The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[ a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions

doi:10.1016/j.jmb.2008.12.082 J. Mol. Biol. (2009) 386, 1193–1203

Available online at www.sciencedirect.com

COMMUNICATION

The Sequence Dependence of Human NucleotideExcision Repair Efficiencies of Benzo[a]pyrene-derivedDNA Lesions: Insights into the Structural Factors thatFavor Dual Incisions

Konstantin Kropachev1, Marina Kolbanovskii1, Yuqin Cai2,Fabian Rodríguez1, Alexander Kolbanovskii1, Yang Liu1, Lu Zhang1,ShantuAmin3, DinshawPatel4, SuseBroyde2andNicholasE.Geacintov1⁎
1Department of Chemistry,New York University,New York, NY 10003, USA2Biology, New York University,New York, NY 10003, USA3Department of Pharmacology,Penn State College of Medicine,Hershey, PA 17033, USA4Structural Biology Program,Memorial Sloan-KetteringCancer Center, New York,NY 10021, USA
Received 29 September 2008;received in revised form17 December 2008;accepted 22 December 2008Available online8 January 2009

*Corresponding author. E-mail addrAbbreviations used: NER, nucleot

hydrocarbons; anti-BPDE, 7r,8t-dihymolecular dynamics.

0022-2836/$ - see front matter © 2009 E

Nucleotide excision repair (NER) is a vital cellular defense system againstcarcinogen–DNA adducts, which, if not repaired, can initiate cancer dev-elopment. The structural features of bulky DNA lesions that account fordifferences in NER efficiencies in mammalian cells are not well under-stood. In vivo, the predominant DNA adduct derived from metabolicallyactivated benzo[a]pyrene (BP), a prominent environmental carcinogen, is the10S (+)-trans-anti-[BP]-N2-dG adduct (G*), which resides in the B-DNA minorgroove 5′-oriented along the modified strand. We have compared the structuraldistortions in double-stranded DNA, imposed by this adduct, in the differentsequence contexts 5′-…CGG*C…, 5′-…CG*GC…, 5′-…CIG*C… (I is 2′-deoxyino-sine), and 5′-…CG*C…. On the basis of electrophoretic mobilities, all duplexesmanifest moderate bends, except the 5′-…CGG*C…duplex, which exhibits ananomalous, slow mobility attributed to a pronounced flexible kink at the site ofthe lesion. This kink, resulting from steric hindrance between the 5′-flankingguanine amino group and the BP aromatic rings, both positioned in the minorgroove, is abolished in the 5′-…CIG*C…duplex (the 2′-deoxyinosine group, I,lacks this amino group). In contrast, the sequence-isomeric 5′-…CG*GC…duplexexhibits only amoderate bend, but displays a remarkably increased opening rateat the 5′-flanking base pair of G*, indicating a significant destabilization ofWatson–Crick hydrogen bonding. The NER dual incision product yields werecompared for thesedifferent sequences embedded inotherwise identical 135-merduplexes in cell-free humanHeLa extracts. The yields of excisionproducts variedby a factor of as much as ∼4 in the order 5′-...CG*GC…N5′...CGG*C…≥5′...CIG*C…≥5′-…CG*C…. Overall, destabilizedWatson–Crick hydrogen bonding,manifested in the 5′-...CG*GC...duplex, elicits themost significantNERresponse,while the flexible kink displayed in the sequence-isomeric 5′-...CGG*C...duplexrepresents a less significant signal in this series of substrates. These resultsdemonstrate that the identical lesion can be repaired with markedly variableefficiency in different local sequence contexts that differentially alter thestructural features of the DNA duplex around the lesion site.

© 2009 Elsevier Ltd. All rights reserved.

Keywords: nucleotide excision repair susceptibility; benzo[a]pyrenyl-guanine lesion; structure–function relationship; flexible DNA bend;sequence-dependence
Edited by J. Karn
ess: [email protected] excision repair; CPD, cyclobutane pyrimidine dimer; PAH, polycyclic aromaticdroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BP, benzo[a]pyrene; MD,

lsevier Ltd. All rights reserved.

1194 Sequence Dependence in NER of BP-DNA Lesions

The global genome nucleotide excision repair(NER) mechanism is a repair pathway in eukaryotesthat removes bulky DNA adducts in vivo.1–3 Theeukaryotic NER system consists of a set of ∼30distinct subunits4 that collaborate in the excision oflesion-containing oligonucleotides 24–32 nt longfrom the damaged strand. One of the key and vitalfeatures of the NER machinery is its ability to re-cognize and excise an astounding variety of bulkyDNA lesions. The protein XPC, tightly associatedwith another protein, HR23B, in vivo5 and in vitro,6–9

has an initial role in recognizing DNA lesions incellular environments.7,10–16 The XPC/HR23B com-plex initiates the recruitment of other NER factorsthat are essential for ultimately stimulating the dualincisions of the damaged strand.4,12,13,17 The firstamong these other factors is the multi-protein trans-cription factor TFIIH.18–20 The helicases XPB andXPD, components of TFIIH, cause the unwinding ofa 20–25 nt patch around the site of the lesion in anATP-dependent manner.11,14 The factor XPA thenbinds to this complex, presumably acting as a wedgeto stabilize this “bubble”-like structure, and XPC/HR23B is released. Another factor, RPA, a single-strand binding protein, further stabilizes this nucleo-protein complex. Subsequently, the structure-specificendonucleases XPG and XPF/ERCC1 bind to thecomplex and incise the damaged strand on the 3′ and5′ sides of the lesion, respectively, thus releasing the24–32 nt long fragments that are the hallmark of suc-cessful mammalian NER incision activity.11,13,18,21,22

The rate of repair of chemically and conformation-ally different lesions by the mammalian NER appa-ratus varies over several orders of magnitude.2,21,23The structures of the DNA lesions that elicit efficientor inefficient NER repair have been the subject of con-siderable interest over the years.1–3,21,24 It has beensuggested that the NER factors do not recognizethe lesion itself, but the local distortions in the DNAthat are associated with the lesions.7,13,18,23,25 Furtherinsights into these issues are provided by the X-raycrystallographic structure of a truncated form ofRad4/Rad23 (the Saccharomyces cerevisiae homologsof XPC and HR23B, respectively) in a complex withan oligonucleotide containing a cyclobutane pyrimi-dine dimer (CPD) lesion.26 One of the three β-hairpindomains of Rad4 is inserted into the DNA helix, thusseparating the CPD lesion from the unmodifiedstrand. The CPD lesion is positioned in a disorderedregion of the crystal where it has no visible contactswith the protein.26 In contrast, the two thymines inthe complementary strand that were positionedopposite the CPD in the duplex, interact with Rad4amino acid residues. As discussed by Schärer,24,27 thisstructure indicates that the interaction of the unmo-dified complementary strand with the protein has animportant role in lesion recognition, which had beensuggested by the results of experiments reported byButerin et al.28 The structure points to the necessity oflocal strand separation that should be facilitated bylesions causing significant local thermodynamic de-stabilization of duplex DNA. In turn, this local desta-bilization is affected by multiple factors such as the

interactions of the lesions themselves with the localDNA residues that can cause the weakening of localhydrogen bonding and base stacking interactions.Thus, multiple structural deviations, or a multipartitemechanism, may be involved in the recognition ofDNA lesions.29,30 Other structural perturbations thatlead to the recognition of DNA lesions have beenconsidered. These include the recognition of basesflipped out from the complementary strand,28 DNAflexibility around the lesion site,31 and oscillatory mo-tions of the unmodified complementary strand.3,32,33

All of these phenomena involve lesion-induced de-viations from the normal B-DNA duplex struc-tural parameters.34 Many of these parameters arecoupled,35,36 and the various deviations are there-fore not mutually exclusive. Our interests have beendirected towards an understanding of the multi-partite changes in the DNA structural parametersand how they affect the recognition and subsequentdual incisions that characterize both prokaryo-tic29,30,37–39 and eukaryotic NER.29

In order to elucidate the relationship betweenNER efficiency and the conformation of bulky DNAadducts and the DNA distortions they cause, wehave been studying lesions derived from the bind-ing of metabolites of polycyclic aromatic hydro-carbons (PAH) to DNA.40 Adducts derived fromthe binding of the diol epoxide reactive intermediate7r,8t-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), a metabolite of the cancer-causing compound benzo[a]pyrene (BP),41,42 are ex-cellent substrates for probing the features of DNAlesions that are recognized by the NER apparatus.The most abundant stable adduct derived from thereaction of BPDE with DNA in mammalian cells43,44

is the 10S (+)-trans-anti-[BP]-N2-dG adduct (Fig. 1a).However, the reactions of BPDE with DNA alsoyield other, stereoisomeric adducts to dG and dA,with varying conformations and differences instructural perturbation of double-stranded DNA.45

The susceptibilities of the different PAH–DNAadducts to NER depend on adduct stereochemis-try46,47 and PAH topology.48 The [BP]–DNA adducts,unless removed by DNA repair mechanisms,49 arehighly mutagenic.50,51We have shown recently by NMR studies that the

identical 10S (+)-trans-anti-[BP]-N2-dG adduct (G*,Fig. 1a) positioned either at G6 or at G7 in the G6G7duplex (Fig. 1b) exhibits remarkably different, basesequence-dependent structural characteristics.52

The G6G7 dinucleotide sequence was initiallyselected for study because it constitutes a muta-tional hotspot in Escherichia coli53,54 and in mamma-lian cells.50 The bulky aromatic residue inG* adoptsa minor groove adduct conformation in both theG6G7* and the G6*G7 12-mer duplexes. However,the characteristics of these two sequence-isomericduplexes differ in significant aspects. Specifically, inthe G6*G7 case, the duplex exhibits a moderatebend, and the flanking C5:G20 base pair isdestabilized at ambient temperatures, while allother base pairs flanking the G6*:C19 base pairremain intact.52 In contrast, the G6G7* 12-mer

Fig. 1. (a) Structure and stereo-chemical properties of the 10S (+)-trans-anti-[BP]-N2-dG adduct (top).(b) The base sequence contexts of thedifferent duplexes studied and theirabbreviations are defined here,where G*=10S (+)-trans-anti-[BP]-N2-dG. The NMR properties52,58,70and the gel electrophoresis results(Fig. 2) were obtained with the dup-lexes shown. The NER studies wereconducted with the same duplexesembedded in 135-mer duplexes thatotherwise had the same base se-quence (see Supplementary Data).

1195Sequence Dependence in NER of BP-DNA Lesions

duplex manifests a pronounced flexible bend orkink at the site of the lesion, but all base pairsflanking the G7*:C18 pair are intact.52 Since theseduplexes are identical except for the positions of themodified guanine residues, we wished to determinewhether the NER dual incision efficiencies areaffected by these physical differences.The relative NER efficiencies were determined by

incubating the G6*G7 and the G6G7* 12-mer dup-lexes (Fig. 1b) embedded in otherwise identical135-mer duplex sequences, in cell-free extracts fromhuman HeLa cells, as described.46,47 The yields ofdual incision products were also investigated usingthe G6G7* duplex analog I6G7*. In the latter, G6 inthe G6G7* duplex was replaced by inosine (I6)†and is of interest because the I6G7* duplex exhibitsa moderate bend, rather than the pronouncedkink/bend exhibited by the G6G7* duplex. Finally,the new NER results reported here are compared tothe dual incision yields obtained with the CPD and6-4bTTN photodimers,7,13,22,55–57 and the sameminor groove 10S (+)-trans-anti-[BP]-N2-dG adduct58

in the CG*C sequence context (Fig. 1b) reportedearlier.46,47

†Formally, inosine is the nucleoside,while hypoxanthineis the correct name for the corresponding base. Forsimplicity we utilize the term inosine only.

An unusual flexible kink in the G6G7* duplex

The impact of the 10S (+)-trans-anti-[BP]-N2-dGadduct on the three-dimensional characteristicsof the modified oligonucleotide duplexes can beassessed by comparing the electrophoretic mobili-ties of the BP-modified and unmodified duplexes innative (non-denaturing) polyacrylamide gels.59–62

The G6*G7, G6G7* and I6G7* duplexes (Fig. 1b)exhibit extraordinary differences in electrophoreticmobilities (Fig. 2). The mobility of the 12-merG6*G7duplex is about 10% lower than that of the unmo-dified G6G7 duplex (Fig. 2, lanes 3 and 4, respec-tively). These results are comparable to the mobilityof the CG*C duplex (∼5% lower mobility) and withG* in other sequence contexts.59–62 Remarkably, themobility of the G6G7* duplex is lowered by 42%(Fig. 2, lanes 2 and 4, respectively). This result indi-cates that the lesion atG7* induces an usually strongbend59 or kink in the G6G7* sequence context.Interestingly, the anomalous slow electrophoreticmobility of the G6G7* duplex is abolished by subs-tituting the guanine base G6 by inosine. The mobi-lity of the I6G7* duplex is identical with the mobilityof the G6*G7 duplex, suggesting that the overallthree-dimensional shapes of the G6*G7 and I6G7*duplexes are similar. Since inosine is identical withguanine except that it lacks the latter’s exocyclicamino group, these results indicate that the exo-

Fig. 2. Relative electrophoretic mobilities of unmodi-fied and the 10S (+)-trans-anti-[BP]-N2-dG adduct-mod-ified oligonucleotide duplexes defined in Fig. 1 with theirnatural complementary strands in native 8% (w/v) poly-acrylamide gels at room temperature. The faint bands atthe top of the gel represent the wells. Table: percentagedecrease in electrophoretic mobilitya: (Mobility of themodified duplex − mobility of the unmodified duplex)×100/mobility of the unmodified duplex. bData are takenfrom Xu et al.66


cyclic amino group of guanine at G6 plays a domi-nant role in causing the prominent kink in theG6G7* duplex.52,63

The difference between rigid, directed bends andflexible, hinge-like joints can be established byexamining the relationships between the electro-phoretic mobility patterns of self-ligation productsand the phasing of the bends with respect to thehelical repeat64 (10.3–10.6 base pairs65 per helicalturn), as discussed by Hagerman.64 These techni-ques were previously employed to demonstrate thatthe prominent kink/bend induced by the 10S (+)-trans-anti-[BP]-N2-dG adduct in the G6G7* duplex ishighly flexible.66 In contrast, for the same 10S (+)-trans-anti-[BP]-N2-dG adduct in the G6*G7 andI6G7* duplexes, the bending is moderate (Fig. 2)and the bends are rigid rather than flexible, asshown earlier in our laboratory.66 Furthermore,when G* is flanked by cytosine on both sides, asin the CG*C sequence context, the bend is also rigid,as shown by phasing methods.60,62 In the case of thestereoisomerically different 10R (+)-cis-anti-[BP]-N2-dG adduct in the CG*C duplex, which adopts abase-displaced/intercalated conformation (Supple-

mentary Data Fig. S1),67 the extent of bending issmaller than in the case of the (+)-trans- adduct in thesame sequence context.68

Relative NER efficiency

The relative efficiency of dual incisions of themodified strand ofG6*G7, G6G7*, I6G7* and CG*C135-mer duplexes containing identical single 10S(+)-trans-anti-[BP]-N2-dG adducts were compared incell-free HeLa cell extracts (details are given in theSupplementary Data). Briefly, the NER activitieswere assessed using denaturing polyacrylamide gelelectrophoresis to resolve the characteristic 24–32oligonucleotide dual incision products. Phosphor-imager methods were then used to evaluate theyields of these products (radioactivity in the 24–32band region divided by the total radioactivity in thesame lane). These yields varied in different cell ex-tracts (2–6%), and this variability was accounted forby including experiments with a standard stereo-isomeric 10R (+)-cis-anti-[BP]-N2-dG adduct in theCG*C sequence context, in each set of experiments.Thus, in experiments conducted with different cellextract preparations, the dual incision efficiencies forthe 10S (+)-trans adduct in different sequence con-texts were normalized with respect to the 10R (+)-cispositive control standard. The 10R (+)-cis-adduct,with its base-displaced/intercalated conformation,67

is removed about five times more efficiently than theminor groove 10S (+)-trans-adduct58 in the sameCG*C sequence context in HeLa cell extracts, and bya factor of∼2.5 by a set of reconstituted and purifiedNER factors.47 We compared the time dependence ofNER dual incision efficiencies of the 10S (+)-trans-anti-[BP]-N2-dG adduct in the CG*C duplex withthose characterizing the extensively studied CPDand the 6-4 UV photodimer under identical con-ditions.13,22,55,57,69 Typical densitometry traces areshown in Fig. 3a and were used for the quantitativeevaluation of relative NER efficiency. The timedependence is linear up to 40 min (Fig. 3b). Therelative rates decreased in the order (6-4 bTTN)∼10R (+)-cis-anti-[BP]-N2-dGN10S (+)-trans-anti-[BP]-N2-dGNCPD bTTN. The [6-4 bTTN]/[CPDbTTN] ratio of dual incision rates is ∼19. This com-pares with ratios of ≥10 found also in human cellextracts in vitro57 and in vivo in UVB/UVA-irradiated human skin,56 and a ratio of ∼8 observedin CHO cell extracts.22

Sequence context-dependent differentialexcision

The excision efficiencies of the CG*C, G6*G7, andG6G7* sequences embedded in otherwise identical135-mer duplexes are compared in Fig. 4a. The same10S (+)-trans-anti-[BP]-N2-dG adduct embedded indifferent sequence contexts is excised with differentefficiencies in the order G6*G7NG6G7*NCG*C(Fig. 4). Both XPC, a critical component of the global

Fig. 3. (a) Comparison of nucleotide excision repair results in HeLa cell extracts for 135-mer duplexes containing theUV photodimer CPD bTTN or 6–4 bTTN duplexes, the 10S (+)-trans-anti-[BP]-N2-dG (CG*C), or the stereoisomeric (+)-cisadducts in the same CG*C sequence context (cis-CG*C). The (+)-cis-duplex was used as a reference standard in each set ofexperiments to adjust for variability in the NER activities of cell extracts prepared at different times (see the text fordetails). The overall radioactivity levels in each lane were comparable, to allow for a visual inspection of the differences indual incision efficiencies. Lanes 1, 2, 3, and 4 represent incubation times of 10, 20, 30, and 40 min, respectively. The lanemarked bTTN represents parallel incubations with unmodified bTTN duplexes, while lane M contains unmodifiedoligonucleotide markers of 32 nt, 30 nt, 28 nt, 26 nt, and 24 nt. In this experiment, the maximum extent of NER incisions(observed in the case of the 10R (+)-cis-anti-[BP]-N2-dG adduct in the cis-CG*C duplex at 40 min), was 4.6%. (b) Timedependence of formation of dual incision products. The average values and standard deviations are based on fiveindependent experiments in the case of the two UV photodimers, and eight experiments in the case of the 10S (+)-trans-anti-[BP]-N2-dG adduct in the CG*C sequence. The straight lines are the least-squares fits to the data points (the relativevalues of the slopes and the standard errors are summarized in Supplementary Data Table S1). In all these independentexperiments, the data points for the CPD bTTN, 6-4 bTTN, and CG*C (10S (+)-trans-anti-[BP]-N2-dG adduct) werenormalized to the 40 min value of the 10R (+)-cis-anti-[BP]-N2-dG adduct-containing CG*C duplex (open circles) obtainedin the same experiment. The inset in b shows the densitometric tracings of the 40 min lanes shown in the gel (a).


genome NER apparatus, and XPA, are essential tothe NER activities shown. In XPC-, or XPA-deficientcell lines, the NER dual incision efficiencies arereduced by 90% or more, but are restored in mix-tures of cell extracts from XPA and XPC cells, asexpected if an NER mechanism is operative (Sup-plementary Data Fig. S2).In order to compare the initial rates of excision of

these sequences, and to take into account possiblevariations obtained with different cell extracts, weexamined the time course of incisions in the CG*C,G6*G7, G6G7* and I6G7* 135-mer duplexes. A re-presentative example of a gel is shown in Fig. 5a.Results from multiple experiments with differentcell extracts and substrate samples are summarizedin Fig. 5b, together with the standard deviations ofeach set of time-dependent data points. The keyfeature of the findings (Supplementary Data TableS1) is that the NER efficiencies of the identical 10S(+)-trans-anti-[BP]-N2-dG lesions are markedlydependent on the bases flanking the lesion, span-ning a factor of as much as ∼4 (G6*G7 versus CG*Cduplexes). Particularly remarkable are the differencesbetween the sequence isomers G6*G7 and G6G7*;the G6*G7/G6G7* ratio of dual incision was 2.4±0.3(averages of 5-8 independent experiments).

Differences in conformations dictated by basesequence context

The NMR solution structures of the 10S (+)-trans-anti-[BP]-N2-dG adduct embedded in the G6*G7,52

G6G7*,52 I6G7*,70 and CG*C58 duplexes have beeninvestigated and the results show that there aresubtle sequence-dependent conformational differ-ences among double-stranded DNA duplexes. In allcases, the bulky BP aromatic ring system is positionedin the minor groove, pointing in the 5′-directionrelative to the modified guanine base. However,only the G6*G7 duplex manifests a remarkabledestabilization of the base pair flanking the lesion onits 5′-side in a temperature range much lower thanthe global melting temperature of the modifiedduplex. Specifically, the overall thermal stabilities ofthe G6*G7 and G6G7* 12-mer duplexes (Tm=55±1 °C and 57±1 °C, respectively) are not very diffe-rent from one another.52 But the temperature-dependence of the line widths and chemical shiftsof the G20 imino proton NMR resonances in theG6*G7 duplex suggest that the C5:G20 base pair isdestabilized within the temperature interval of 18–30 °C. Furthermore, analysis of the molecular dyna-mics (MD) refined structure based on the NMR data

Fig. 4. Comparison of dual incisions elicited by the 10S(+)-trans-anti-[BP]-N2-dG adduct in CG*C, G6G7* (abbre-viated as G7*), and G6*G7 (abbreviated as G7*) 135-merduplexes in HeLa cell extracts after incubation for 40 min.Control samples: G6G7, unmodified duplex without orafter treatment with cell extracts: CG*C, G6G7*, andG6*G7: untreated controls. The overall radioactivity levelsin each lane were comparable, to allow for a visualinspection of the differences in dual incision efficiencies.(Densitometry tracings of the lanes in the panel comparingdual incision efficiencies of the same 10S (+)-trans-anti-[BP]-N2-dG adduct embedded in the different sequencecontexts are shown in Supplementary Data Fig. S4). Therelative NER efficiency of the (+)-cis-anti-[BP]-N2-dGadduct used as a standard (see the text) is also shown.


shows that the G6G7* duplex shows a unique localuntwisting accompanied by an enlarged Roll52 (Rollis the angle between two neighboring base pairplanes and produces bending into the major or

minor groove.71 See also Supplementary DataFig. S5). Enlarged Roll coupled with untwisting isknown to be largely responsible for DNA bending orkink formation.35,36,72 There are also changes in localbase-base stacking interactions, but without a strongimpact onWatson–Crick pairing except for a modestperturbation at the lesion site itself.63 The propertiesof the I6G7* duplex were also studied by NMRmethods.70 Multiple conformations were found,although twomajor conformers with the BP residuespositioned in theminor groove and oriented towardsthe 5′-direction of the modified strand could beidentified.70 The CG*C and G6G7* duplexes exhibitonly a single conformer and all neighboringWatson–Crick base pairs remain intact.52,58Extensive structural analyses,63 utilizing MD simu-

lation methods, of the 10S (+)-trans-anti-[BP]-N2-dGadduct in the duplexes investigated here haveprovided further molecular insights into the originsof the experimentally observed sequence-depen-dent structural properties.63 The critical elementsthat produce differences in the structural perturba-tions are the variable steric hindrance effects fromthe guanine amino groups that are positioned inthe vicinity of the bulky aromatic BP ring system.Both compete for accommodation in the duplexminor groove. For the G6*G7 duplex, sterichindrance from the guanine amino group of G7causes the episodic rupturing of the Watson–Crickhydrogen bonds of the C5:G20 base pair 5′-flanking the lesion. The G7 guanine amino groupcrowds the BP benzylic ring on the 3′-side of thelesion, causing the BP moiety to rotate about thelinkage site; as a result, the BP distal aromatic ringsystem episodically intrudes into the hydrogen-bonded region of the C5:G20 base pair, flankingthe lesion on its 5′-side (Supplementary Data Fig.S6). Furthermore, in the G6G7* duplex, theanomalously slow electrophoretic mobility asso-ciated with the flexible kink, manifested in our MDsimulation63 as a dynamic untwisting and enlargedRoll at the C5:G20/G6:C19 base pair step, is causedby steric hindrance between the G6 guanine aminogroup and the BP aromatic ring system (Supple-mentary Data Fig. S6). This role of the G6 guanineamino group was also substantiated by MD studiesof the dynamics of the I6G7* duplex,63 which doesnot exhibit untwisting or enlarged Roll. In contrast,in the CG*C sequence, the amino groups of theguanines G18 and G16 in the C5:G18 and C7:G16base pairs, respectively, flanking the lesion, tend toplace the BP moiety in a more confined region ofthe minor groove, thus causing the bending to berigid.

Structural DNA distortions/destabilizationsrecognized by the human NER apparatus

The highest NER efficiency in this series ofminor groove-aligned, stereochemically identical10S (+)-trans-anti-[BP]-N2-dG adducts in the differ-ent sequence contexts considered is observed in the

Fig. 5. (a) Denaturing gel showing the appearance of dual incisions elicited by the 10S (+)-trans-anti-[BP]-N2-dGadducts in the I6G7*, G6*G7, G6G7* and CG*C duplexes as a function of incubation time in HeLa cell extracts. Lanes 1,2, 3, and 4 represent incubation times of 10, 20, 30, and 40 min, respectively. Representative densitometry tracings,adjusted for the total radioactivity in each lane to compensate for loading factors and differences in the radioactivity ofthe samples. (b) Time course of dual incision product formation elicited by the 10S (+)-trans-anti-[BP]-N2-dG adduct inthe G6*G7, G6G7*, I6G7* and CG*C duplexes. The experimental points are averages of seven independent experiments,and the error bars represent the standard deviations. The error bars for the I6G7* data points are omitted for clarity, butthey do overlap with the error bars of the G6G7* and CG*C data points. The straight lines are the least-squares fits to thedata points (the relative values of the slopes and the standard error are summarized in Supplementary Data Table S1). Inall these experiments, the data points were normalized to unity based on the 40 min value of the 10R (+)-cis-anti-[BP]-N2-dG in the CG*C duplex obtained in each individual experiment with a given cell extract (not shown). The lanes for theI6G7* duplex are uniformly lighter (a, left) than in the case of the other sequences; this was due to the smaller quantitiesof I6G7* sample available. However, this difference is not reflected in b, since these data represent ratios of (dualexcision products)/(total DNA substrates) (see the text).


G6*G7 duplex; in this case, the efficiency is ∼2.4times greater than in the sequence-isomeric G6G7*duplex and ∼4.1 times greater than in the CG*Csequence context (Supplementary Data Fig. 6 andTable S1). In terms of structural distortions, only theG6G7* duplex displays a significant destabilizationand episodic denaturation of Watson–Crick hydro-gen bonding at the 5′-side base pair flanking thelesion modification site; this is correlated with thehighest NER efficiency in this series of BP-modifiedduplexes. In contrast, only the G6G7* duplex hasthe hallmarks of a flexible kink or bend,66 mani-fested in the anomalous electrophoretic mobilityobserved in Fig. 2 (lane 2); this is correlated with thesecond highest repair efficiency in this series ofsubstrates. Therefore, in this set of minor-groovealigned sequence-isomeric BP-modified guaninelesions, the weakening of Watson–Crick base pair-ing seems to be a more effective factor for elicitingmammalian NER than a flexible, sharply bent orkinked duplex. The I6G7* duplex lacks this flexiblekink, but its repair efficiency is similar to that of thesharply bent G6G7* duplex substrate with theflexible kink. However, replacing G6 by I6 changesother, local interactions that could affect NER, sincethe conformation of the I6G7* duplex is conforma-tionally heterogeneous and displays at least twodifferent minor groove conformations.70 By contrast

the G6G7* duplex has only one predominant con-formation.52 The conformational heterogeneity ofI6G7* is attributed to a weaker I6:C19 base pair,with only two hydrogen bonds flanking the lesionon the 5′-side. Furthermore, MD studies63 show thatthis I6:C19 base pair has one of the two hydrogenbonds episodically ruptured, which could accountfor its moderate repair susceptibility.63 The leaststructurally distorted CG*C duplex, without dis-rupted Watson–Crick hydrogen bonding or flexiblekink, exhibits the lowest susceptibility to NER in thisseries. The repair in this case seems to be related toits enlarged minor groove,47 and modestly per-turbed Watson–Crick hydrogen bonding at thelesion site;63 these two features are common to allof the substrates we considered here. These struc-ture/function correlations are summarized in Fig. 6and Supplementary Data Table S1.The DNA duplex with the stereoisomerically

different 10R (+)-cis-anti-[BP]-N2-dG adduct, withbase-displaced/intercalated conformation is excisedwith similar efficiencies as the 6-4 bTTN dimer, andabout five times better than the stereoisomeric 10S(+)-trans-anti-[BP]-N2-dG adduct in the CG*C se-quence context.46,47 The large distortions associatedwith this conformation, namely, the prominentlyflipped out base pair,28 with completely rupturedWatson–Crick hydrogen bonding as well as the

Fig. 6. Summary and comparisons of structural effects on relative NER efficiency. Differential steric hindrance bynearby guanine amino groups governs sequence-dependent distortions and duplex destabilization63,70 that influence theDNA sequence-governed NER dual incision efficiencies. In the series of minor groove lesion-containing duplexes studiedhere, the strongest signal that elicits the NER dual incision response is a disrupted Watson–Crick base pair in the G6*G7duplex. The flexible kink/bend in G6G7* appears to be less important, since the bends are more rigid in I6G7* and theother two duplexes studied (see the text). Minor groove enlargement,47,63 together with modest perturbation of Watson–Crick base pairing at the lesion site,63 is common to all DNA sequences examined here.


enlarged minor groove,47 can provide the significantrecognition signals in the family of base-displaced/intercalated [BP]-N2-dG adducts; this also fits wellwith the hierarchy of NER susceptibilities for thecases investigated here.

Conclusions

Analysis of the structural properties of the seriesof minor groove 10S (+)-trans-anti-[BP]-N2-dGadducts positioned in different sequence contextsof double-stranded DNA by NMR methods, electro-phoretic mobilities and MD simulations,63 hasprovided new insights into the structural distor-tions/destabilizations that elicit and modulate theefficiencies of the initial steps in NER (Fig. 6). In thecase of the [BP]-N2-dG adduct studied in this work,the episodic denaturation of Watson–Crick hydro-gen bonding in the base pair flanking the lesion onthe 5′-side provides a stronger recognition signalthan a flexible kink/bend. The origins of thesestructural differences stem from steric hindrancebetween the exocyclic amino groups of unmodified

guanines in the vicinity of the bulky [BP]-N2-dGadducts in the minor groove. The impact of theseexocyclic amino groups on the physical properties ofthe duplexes depends on the positions of theunmodified guanines in the duplexes. Our resultsare consistent with the finding that in this family ofbulky lesions, differences in the local structuraldistortions and destabilizations caused by theadducts are correlated with local helix openingpatterns caused by the XPC/HR23B binding andtheir differential dual incision efficiencies.47

Depending on the sequence context, the lesion-containing duplexes manifest different structuraldistortions/destabilizations.63 In turn, strand sepa-ration, base flipping, and β-hairpin insertion bythe Rad426 or XPC recognition factors may dependon the local base sequence context. Future detailedstudies of the structural properties of differentPAH-derived lesions with different conformationsin selected base sequence contexts should providefurther clues to the mechanisms underlying the re-cognition and excision of bulky DNA lesions by themammalian NER apparatus, and their mutagenicand tumorigenic properties.


Acknowledgements

This work was supported by NIH grant CA-099194 (to N.E.G.) and by grant CA-28038 (to S.B.),and partial support for computational infrastructureand systems management was provided by grantCA75449 (to S.B.). Support for this work wasprovided by grant CA-046533 (to D.J.P.). The contentis solely the responsibility of the authors and doesnot necessarily represent the official views of theNational Cancer Institute or the National Institutesof Health.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.12.082

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The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[ a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions