Critical effect of the N2 amino group on structure, dynamics, and elasticity of DNA polypurine tracts

Unrestrained 5-20-ns explicit-solvent molecular dynamics simulations using the Cornell et al. force field have been carried out for d[GCG(N)11GCG]2 (N, purine base) considering guanine*cytosine (G*C), adenine*thymine (A*T), inosine*5-methyl-cytosine (I*mC), and 2-amino-adenine*thymine (D*T) basepairs. The simulations unambiguously show that the structure and elasticity of N-tracts is primarily determined by the presence of the amino group in the minor groove. Simulated A-, I-, and AI-tracts show almost identical structures, with high propeller twist and minor groove narrowing. G- and D-tracts have small propeller twisting and are partly shifted toward the A-form. The elastic properties also differ between the two groups. The sequence-dependent electrostatic component of base stacking seems to play a minor role. Our conclusions are entirely consistent with available experimental data. Nevertheless, the propeller twist and helical twist in the simulated A-tract appear to be underestimated compared to crystallographic studies. To obtain further insight into the possible force field deficiencies, additional multiple simulations have been made for d(A)10, systematically comparing four major force fields currently used in DNA simulations and utilizing B and A-DNA forms as the starting structure. This comparison shows that the conclusions of the present work are not influenced by the force field choice.     

The effect of methylation of the 6 oxygen of guanine on the structure and stability of double helical DNA

The effect of methylation of the O-6 position of guanine in short segments of double helical DNA has been investigated by molecular mechanical simulations on the sequences d(CGCGCG)2, d(CGC[OMG]CG)2, d(CGT[OMG]CG)2, d(CGC[OMC]CG/(CGCGCG), d(CGC[OMG]CG/d(CGTGCG), d(CGCGAATTCGCG)2 and d(CGCGAATTC[OMG]CG)2. Guanines methylated at the O-6 position are found to form hydrogen bonds of roughly equal strength to cytosine and thymine. The optimum structure of these modified base pairs are not dramatically different from normal GC pairs, but both involve some bifurcation of the proton donors of cytosine (4NH2) or thymine (3NH) between the guanine N3 and O6 groups.     

Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5'-d(…G*pT*…) and 5'-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a modified thymine on the structure and stability of [d(TGGGT)]4 quadruplex

Telomeric guanine-rich sequence can adopt quadruplex structures that are important for their biological role in chromosomal stabilisation. G quartets are characterised by the cyclic hydrogen bonding of four guanine bases in a coplanar arrangement and their stability is ion-dependent. In this work we compare the stability of [d(TGGGT)]₄ and [d(T*GGGT)]₄ quadruplexes. The last one contains a modified thymine, where the hydroxyl group substitutes one hydrogen atom of the methyl group of the thymine in the [d(TGGGT)]₄ sequence. We used a combination of spectroscopic, calorimetric and computational techniques to characterise the G-quadruplex formation. NMR and CD spectra of [d(T*GGGT)]₄ were characteristic of parallel-stranded, tetramolecular quadruplex. CD and DSC melting experiments reveal that [d(T*GGGT)]₄ is less stable that unmodified quadruplex. Molecular models suggest possible explanation for the observed behaviour.     

A simple method for the solid phase synthesis of oligodeoxynucleotides containing O4-alkylthymine.

A route to prepare the cyanoethyl-phosphoramidite monomer of O4-alkylthymine and a method for the routine solid-phase synthesis of oligodeoxynucleotides containing O4-alkylthymine are described. This method has been used to make DNA sequences up to 48 bases in length. The amino function of the adenine and guanine in the sequence were protected with the phenoxyacetyl group, and that of cytosine with the isobutyryl group. The phosphodiesters were protected with the cyanoethyl group. This allowed complete deprotection of the oligomer with alkoxide ions (methanol/1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) for the oligomers containing O4-methylthymine, or ethanol/DBU for those containing O4-ethylthymine) thus avoiding the use of ammonia which is known to attack the O4-alkylthymine to form 5-methylcytosine. There was no detectable loss of the alkyl group to form thymine.     

Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: quantitative and qualitative distribution within DNA

As after irradiation with 254-nm UV light, exposure of thymidine and three isomeric pyridopsoralen derivatives to UVA radiation, in the dry state, leads to the formation of the six diastereomers of cyclobutadithymidine as the predominant reaction. This unexpected photosensitized reaction, which also gives rise to both 5R* and 5S* diastereomers of 5,6-dihydro-5-(alpha-thymidylyl)thymidine (or "spore" photoproduct), is selective since [2 + 2] dimerization of 2'-deoxycytidine was not detected under the same experimental conditions. The cis-syn isomer of cyclobutadithymine was also found to be produced within isolated DNA following UVA irradiation in aqueous solutions containing 7-methylpyrido[3,4-c]psoralen. Quantitatively, this photoproduct represents about one-fifth of the overall yield of the furan-side pyridopsoralen [2 + 2] photocycloadducts to thymine. DNA sequencing methodology was used to demonstrate that pyridopsoralen-photosensitized DNA is a substrate for T4 endonuclease V and Escherichia coli photoreactivating enzyme, two enzymes acting specifically on cyclobutane pyrimidine dimers. Furthermore, the dimerization reaction of thymine is sequence dependent, with a different specificity from that mediated by far-UV irradiation as inferred from gel sequencing experiments. Interestingly, adjacent thymine residues are excellent targets for 7-methylpyrido[3,4-c]psoralen-mediated formation of cyclobutadithymine in TTTTA and TTAAT sites, which are also the strongest sites for photoaddition. The formation of cyclobutane thymine dimers concomitant to that of thymine-furocoumarin photoadducts and their eventual implication in the photobiological effects of the pyridopsoralens are discussed.     

Effect of strand orientation on the interaction of berenil with DNA triple helices

Using circular dichroism spectroscopy the ability of berenil, a minor groove binding drug, to induce triple helix formation was investigated with two oligonucleotides designed to form two intramolecular triplexes containing T*A:T and G*G:C triplets, which differ only by the orientation of their third strand: 5'-d(G4A4G4-[T4]-C4T4C4-[T4]-G4T4G4), and 5'-d(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4), where [T4] represents a stretch of four thymine residues. We demonstrate that when added to the duplex form of these oligonucleotides, berenil induces triplex structure formation only if the orientation of third strand is anti-parallel to the purine strand.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.     

Recognition of GT mismatches by Vsr mismatch endonuclease

The Vsr mismatch endonuclease recognises the sequence CTWGG (W = A or T) in which the underlined thymine is paired with guanine and nicks the DNA backbone on the 5'-side of the mispaired thymine. By using base analogues of G and T we have explored the functional groups on the mismatch pair which are recognised by the enzyme. Removal of the thymine 5-methyl group causes a 60% reduction in activity, while removing the 2-amino group of guanine reduces cleavage by 90%. Placing 2-amino-purine or nebularine opposite T generates mis-matches which are cut at a much lower rate (0.1%). When either base is removed, generating a pseudoabasic site (1', 2'-dideoxyribose), the enzyme still produces site-specific cleavage, but at only 1% of the original rate. Although TT and CT mismatches at this position are cleaved at a low rate (approximately 1%), mismatches with other bases (such as GA and AC) and Watson-Crick base pairs are not cleaved by the enzyme. There is also no cleavage when the mismatched T is replaced with difluorotoluene.     

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.     

Sequence preferences of covalent DNA binding by anti-(+)- and anti-(-)-benzo[a]pyrene diol epoxides

The sequence preferences of formation of piperidine-labile adducts of guanine by individual (+)- and (-)-isomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [anti-(+)- and anti-(-)-BPDE] were examined by techniques analogous to chemical DNA sequencing. Data were obtained on over 1200 bases with anti-(-)-BPDE and 1000 bases with anti-(+)-BPDE. Guanines on average yielded more labile adducts than other bases, and the reactivities of guanines with both anti-(+)- and anti-(-)-BPDE isomers were found to be distinctly nonrandom with respect to DNA sequence. The most and least reactive guanines, defined in terms of the upper and lower 10 percentiles of reactivity, differed on average by a factor of 17. This range of guanine reactivities was correlated with distinct sequence preferences, which differed in part for the two isomers. The strongest determinant for preferred reaction of anti-(-)-BPDE to form a labile adduct at a guanine was the presence of a 3'-flanking guanine, but a thymine 5'-flanking a guanine also generally enhanced reactivity. The triplets containing central guanines most preferred by anti-(-)-BPDE were AGG, CGG, and TG(G greater than T greater than C,A). anti-(+)-BPDE also formed labile adducts preferentially at AGG and CGG triplets, but not at TGN triplets. Significant effects of next-nearest-neighbor bases on guanine reactivities were also noted.     

32P-base analysis of DNA

A ³²P-labeling method for the base composition analysis of nonradioactive DNA was developed consisting of the digestion of DNA to deoxynucleoside 3′-monophosphates by incubation with a mixture of micrococcal nuclease and spleen phosphodiesterase, transfer of ³²P-label from [γ-³²P]ATP to the 5′-hydroxyl groups of the mononucleotides by T4 polynucleotide kinase, two-dimensional anion-exchange thin-layer chromatography on PEI-cellulose of the resultant [5′-³²P]deoxynucleoside 3′,5′-bisphosphates, autoradiography, and scintillation counting. The method was standardized to afford quantitative digestion of DNA to mononucleotides as well as to give quantitative incorporation of ³²P-label into the nucleotides in the DNA hydrolysate so as to make the method an accurate means for determining the base composition of eucaryotic DNA containing adenine, guanine, thymine, cytosine, and 5-methylcytosine.     

Site-resolved energetics in DNA triple helices containing G*TA and T*CG triads

Recognition of specific sites in double-helical DNA by triplex-forming oligonucleotides has been limited until recently to sites containing homopurine-homopyrimidine sequences. G*TA and T*CG triads, in which TA and CG base pairs are specifically recognized by guanine or by thymine, have now extended this recognition code to DNA target sites of mixed base sequences. In the present work, we have obtained a characterization of the stabilities of G*TA and T*CG triads, and of the effects of these triads upon canonical triads, in triple-helical DNA. The three DNA triplexes investigated are formed by the folding of the 31-mers d(GAAXAGGT(5)CCTYTTCT(5)CTTZTCC) with X = G, T, or C, Y = C, A, or G, and Z = C, G, or T. We have measured the exchange rates of imino protons in each triad of the three triplexes using nuclear magnetic resonance spectroscopy. The exchange rates are used to map the local free energy of structural stabilization in each triplex. The results indicate that the stability of Watson-Crick base pairs in the G*TA and T*CG triads is comparable to that of Watson-Crick base pairs in canonical triads. The presence of G*TA and T*CG triads, however, destabilizes neighboring canonical triads, two or three positions removed from the G*TA/T*CG site. Moreover, the long-range destabilizing effects induced by the T*CG triad are larger than those induced by the G*TA triad. These findings reveal the molecular basis for the lower overall stability of G*TA- and T*CG-containing triplexes.     

Synthesis and hybridization of dodecadeoxyribonucleotides containing a fluorescent pyridopyrimidine deoxynucleoside.

Partially self-complementary dodecadeoxyribonucleotides containing a fluorescent nucleoside, 3-beta-D-2'-deoxyribofuranosyl-2, 7-dioxopyrido[2, 3-d]pyrimidine (pyridopyrimidine deoxynucleoside, dF) were synthesized by the phosphotriester solidphase method. A dodecanucleotide d(GGGAAFGTTCCC) pairing the analog and guanine at the centre of the chain showed a higher melting temperature than the corresponding G-C paired duplex. A similar comparison between A-T and A-F suggested that weaker hydrogen bonds exist when adenine and pyridopyrimidine residues are paired.     

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.     

The main role of human thymine-DNA glycosylase is removal of thymine produced by deamination of 5-methylcytosine and not removal of ethenocytosine

Metabolites of vinyl chloride react with cytosine in DNA to form 3,N(4)-ethenocytosine. Recent studies suggest that ethenocytosine is repaired by the base excision repair pathway with the ethenobase being removed by thymine-DNA glycosylase. Here single turnover kinetics have been used to compare the excision of ethenocytosine by thymine-DNA glycosylase with the excision of thymine. The effect of flanking DNA sequence on the excision of ethenocytosine was also investigated. The 34-bp duplexes studied here fall into three categories. Ethenocytosine base-paired with guanine within a CpG site (i.e. CpG.(epsilon)C-DNA) was by far the best substrate having a specificity constant (k(2)/K(d)) of 25.1 x 10(6) m(-1) s(-1). The next best substrates were DNA duplexes containing TpG.(epsilon)C, GpG.(epsilon)C, and CpG.T. These had specificity constants 45-130 times smaller than CpG.(epsilon)C-DNA. The worst substrates were DNA duplexes containing ApG.(epsilon)C and TpG.T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG.(epsilon)C-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G.T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G.(epsilon)C base pair more easily than it can flip out thymine from a G.T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.     

Crystal structure of human methyl-binding domain IV glycosylase bound to abasic DNA

The mammalian repair protein MBD4 (methyl-CpG-binding domain IV) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC), and downstream base excision repair proteins restore a G·C pair. MBD4 is also implicated in active DNA demethylation by initiating base excision repair of G·T mispairs generated by a deaminase enzyme. The question of how mismatch glycosylases attain specificity for excising thymine from G·T, but not A·T, pairs remains largely unresolved. Here, we report a crystal structure of the glycosylase domain of human MBD4 (residues 427-580) bound to DNA containing an abasic nucleotide paired with guanine, providing a glimpse of the enzyme-product complex. The mismatched guanine remains intrahelical, nestled into a recognition pocket. MBD4 provides selective interactions with the mismatched guanine (N1H, N2H(2)) that are not compatible with adenine, which likely confer mismatch specificity. The structure reveals no interactions that would be expected to provide the MBD4 glycosylase domain with specificity for acting at CpG sites. Accordingly, we find modest 1.5- to 2.7-fold reductions in G·T activity upon altering the CpG context. In contrast, 37- to 580-fold effects were observed previously for thymine DNA glycosylase. These findings suggest that specificity of MBD4 for acting at CpG sites depends largely on its methyl-CpG-binding domain, which binds preferably to G·T mispairs in a methylated CpG site. MBD4 glycosylase cannot excise 5-formylcytosine (fC) or 5-carboxylcytosine (caC), intermediates in a Tet (ten eleven translocation)-initiated DNA demethylation pathway. Our structure suggests that MBD4 does not provide the electrostatic interactions needed to excise these oxidized forms of mC.     

Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.