflect the presence of a -2 bulge duplex formation. Figure 26b shows FAAF-modified -2 SMI bulge structure with increasing length of primers (n-1 to n+6). The major difference compared with the unmodified control ( Figure 26a) indicated that Tm and ΔH increased progressively with increasing primer elongation from n-1 to n+3 TLS. In contrast with the unmodified control of -2 SMI, the curves for n to n+3 were all clustered together around the Tm of 48 °C (Figure 26a). These DSC patterns resemble those obtained from melting of a regular full-length unmodified DNA duplex. These results support a unique stabilizing effect of the bulky FAAF though insertion and hydrophobic stacking. Figure 26c shows the DSC profiles for the unmodified -2 SMI TLS models in the dT series. The DSC profile trend was similar to that of the corresponding dC series (Figure 26a) with slightly better Tm dispersion for n to n+3. A major exception demonstrated that the Tm and ΔH values are generally smaller in the dT series. Figure 26d shows the DSC curves for the FAAFmodified -2 SMI TLS models. The profile trend was very similar to that of the corresponding FAAF-modified -2 SMI dC series with consistently smaller Tm and ΔH values. The increase in ΔH was not as incremental as Tm in the dC series above. The melting Tm of the n-1 duplex was relatively lower (32.3 °C) than that (41.9°C) ( Table 5) of the dC series. This finding is ascribed to the presence of a weak T: A base pair instead of a more stable C: G base pair at the 3'-next flanking base, i.e., dT versus dC at N position (5'-CGGCG*CN-3').  Interestingly, FABP-modified SMI showed a gradual decrease of intensity at 270 nm in the n-1 → n → n+1 sequence, whereas the dT series exhibited slight changes in intensity.

NarI-FAAF-SMI-2-dT:
These CD results indicate that FABP and FAAF are involved in uniquely different mechanisms in the formation of -2 bulge adduct structures. Figure 28 shows the overlays of the CD spectra of FAAF-modified duplexes (red) with those of the unmodified controls (blue) for all TLS steps from n-1 to n+6 in both dC and dT series. In every case, FAAF-modified duplexes exhibited significant blue shifts compared with the unmodified controls. The effects were also greater for dC over dT series (dC series: 6 nm at n-1, 5 nm at n to n+3, 7 nm at n+6; dT series: 4 nm at n-1, 2 nm at n to n+3, 1 nm at n+6) ( Table 6). These data suggest an adduct-induced DNA backbone bending. No significant changes existed in the CD intensity at 270 nm in both series throughout TLS except for the n+3/n+6 in the dT series, indicating a different pathway for the formation of -2 bulge adduct structures. Figure 29 shows similar CD overlays for the FABP-modified duplexes (red) with those of the unmodified controls (blue) in both dC and dT series. We observed FABPinduced blue shifts. However, they were generally smaller (dC series: 4 nm at n-1, 5 nm at n to n+3, 3 nm at n+6; dT series: 1 nm at n-1, 2 nm at n to n+3, 1 nm at n+6) ( Table 6) than the FAAF series. These data indicated a relatively smaller DNA backbone bending in the FAAF case. In the dC series, the intensity at 270 nm was greater than that in the controls during the early stage of bulge formation (n-1 to n+1). However, subsequent TLS decreased from n+2 to n+6. By contrast, FABP in the dT series showed consistently low intensity at 270 nm relative to the unmodified controls with minimal blue shifts (1 nm). Figure 30 shows dynamic 19 F NMR spectra of FAAFmodified -2 SMI TLS models (n-1, n, n+1, n+3 and n+6) for the dC series (see Figure 2 for all sequences). These -2 SMI duplexes exhibited a mixture of 19 F signals, each representing a unique conformation with different electronic environments. The n+6
The 19 F NMR measurements were performed at 5 °C to 70 °C temperature range.
All 19 F signals coalesce into a sharp single peak above 60 °C at around −115 ppm, which represents a fast averaging FAAF-modified single-stranded 16-mer template. The data indicated that conformational heterogeneity exists at the n-1 stage, where the 3'-end of primer was located at one base before the lesion site and the heterogeneity was maintained even at 40 °C. The heterogeneity became more complex as bulge formation was about to occur at n and n+1. The bulge structure began maturing at n+3, and was completed at n+6. We have previously shown the 19 F signals owing to the B-, S-, and Wconformation of a fully paired FAAF-modified duplex to appear at −115.0 to −115.5 ppm, −115.5 to −117.0 ppm and −116.5 to −118.0 ppm ranges, respectively. [11] As mentioned above, the −115 ppm signals at the coalescence temperatures are attributed to the denatured single strand in which the 19 F tag is fully exposed to the solvent. This signal is usually in sync with B-type conformer, in which the 19 F tag is exposed and thus shifted to downfield. The shielded signal at −116.3 ppm can arise from the Van der Waals interactions between the 19 F tag and neighboring base pair as in the S-or W-conformer.
However, the current model is a -2 bulge structure without discernible major or minor groove configurations. As a result, two major 19 F signals at −115.5 and −116.4 ppm at 20 °C in the n+6 duplex (e.g., completed -2 bulge structure) could be assigned to either "lesion-exposed" (B-SMI) or "lesion-stacked" (S-SMI) conformers ( Figure 1). A small signal at −114.8 ppm was observed at lower temperatures (5 °C to 10 °C) and coalesced with the B-SMI signal at 20 °C. The identity of this minor thermally unstable conformer could not be characterized. The B-and S-SMI designation can only be made at the n+1, n+3, and n+6 duplexes, where two well-defined signals were obtained. The conformers observed for the n-1 to n+1 duplexes comprise a mixture of narrow and broad signals, which could be assigned to various conformationally flexible species, including the Band S-SMI originated from the immaturity of the corresponding -2 bulge structures.

NarI-FAAF-SMI-2-dT: Similar dynamic 19F NMR experiments were performed
for the dT series ( Figure 31). We observed a much greater heterogeneity at the n-1 and n duplexes than at the dC series; at least four different conformations were found in the −113 ppm to −117 ppm range. This 19 F signal complexity could be ascribed to numerous intermediate conformers possibly near the lesion at the beginning of bulge formation.
The spectral pattern was simplified at n+1 presumably because of an increased conformational stability, indicating a near completion of the G 3 *C bulge structure. A similar pattern persisted at n+3 with one major and one minor signal at −115.3 and −116.2 ppm, respectively. These results indicate that a primer elongation of three bases after the lesion site is enough to produce a stable -2 SMI and the pattern continue into a full bulge duplex at n+6 with slight changes.
In the dC series, the major downfield and minor upfield signals in the n+6 duplex were assigned to the solvent exposed B-and inserted stacked S-SMI conformers, respectively. As expected, the minor upfield S-SMI signal gradually coalesces into the B-  Figures 35 and 36 show the dynamic 19 F NMR spectra of the FABP-TLS for the formation of a -2 SMI (n-1, n, n+1, n+3, n+6) in the dC and dT series. Unlike the FAAF duplex cases, FABP exhibited a relatively simple conformational heterogeneity throughout TLS. The simplicity is more pronounced in the dC series relative to the dT. The n+6 duplex exhibited 86: 14 ratio of the B-and S-SMI conformers. One major signal dominated in the n+1to n+6 sequence at 5 °C. However, a small peak at around −117 ppm increased along with the temperature increase in n+3 and n+6 duplexes. In particularly, two major peaks existed in n+6 at 50 °C, then exchanged, and eventually merged at 65 °C. Unlike the dC series, the dT series simplified as one major peak from n-1 to n+6. Except at 40 °C, a second minor peak showed up at n+1, n+3, and n+6, although two peaks merged at 60 °C.  (Figures 37 and 38). In summary, the imino proton NMR results generally support the sequence dependent conformational heterogeneity observed in the 19 F NMR experiments.

Surface plasmon resonance
We used surface plasmon resonance (SPR) to examine the binding interactions of the modified 16-mer templates as a function of primer length during TLS involvement in the formation of -2 bulge structures. The FAAF/FABP modified sequences were either full length or -2 SMI duplexes in the dC and dT series. Figures 44 and 45 show the SPR set up used in the present study. The procedure is similar to that reported previously, [38] which involves modified biotinylated 16-mer sequence on a streptavidin-coated carboxymethylated surface with addition of various primers as flow-through analytes. The modified biotinylated 16-mer sequence templates were characterized by 3'-5' exonuclease digestion followed by MALDI-TOF. Figure 42a shows the spectra obtained from peak 2 of the dC series, which displayed the parent ion at 5424 m/z before digestion and major fragments at 3625 and 3336 m/z upon digestion. These findings indicated FAAF modification at G 3 . Figure 42b shows the spectra obtained for peak 2 in the dT series, which exhibited the parent ion at 5439 m/z and major persistent fragments at 3625 and 3336 m/z. The results indicated FAAF modification at G 3 . FABP modified dC/dT series was similarly characterized. Figure 43a shows the following spectra of the dC series: the parent ion at 5370 m/z before digestion and a persistent peak at 3282 m/z, which indicated G 3 modification. Figure 43b shows the following spectra of the FABP modified dT series: the parent ion at 5385 m/z and a persistent fragment ion 3283 m/z, which indicated G 3 modification.  Tables 7 and 8.

Unmodified control models:
In the fully extended model (Figures 44a and 44b),

FAAF modified -2 SMI TLS:
The FAAF-modified -2 SMI models (Figures 44g and 44h) showed fairly gradual increment of RU with increasing primer lengths. The overall RU intensities were greater for dC (Figure 44g) over the dT (Figure 44h) series. Interestingly, the overall sensorgram patterns of the FAAF -2 SMI are quite similar to those of the unmodified fully extended (Figures 44a and 44b). Significant increases also occurred in dissociations rates. These results suggested the strengthening of the template-primer binding affinities by the FAAF-lesion at G 3

Discussion
We conducted a series of systematic structural studies to probe the conformational mechanisms of arylamine-induced -2 frameshift mutation frequently observed in the E.
coli NarI sequence ( The AAF-induced conformational heterogeneity was largely dependent on the nature of flanking bases around the lesion (NG*N sequence context), which in turn led to different mutational and repair outcomes. Earlier, we showed that the AF-modified -2 SMI 12-mer duplex with N=C (CTCG 1 G 2 CG 3 *CCATC) adopts exclusively an "inserted" stacked S conformer (S-SMI), whereas the same duplex with N=T (CTCG 1 G 2 CG 3 *CTATC) exists in a mixture of S-SMI and the "solvent exposed" B-type B-SMI conformers. [28] These results explain why the unusual frameshift vulnerability of the AF lesion at G 3 is dictated by the nature of the next flanking base N (C >> T). However, the detailed conformational mechanisms of SMI formation have yet to be elucidated, which is the subject of the present study.
We hypothesized that the conformational, thermodynamic, and binding stabilities of -2 SMI are critical factors to determine the efficacy of frameshift mutations in the NarI sequence context. In this study, we examined the conformational details of how a bulky lesion favors a certain specific type of bulge structure during TLS. As mentioned in the Results section, the Streisinger-based -2 bulge formation in the NarI sequence would allow two possible -2 SMI structures, G 3 *C or CG 3 * looped out, albeit producing an identical -2 deletion daughter strand. Basing on the NMR and fluorescence results, we selected the G 3 *C and CG 3 * models for FAAF or FABP, respectively ( Figure 2). We  Tables 1-5 and Tables 7-8).
Conformation rigidity improves as the length of the primer increases, which is again evidenced by the dynamic 19 F NMR spectra. The continued progressive TLS from n+1 produced two very different -2 SMI conformers at n+3 and ultimately in the fully matured n+6 (F and G). They are the B-SMI conformer (F1 and G1 for FABP and FAAF, respectively) in which the lesion is solvent exposed as in the B-type conformation, or S-SMI (F2 and G2 for FABP and FAAF, respectively) in which the lesion is solvent protected and inserted/stacked with the presumed syn-glycosidic G 3 *. Figure Tables 1-5 and Table 7-8).
Lesion Effect: The structures of FABP and FAAF are generally similar in that both are C8-substituted dG lesions, but they differ in two major ways: 1) FABP lacks a bridging methylene carbon, so it is less coplanar than FAAF, and 2) FAAF is Nacetylated and is thus steric near the adduction point and perturbs the DNA helix. We found that FABP prefers, on average, B-SMI (~90% B) over S-SMI (5-10%) regardless of the nature of the 3'-flanking base N (dC or dT). This is in contrast to FAAF, which showed a mixture of B-SMI (59% and 86%) and S-SMI (41% and 14%) for the dC and dT series, respectively. These results indicate the importance of the relative nonplanarity of FABP over FAAF in producing a great amount of S-SMI. Our 19 F NMR data are in general agreement with those of Milhe's 1 H NMR study of an AAF-modified NarI-based 11/9-mer duplex (5'-ACCGGCG*CCACA-3')(5'-TGTG--GCCGGT-3'), which showed 80% of syn-modified dG* S-SMI conformation. [36] A similar study by Mao et al. [5] showed an exclusive presence of stacked S-SMI conformation for N-deacetylated AF in the NarI-based 12/10-mer duplex (5'-CTCGGCG*CCATC-3') (5'-GATGG--CCGAG-3'). The latter appears to be in direct contrast to that observed for the similarly N-  (Tables 1-4). A gradual increase in thermal stability, however, seems to be inconsistent with the striking conformational differences in B-and S-SMI observed between FAAF and FABP.
The result indicates that lesion stacking and bulge formations are both important factors that contribute to the stability of -2 bulge duplexes.

Sequence Effect on Bulge Formation:
Another interesting finding is the effect of the 3'-next flanking N base (dC vs. dT series, Figure 39) on bulge formation, i.e., FABP at n+6 (F) prefers, on average, B-SMI (86% and 94% B) over S-SMI (14% and 6%) for the dC and dT series, respectively. In other words, no discernible difference in conformational population was observed for FABP between the dC and dT series.
However, that was not the case for FAAF, which showed a significant S/B-population difference between the two series: 59%: 41% of B-and S-SMI for the dC series and 86%:14% of B-and S-SMI for the dT series. These results are consistent with our proposed model (Figure 39), which contends the importance of the "lesion coplanarity" and "N-acetyl" factor. For example, the planar and hydrophobic lesion in the FAAF-                                                            (1). Here we describe a protocol that is designed to address some of those issues. It encompasses procedural steps beginning with immobilization of streptavidin on CM5 chips to the final step of data reporting on DNApolymerase interaction binding kinetics. In evaluating the protocol, we carried out experiments using a simple methodology developed in our laboratory, taking advantage of the high sensitivity and superior signal-to-noise ratio of Biacore T200. We probed the binary and ternary binding affinities between exonuclease-deficient Klenow fragment (Kf-exo -) and various arylamine DNA lesions. We employed unmodified and carcinogenmodified oligonucleotides in the presence and absence of dNTPs. The total time required to carry out the method to completion is between one and two weeks, approximately two days for the SPR binding assays and one week for synthesis, purification, and characterization of modified oligonucleotides. Though the protocol presented here is meant for Biacore T100 or T200 model, the overall methodology can be applied for other instruments also.

35.
In the calibration window, enter 4 standards' mass and name.

36.
Fire one standard a time, place the cursor to the required peak and update in the calibration window.

37.
Repeat this step to finish the rest of the standards, and click the "Calibrate" button twice.

38.
Save the calibration method in the calibration files.

43.
For 31 mer DNA (MW < 10,000 Da), linear negative mode is not applicable because of large signal to noise ratios, reflectron positive mode and peptide calibration profile can be used.

44.
In the peak processing part, advanced scenario is used, along with 1 channel peak width, average smoothing method, 20 channels smoothing filter width, subtract the baseline, 80 channels of baseline filter width, 25 % Centroid threshold peak detection method, double threshold, 1 mass range.
Step 2: DNA coating i. How well does the fitted curve overlay with the experimental data.
ii. Does the random injection of same concentration of analyte overlay.
iii. Check the residual range (between the green lines in Biaevaluation software).
iv. Does  2 fall within 1% of highest signal response. vi. Mass transport limitation: Check whether data is limited by mass transport (step 96).
vii. Check the U value (this feature present in Biacore T200 not in T100).

97.
Once k a and k d values are determined, input these parameters in BIAsimulation Basic kinetics module.

98.
Compare the curves between simulated and experimental curves ( Figure 3).
Step 9: Preparation of reports

99.
The fitted curves can be plotted by exporting the file in ASCII format by rightclick over the curves and imported it in any plotting software.

Anticipated results
Due to the high sensitivity of Biacore T200, the DNA coated on the surface and polymerase used in this study was as low as 0.7-3.5 RU and 10 nM, respectively. The amount of DNA and polymerase required for this assay is 20-100 fold lower than that required by previously reported methods (2). With low DNA concentration potentially confounding complexities of mass transport limitation could be minimized and possibly