Conformation-dependent lesion bypass of bulky arylamine-dG adducts generated from 2-nitrofluorene in epigenetic sequence contexts.

Sequence context influences structural characteristics and repair of DNA adducts, but there is limited information on how epigenetic modulation affects conformational heterogeneity and bypass of DNA lesions. Lesions derived from the environmental pollutant 2-nitrofluorene have been extensively studied as chemical carcinogenesis models; they adopt a sequence-dependent mix of two significant conformers: major groove binding (B) and base-displaced stacked (S). We report a conformation-dependent bypass of the N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (dG-FAF) lesion in epigenetic sequence contexts (d[5'-CTTCTC#G*NCCTCATTC-3'], where C# is C or 5-methylcytosine (5mC), G* is G or G-FAF, and N is A, T, C or G). FAF-modified sequences with a 3' flanking pyrimidine were better bypassed when the 5' base was 5mC, whereas sequences with a 3' purine exhibited the opposite effect. The conformational basis behind these variations differed; for -CG*C- and -CG*T-, bypass appeared to be inversely correlated with population of the duplex-destabilizing S conformer. On the other hand, the connection between conformation and a decrease in bypass for flanking purines in the 5mC sequences relative to C was more complex. It could be related to the emergence of a disruptive non-S/B conformation. The present work provides novel conformational insight into how 5mC influences the bypass efficiency of bulky DNA damage.

Developing virtual and augmented reality applications for science, technology, engineering and math education.

The Rhode Island IDeA Network of Biomedical Research Excellence Molecular Informatics Core at the University of Rhode Island Information Technology Services Innovative Learning Technologies developed virtual and augmented reality applications to teach concepts in biomedical science, including pharmacology, medicinal chemistry, cell culture and nanotechnology. The apps were developed as full virtual reality/augmented reality and 3D gaming versions, which do not require virtual reality headsets. Development challenges included creating intuitive user interfaces, text-to-voice functionality, visualization of molecules and implementing complex science concepts. In-app quizzes are used to assess the user's understanding of topics, and user feedback was collected for several apps to improve the experience. The apps were positively reviewed by users and are being implemented into the curriculum at the University of Rhode Island.

Probing the Interactions of Perfluorocarboxylic Acids of Various Chain Lengths with Human Serum Albumin: Calorimetric and Spectroscopic Investigations.

Despite an exponential increase in PFAS research over the past two decades, the mechanisms behind how PFAS cause adverse health effects are still poorly understood. Protein interactions are considered a significant driver of bioaccumulation and subsequent toxicity from re-exposure; however, most of the available literature is limited to legacy PFAS. We utilized microcalorimetric and spectroscopic methods to systematically investigate the binding between human serum albumin (HSA) and perfluorocarboxylic acids (PFCAs) of varying chain lengths and their nonfluorinated fatty acid (FA) counterparts. The results reveal the optimal chain length for significant PFCA-HSA binding and some fundamental interactions, i.e., the polar carboxylic head of PFCA is countered by ionizable amino acids such as arginine, and the fluorocarbon tails stabilized by hydrophobic residues like leucine and valine. Additionally, fluorine's unique polarizability contributes to PFCA's stronger binding affinities relative to the corresponding fatty acids. Based on these observations, we posit that PFCAs likely bind to HSA in a "cavity-filling" manner, provided they have an appropriate size and shape to accommodate the electrostatic interactions. The results reported herein widen the pool of structural information to explain PFAS bioaccumulation patterns and toxicity and support the development of more accurate computational modeling of protein-PFAS interactions. TOC graphic created with Biorender.com.

Characterization of molecular interactions between cannabidiol and human plasma proteins (serum albumin and γ-globulin) by surface plasmon resonance, microcalorimetry, and molecular docking.

A cannabidiol (CBD) oral solution (Epidiolex®) has been approved by the United States Food and Drug Administration to treat seizure conditions. However, the biomedical and pharmaceutical applications of CBD are hindered partially due to a limited understanding of CBD's pharmacokinetic behaviors, such as its interactions with plasma proteins. Herein, we investigated the molecular interactions between CBD and two plasma proteins, namely, human serum albumin (HSA) and γ-globulin, using biophysical techniques including surface plasmon resonance (SPR), isothermal titration calorimetry, and differential scanning calorimetry, as well as molecular docking. CBD bound to HSA and γ-globulin in an exothermic manner (enthalpy: -9.3 ×104 and -3.7 ×104 kcal/mol, respectively) with a binding affinity of 1.8 × 10-5 and 1.3 × 10-5 M, respectively. The binding ratio between CBD and HSA or γ-globulin was approximately 1:1 and 3:1, respectively. Furthermore, computational modeling suggested that CBD and warfarin may bind to HSA independently, supported by data from a competitive SPR binding assay. Findings from the current study elucidate CBD's plasma protein binding characteristics and shed light on their impact on CBD's pharmacokinetic property.

Inhibitory Effects and Surface Plasmon Resonance-Based Binding Affinities of Dietary Hydrolyzable Tannins and Their Gut Microbial Metabolites on SARS-CoV-2 Main Protease.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (Mpro) inhibitors are considered as potential treatments for coronavirus disease 2019, and dietary polyphenols show promise in SARS-CoV-2 Mpro inhibition based on in silico studies. In the present study, we utilize a combination of biochemical-, surface plasmon resonance-, and docking-based assays to evaluate the inhibition and binding affinities of a series of tannins and their gut microbial metabolites on SARS-CoV-2 Mpro. The tested compounds (2-50 µM) were hydrolyzable tannins, including ellagitannins (punicalagin and ellagic acid) and gallotannins (tannic acid, pentagalloyl glucose, ginnalin A, and gallic acid), and their gut microbial metabolites, urolithins and pyrogallol, respectively. They inhibited SARS-CoV-2 Mpro (by 6.6-100.0% at 50 µM) and bound directly to the Mpro protein (with dissociation constants from 1.1 × 10-6 to 5.3 × 10-5 M). This study sheds light on the inhibitory effects of tannins and their metabolites on SARS-CoV-2 Mpro.

RI-INBRE: A Statewide NIH Program Grant to Improve Institutional Biomedical Research Capacity in Rhode Island.

The overarching goal of the Rhode Island-IDeA Network of Biomedical Research Excellence (RI-INBRE) is to improve institutional capacity for biomedical research excellence and expand student experiential training opportunities in the State of Rhode Island. RI-INBRE comprises five major core components: The Administrative Core, the Bioinformatics Core, the Centralized Research Core Facility, the Training Core, and the Developmental Research Project Program Core. Since its inception in 2001, RI-INBRE has made significant investments and marked advancements in the biomedical research infrastructure of Rhode Island. RI-INBRE funding has increased the scale and quality of faculty research and engaged undergraduate students, graduate students, and postdoctoral fellows in structured and mentored research training experiences. Over the last 19 years, RI-INBRE has supported 212 faculty researchers and over 533 projects and has provided research-training opportunities for nearly 2,000 students, resulting in 757 publications. Through its student-training program, RI-INBRE has contributed to regional workforce development by engaging students and encouraging them to pursue careers in biomedical fields. Many of these students have been admitted to graduate or medical schools and obtained biomedical industry jobs following graduation. RI-INBRE has been particularly influential in building the research infrastructure at primarily undergraduate institutions, which have seen significant improvements in research quality and output, student training, and research infrastructure.

Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using 4-Aminobiphenyl-Modified DNA.

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356-6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

DNA base sequence effects on bulky lesion-induced conformational heterogeneity during DNA replication.

4-Aminobiphenyl (ABP) and its structure analog 2-aminofluorene (AF) are well-known carcinogens. In the present work, an unusual sequence effect in the 5'-CTTCTG1G2TCCTCATTC-3' DNA duplex is reported for ABP- and AF-modified G. Specifically, the ABP modification at G1 resulted in a mixture of 67% major groove B-type (B) and 33% stacked (S) conformers, while at the ABP modification at G2 exclusively resulted in the B-conformer. The AF modification at G1 and G2 lead to 25%:75% and 83%:17% B:S population ratios, respectively. These differences in preferred conformation are due to an interplay between stabilizing (hydrogen bonding and stacking that is enhanced by lesion planarity) and destabilizing (solvent exposure) forces at the lesion site. Furthermore, while the B-conformer is a thermodynamic stabilizer and the S-conformer is a destabilizer in duplex settings, the situation is reversed at the single strands/double strands (ss/ds) junction. Specifically, the twisted biphenyl is a better stacker at the ss/ds junction than the coplanar AF. Therefore, the ABP modification leads to a stronger strand binding affinity of the ss/ds junction than the AF modification. Overall, the current work provides conformational insights into the role of sequence and lesion effects in modulating DNA replication.

Characterization of Byproducts from Chemical Syntheses of Oligonucleotides Containing 1-Methyladenine and 3-Methylcytosine.

Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact MW by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a MW identical to uncharged m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged m3C in the ∼4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted.

Anti-glycation and anti-oxidative effects of a phenolic-enriched maple syrup extract and its protective effects on normal human colon cells.

Oxidative stress and free radical generation accelerate the formation of advanced glycation endproducts (AGEs) which are linked to several chronic diseases. Published data suggest that phenolic-rich plant foods, show promise as natural anti-AGEs agents due to their anti-oxidation capacities. A phenolic-enriched maple syrup extract (MSX) has previously been reported to show anti-inflammatory and neuroprotective effects but its anti-AGE effects remain unknown. Therefore, herein, we investigated the anti-glycation and anti-oxidation effects of MSX using biochemical and biophysical methods. MSX (500 µg mL-1) reduced the formation of AGEs by 40% in the bovine serum albumin (BSA)-fructose assay and by 30% in the BSA-methylglyoxal (MGO) assay. MSX also inhibited the formation of crosslinks typically seen in the late stage of glycation. Circular dichroism and differential scanning calorimeter analyses demonstrated that MSX maintained the structure of BSA during glycation. In the anti-oxidant assays, MSX (61.7 µg mL-1) scavenged 50% of free radicals (DPPH assay) and reduced free radical generation by 20% during the glycation process (electron paramagnetic resonance time scan). In addition, the intracellular levels of hydrogen peroxide induced reactive oxygen species were reduced by 27-58% with MSX (50-200 µg mL-1) in normal/non-tumorigenic human colon CCD-18Co cells. Moreover, in AGEs and MGO challenged CCD-18Co cells, higher cellular viabilities and rapid extracellular signal-regulated kinase (ERK) phosphorylation were observed in MSX treated cells, indicating its protective effects against AGEs-induced cytotoxicity. Overall, this study supports the biological effects of MSX, and warrants further investigation of its potential as a dietary agent against diseases mediated by oxidative stress and inflammation.

DNA-binding studies of the natural ß-carboline eudistomin U.

Eudistomin U is a member of the ß-carboline class of heterocyclic amine-containing molecules that are capable of binding to DNA. The structure of eudistomin U is unique since it contains an indole ring at the 1-position of the pyridine ring. While simple ß-carbolines are reported to intercalate DNA, an examination of the mode of binding of eudistomin U has been lacking. We report preliminary spectroscopic (UV-Vis, thermal denaturation, CD) and calorimetric (DSC) data on the binding of eudistomin U to DNA, which suggest that eudistomin U binds weakly according to a mechanism that is more complicated than other members of its class.

Dissociation Dynamics of XPC-RAD23B from Damaged DNA Is a Determining Factor of NER Efficiency.

XPC-RAD23B (XPC) plays a critical role in human nucleotide excision repair (hNER) as this complex recognizes DNA adducts to initiate NER. To determine the mutagenic potential of structurally different bulky DNA damages, various studies have been conducted to define the correlation of XPC-DNA damage equilibrium binding affinity with NER efficiency. However, little is known about the effects of XPC-DNA damage recognition kinetics on hNER. Although association of XPC is important, our current work shows that the XPC-DNA dissociation rate also plays a pivotal role in achieving NER efficiency. We characterized for the first time the binding of XPC to mono- versus di-AAF-modified sequences by using the real time monitoring surface plasmon resonance technique. Strikingly, the half-life (t1/2 or the retention time of XPC in association with damaged DNA) shares an inverse relationship with NER efficiency. This is particularly true when XPC remained bound to clustered adducts for a much longer period of time as compared to mono-adducts. Our results suggest that XPC dissociation from the damage site could become a rate-limiting step in NER of certain types of DNA adducts, leading to repression of NER.

Conformational Insights into the Mechanism of Acetylaminofluorene-dG-Induced Frameshift Mutations in the NarI Mutational Hotspot.

Frameshift mutagenesis encompasses the gain or loss of DNA base pairs, resulting in altered genetic outcomes. The NarI restriction site sequence 5'-G1G2CG3CX-3' in Escherichia coli is a well-known mutational hotspot, in which lesioning of acetylaminofluorene (AAF) at G3* induces a greater -2 deletion frequency than that at other guanine sites. Its mutational efficiency is modulated by the nature of the nucleotide in the X position (C ∼ A > G ≫ T). Here, we conducted a series of polymerase-free solution experiments that examine the conformational and thermodynamic basis underlying the propensity of adducted G3 to form a slipped mutagenic intermediate (SMI) and its sequence dependence during translesion synthesis (TLS). Instability of the AAF-dG3:dC pair at the replication fork promoted slippage to form a G*C bulge-out SMI structure, consisting of S- ("lesion stacked") and B-SMI ("lesion exposed") conformations, with conformational rigidity increasing as a function of primer elongation. We found greater stability of the S- compared to the B-SMI conformer throughout TLS. The dependence of their population ratios was determined by the 3'-next flanking base X at fully elongated bulge structures, with 59% B/41% S and 86% B/14% S for the dC and dT series, respectively. These results indicate the importance of direct interactions of the hydrophobic AAF lesion with the 3'-next flanking base pair and its stacking fit within the -2 bulge structure. A detailed conformational understanding of the SMI structures and their sequence dependence may provide a useful model for DNA polymerase complexes.

Structure Activity Related, Mechanistic, and Modeling Studies of Gallotannins containing a Glucitol-Core and α-Glucosidase.

Gallotannins containing a glucitol core, which are only produced by members of the maple (Acer) genus, are more potent α-glucosidase inhibitors than the clinical drug, acarbose. While this activity is influenced by the number of substituents on the glucitol core (e.g. more galloyl groups leads to increased activity), the mechanisms of inhibitory action are not known. Herein, we investigated ligand-enzyme interactions and binding mechanisms of a series of 'glucitol-core containing gallotannins (GCGs)' against the α-glucosidase enzyme. The GCGs included ginnalins A, B and C (containing two, one, and one galloyl/s, respectively), maplexin F (containing 3 galloyls) and maplexin J (containing 4 galloyls). All of the GCGs were noncompetitive inhibitors of α-glucosidase and their interactions with the enzyme were further explored using biophysical and spectroscopic measurements. Thermodynamic parameters (by isothermal titration calorimetry) revealed a 1:1 binding ratio between GCGs and α-glucosidase. The binding regions between the GCGs and α-glucosidase, probed by a fluorescent tag, 1,1'-bis(4-anilino-5-napththalenesulfonic acid, revealed that the GCGs decreased the hydrophobic surface of the enzyme. In addition, circular dichroism analyses showed that the GCGs bind to α-glucosidase and lead to loss of the secondary α-helix structure of the protein. Also, molecular modeling was used to predict the binding site between the GCGs and the α-glucosidase enzyme. This is the first study to evaluate the mechanisms of inhibitory activities of gallotannins containing a glucitol core on α-glucosidase.

Real-time surface plasmon resonance study of biomolecular interactions between polymerase and bulky mutagenic DNA lesions.

Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl (FABP) or N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5'-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo(-)) or polymerase ß (pol ß). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo(-) binds strongly to a double strand/single strand template/primer junction, whereas pol ß binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol ß, Kf-exo(-) binds tightly to lesion-modified templates; however, both polymerases exhibited minimal nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and (19)F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared to that in CG*A. Although Kf-exo(-) was not sensitive to the presence of a DNA lesion, FAAF-induced conformational heterogeneity perturbed the active site of pol ß, weakening the enzyme's ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.

Conformational insights into the lesion and sequence effects for arylamine-induced translesion DNA synthesis: 19F NMR, surface plasmon resonance, and primer kinetic studies.

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3'-next flanking base (G*CT vs G*CA; G*, FABP, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl; FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair ( Jain , V. , Hilton , B. , Lin , B. , Patnaik , S. , Liang , F. , Darian , E. , Zou , Y. , Mackerell , A. D. , Jr. , and Cho , B. P. ( 2013 ) Nucleic Acids Res. , 41 , 869 - 880 ). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo(-)) and Dpo4. We obtained dynamic (19)F NMR spectra for two 19-mer modified templates during primer elongation: G*CA [d(5'-CTTACCATCG*CAACCATTC-3')] and G*CT [d(5'-CTTACCATCG*CTACCATTC-3')]. We found that lesion stacking is favored in the G*CT sequence compared to the G*CA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*CA > G*CT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct's chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo(-) vs Dpo4). Steady-state kinetics analysis with Kf-exo(-) revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.

Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo.

Cartilage destruction is a crucial process in arthritis and is characterized by the degradation of cartilage proteins, proteoglycans, and type II collagen (CII), which are embedded within the extracellular matrix. While proteoglycan loss can be reversed, the degradation of CII is irreversible and has been correlated with an over-expression and over-activation of matrix metalloproteinases (MMPs). Among the various MMPs, the collagenase MMP-13 possesses the greatest catalytic activity for CII degradation. Here we show that the pomegranate-derived polyphenols, punicalagin (PA) and ellagic acid (EA), inhibit MMP-13-mediated degradation of CII in vitro. Surface plasmon resonance studies and molecular docking simulations suggested multiple binding interactions of PA and EA with CII. The effects of PA on bovine cartilage degradation (stimulated with IL-1ß) were investigated by assaying proteoglycan and CII release into cartilage culture media. PA inhibited the degradation of both proteins in a concentration-dependent manner. Finally, the anti-inflammatory effects of PA (daily IP delivery at 10 and 50mg/kg for 14days) were tested in an adjuvant-induced arthritis rat model. Disease development was assessed by daily measurements of body weight and paw volume (using the water displacement method). PA had no effect on disease development at the lower dose but inhibited paw volume (P<0.05) at the higher dose.

Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence.

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.