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DNA Repair 10 (2011) 684– 696

Contents lists available at ScienceDirect

DNA Repair

j ourna l ho me pag e: www.elsev ier .com/ locate /dnarepai r

robing for DNA damage with �-hairpins: Similarities in incision efficiencies ofulky DNA adducts by prokaryotic and human nucleotide excision repairystems in vitro

ang Liua, Dara Reevesa, Konstantin Kropacheva, Yuqin Caib, Shuang Dingb,arina Kolbanovskiya, Alexander Kolbanovskiya, Judith L. Boltonc,

use Broydeb, Bennett Van Houtend, Nicholas E. Geacintova,∗

Chemistry Department, New York University, 31 Washington Pl., New York, NY 10003, USABiology Department, New York University, 31 Washington Pl., New York, NY 10003, USADepartment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USADepartment of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, University of Pittsburgh, Cancer Institute, Pittsburgh, PA 15213, USA

r t i c l e i n f o

rticle history:vailable online 8 July 2011

eywords:ERrokaryoticumanenzo[a]pyrenequilenin

a b s t r a c t

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism thatremoves a large variety of structurally distinct lesions in cellular DNA. While the proteins involved arecompletely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryoticand eukaryotic NER damage-recognition factors have common structural features of �-hairpin intrusionbetween the two DNA strands at the site of the lesion. In the present study, we explored the hypothesisthat this common �-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the twosystems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determinetheir relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with aset of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens indifferent sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similarrelative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% orless from a perfect correlation, although several more distant outliers are also evident. This resemblance is

consistent with the hypothesis that lesion recognition through �-hairpin insertion, a common feature ofthe two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases.In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining thesame UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggestthat, in addition to recognition involving the initial modified duplexes, downstream events involving

in dis

UvrC can also play a role

Abbreviations: NER, nucleotide excision repair; BP, benzo[a]pyrene; 4-HEN, 4-hydroxyequilenin; dG, 2′-deoxyguanosine; dA, 2′-deoxyadenosine;PD, cyclobutane pyrimidine dimer; (+)-anti-BPDE, (+)-7R,8S-dihydroxy-9S,10R-poxy-7,8,9,10-tetrahydrobenzo[a]pyrene; cis-BP-dG, (+)-(7R,8S,9R,10R)-N2-10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2′-deoxyguanosine; trans-BP-dG,+)-(7R,8S,9R,10S)-N2-[10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2′-eoxyguanosine.∗ Corresponding author.

E-mail address: nicholas.geacintov@nyu.edu (N.E. Geacintov).

568-7864/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2011.04.020

tinguishing and processing different lesions in prokaryotic NER.© 2011 Elsevier B.V. All rights reserved.

1. Introduction

1.1. Overview

Nucleotide excision repair (NER) is important for recognitionand removal of a large variety of structurally and chemically dis-tinct DNA lesions in prokaryotic [1] and eukaryotic [2] organisms.While the proteins involved share little sequence homology, themode of action of this repair system in all branches of life is similarin many respects. Since a large range of substrates are removed byboth NER systems, it is widely accepted that the recognition process

involves the sensing of the structural distortions/destabilizationsin the DNA duplex caused by bulky lesions, rather than the lesionsthemselves. While NER can remove a large variety of DNA lesions,the DNA repair capacity, or efficiency, can vary by two orders of

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agnitude or more, depending on the lesion [2–5]. The chemicaltructures of the DNA lesions, the base sequence contexts in whichhe lesions are embedded, as well as their stereochemical proper-ies can affect the DNA repair efficiencies catalyzed by prokaryotic6–12] and eukaryotic [4,5,13–16] repair systems. While the impor-ance of these factors on NER efficiencies is well documented, thexact molecular origins underlying these differences are still notell understood [17].

Recently, crystal structures of several prokaryotic and eukary-tic NER proteins that recognize and bind to DNA lesions haverovided important new insights into the structural featureshat have an impact on lesion-removal by the two NER systems16,18–26] (see also review by Fuss and Tainer in this issue ofNA Repair). Furthermore, we have accumulated a substantial

ibrary of NER substrates consisting of structurally defined DNAesions positioned in similar or different oligonucleotide sequenceontexts. These substrates provide unique opportunities for inves-igating the relationships between the chemical structures of theesions and the impact of base sequence context effects on theirecognition and processing by eukaryotic and prokaryotic NERystems.

These two NER systems (e.g., [1,2,27,28]) share common overalleatures that include: (1) the initial steps involve the recognition ofhe local distortions/destabilizations caused by the lesions, ratherhan the lesions themselves, thus allowing for the processing of aarge range of structurally unrelated DNA lesions; (2) the damagedtrand is incised by endonucleases on both sides of the lesions, thusemoving entire oligonucleotide sequences containing the dam-ge rather than just the lesions; (3) recently published crystaltructures suggest that one of the recognition steps in prokary-tic [20,25,28] and eukaryotic [23,25,28] NER systems involves thensertion of a �-hairpin between the two DNA strands in the imme-iate vicinity of the lesion. These structural findings are particularly

ntriguing because they suggest that thermodynamic destabiliza-ion of the native B-DNA structure can play an important role29,30] in the recognition of DNA lesions in both NER systems, aoncept that had been previously proposed [4,31–33].

In recent years, we have accumulated a body of results on theelationships between the structural properties of DNA lesions andncision efficiencies of prokaryotic and eukaryotic NER systems9,17,34,35]. Here, we examine the similarities and differences inhe processing of bulky DNA lesions by prokaryotic UvrABC NERroteins and the human NER apparatus in vitro. In both cases, theormation of excision products is a result of a complex series ofteps that include the critical initial recognition step. Our hypothe-is is that the ease of insertion of a �-hairpin into the duplex in theicinity of a lesion plays an important role in the recognition stepn both eukaryotic and prokaryotic NER.

In this article, we first consider some of the key features of theseER systems on which this hypothesis is based. We then compare

he relative efficiencies of incision of a series of DNA substratesatalyzed by prokaryotic UvrABC proteins or by the eukaryotic NERystem in HeLa cell extracts in vitro. Our studies are focused on twoifferent types of bulky stereoisomeric DNA lesions that are undertudy in our laboratories. The first type is derived from the reactionsf a diol epoxide derivative of benzo[a]pyrene (BP) to DNA [14,36],nd the second is derived from the binding of 4-hydroxyequilenin4-OHEN), a reactive metabolite of the equine estrogens equilinnd equilenin. Both equine estrogens are important componentsf the hormone replacement therapy preparation Premarin© [37].he catechol 4-OHEN forms a variety of pre-mutagenic DNA lesionshat are suspected to contribute to human cancers by genotoxic

athways [38]. Because of the potential importance of these equinestrogen DNA lesions, we have been interested in their structuralroperties [39,40], as well as their response to nucleotide excisionepair systems [41,42].

0 (2011) 684– 696 685

1.2. Functional and structural characteristics of prokaryotic andeukaryotic NER: similarities and differences

1.2.1. Overview of NER lesion-recognitionIt is generally accepted that the recognition and subsequent pro-

cessing of DNA lesions by NER occur via a two-step process. Thefirst is the recognition of the lesion, and the second is a verificationstep that ensures that a lesion is actually present (see also reviewby Naegeli and Sugasawa in this issue of DNA Repair). Remarkableinsights into the molecular bases of these recognition phenomenahave been obtained from the crystal structures of the key prokary-otic UvrA [20,43,44], UvrB [25], UvrC [21,45], and eukaryotic [23,24]DNA damage-sensing proteins that initiate NER by binding to thesites of the DNA lesions.

In prokaryotic cells, three proteins, UvrA, UvrB, and UvrC areneeded to generate the dual incisions on the two sides of thelesion that generate the 12–13-mer excision products. The distor-tions/destabilizations caused by the lesion are first recognized bya dimer of UvrA that binds to the damaged site in the form of aUvrA2UvrB2 complex [46]. In an ATP-driven mechanism, the UvrAdimer then dissociates and the UvrB–DNA complex is stabilized bythe insertion of a �-hairpin between the two strands in the vicin-ity of the lesion (Fig. 1A). In this damage verification step [28], thelocal destabilization of the duplex at the site of the damage mostlikely facilitates the insertion of the UvrB �-hairpin (see also Sec-tion 1.2.2). If the latter fails to insert between the two strands, asmight be the case in the absence of a lesion, the UvrA–UvrB disso-ciates from the DNA [6,33]. The stable binding of UvrB to damagedDNA stimulates the activity of its ATPase, which is essential for therecruitment of the endonuclease UvrC by this pre-incision complex[28]. The hydrolysis of one phosphodiester bond on the 3′-side andanother on the 5′-side of the lesion results in the 12–13-mer dualincision products.

In the case of the mammalian NER pathway, the presence of thehelix-distorting/destabilizing lesion is sensed by the XPC-RAD23Bheteroprotein complex that opens a ∼6-base pair sequence, asshown with duplexes containing a cisplatin [47] or a bulky BPdiol epoxide-N2-dG adduct [36]. This complex initiates the recruit-ment of other NER factors to the site of the lesion, starting withthe multi-protein TFIIH complex which contains the helicases XPB[18] and XPD [19,22,26]; the latter induce the unwinding of a 20–25nucleotide-long region around the lesion [48,49].

1.2.2. Crystal structures: shared feature of ˇ-hairpin insertionA crystal structure of a complex of the Bacillus caldotenax (Bca)

UvrB bound to double-stranded damaged DNA has been solved[25]. In this structure, one DNA strand, containing a 3′ overhang,threads behind a �-hairpin motif of UvrB, indicating that this hair-pin is inserted between the strands of the double helix, whilethe nucleotides directly behind the �-hairpin are flipped out andinserted into a small, highly conserved pocket in UvrB. Molecularmodeling and molecular dynamics simulations have been utilizedto complete the DNA structure and to include a BP-derived lesion(Fig. 1A) [35].

Although a crystal structure of an XPC-RAD23B complexcontaining damaged DNA is not yet available, the X-ray crystal-lographic structure of a truncated form of the yeast S. cerevisiaeRad4/Rad23 homologue of the mammalian XPC-RAD23B in a com-plex with an oligonucleotide containing a cyclobutane pyrimidinedimer (CPD) lesion has been determined [23]. There are three�-hairpin domains in the Rad4 protein (BHD1, BHD2, and BHD3).While BHD1 binds non-specifically to an unmodified 11-mer

sequence on the 3′-side of the CPD lesion, BHD2 and BHD3 are incontact with the DNA in the vicinity of the lesion, although not indirect contact with the CPD. The BHD3 �-hairpin is inserted into theDNA helix, thus separating the lesion from the unmodified strand.

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he structure shows that this �-hairpin assumes a role similar to thene in the UvrB-damaged DNA complex [25]: it is inserted betweenhe two strands of the DNA duplex and thus assists in the stabiliza-ion of the complex. The CPD lesion is positioned in a disorderedegion of the crystal without any visible contacts with the protein;owever, the two mismatched and flipped out thymine bases in theomplementary strand opposite the CPD lesion interact with therotein; one thymine interacts with BHD3 and the second with bothHD2 and BHD3 (Fig. 1B) [23]. Recently, fluorescence-based imag-

ng techniques have been employed to deduce that the �-hairpinacilitates the mobility of XPC as it scans for DNA damage [50].

.2.3. A common structural theme of lesion recognitionIt is remarkable that the insertion of a �-hairpin between the

wo strands of DNA at or near the site of the lesion is commono both prokaryotic and eukaryotic NER systems even though theroteins do not share a common fold. The insertion of �-hairpinsetween the lesion-containing and undamaged complementarytrand should be more facile in the case of lesions that cause sig-ificant local destabilization of DNA duplexes, and more difficult inhe case of lesions that do not (see also review by Fuss and Tainern this issue of DNA Repair). These considerations suggest that thehermodynamic properties of the lesions are in some way corre-ated with NER efficiencies in both systems [29,30]. In order to testhis hypothesis we have analyzed a series of defined substrates byhe prokaryotic and eukaryotic NER systems.

. Materials and methods

.1. Prokaryotic NER proteins

In all experiments with UvrABC proteins, UvrA and UvrB were from B. caldotenax,hile UvrC subunits were either from B. caldotenax (UvrCBca), Thermatoga maritima

UvrCTma), or from Escherichia coli (UvrCCho). In a few experiments, all three UvrABCroteins were from E. coli. The UvrABca and UvrBBca , as well as all of the UvrC proteins,ere overexpressed in E. coli cells and purified as described by Jiang et al. [51].

.2. DNA substrates

The site-specifically modified single-stranded oligonucleotides (the uppertrands shown in Fig. 2) were synthesized by reacting (±)-r7,t8-dihydroxy-t9,10-poxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) with oligonucleotides I, III–V, as

escribed by Pirogov et al. [52] and by Mao et al. [53]. The 4-hydroxyequilenin (4-HEN)-modified sequences I, VI and VII were synthesized as described [39]. The35-mer duplexes (see Supplementary data Scheme 1), containing the lesions at orear the center of these duplexes, were synthesized by the annealing and ligationethods described in detail elsewhere [15].

ig. 1. (A) Crystal structure of Rad4-Rad23, the yeast homologue of human XPC-RAD23Bre mismatched with the thymines of the T<>T photodimer, and interact with amino acnchoring Rad4 (or XPC) to the site of the damage. (B) Model of UvrB [35] based on a Uvr+)-cis-BP-derived adduct modeled into a conserved pocket behind the �-hairpin. Color coyMOL Molecular Graphics System [75].

0 (2011) 684– 696

2.3. UvrABC and mammalian cell extract incision assays

Aliquots of the UvrA, UvrB, and UvrC stock solutions were stored at −20 ◦C.Before use, the enzymes were diluted with 1× UvrABC buffer (50 mM Tris–HCl atpH 7.5, 10 mM MgCl2, 50 mM KCl, 5 mM dithiothreitol, and 1 mM ATP) to the desiredconcentrations. The buffer and enzymes were kept at 0 ◦C during the dilution. Thediluted enzymes were then preheated to 55 ◦C and were kept at that temperaturefor 5 min in order to inactivate possible E. coli contaminant nucleases. The concen-trations employed in the incision assays were 25 nM UvrA, 40 nM UvrB, and 50 nMUvrC. The concentration of oligonucleotide duplex substrates was 2 nM. The incisionassays were carried out in 40 �L reaction volumes containing 4 �L of 10× UvrABCbuffer, 6 �L of the diluted UvrA, UvrB, and UvrC solutions and DNA substrates. Thereactions were terminated by adding 3 �L of a stop buffer solution (20 mM EDTA,95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol) to 6 �L aliquotsof the reaction mixture taken out at selected time intervals. The solutions werethen heated at 90 ◦C for 3 min, then quickly placed on ice, and loaded into a 15%denaturing polyacrylamide gel (19:1). The gel was operated for 2 h at 45–50 ◦C in1× Tris–borate–EDTA (TBE) buffer (89 mM Tris, 89 mM boric acid, 10 mM EDTA, pH8.4). Upon completion of the gel electrophoresis experiments, the gel was dried andexposed to an imaging screen in a Molecular Dynamics Storm 840 PhosphorImager.The relative radioactivities of the bands were evaluated using Image Quant software.

The eukaryotic NER incision efficiencies were determined in HeLa cell extracts asdescribed earlier [15]. The cell extracts were prepared by standard methods [54], andthe fraction of 135-mer duplexes that were incised were determined by measuringthe amounts of oligonucleotide incision products 24–32 nucleotides in length. Thesources of the data presented below are cited within the individual figure captions.

3. Results and discussion

3.1. Overview of experimental studies

In this study, the incision efficiencies of the same substratescatalyzed by UvrABC proteins and human NER proteins in vitrowere evaluated separately. The stereochemical properties of theDNA lesions and the sequence contexts utilized are summarized inFig. 2, while selected NMR solution structures of the adducts inves-tigated are shown in Fig. 3. In all of these experiments, the UvrAand UvrB subunits were from B. caldotenax, while the UvrC sub-units were either from B. caldotenax, T. maritima, or E. coli (UvrCBca,UvrCTma, and UvrCCho, respectively). Cho is a UvrC homolog of E. colias first reported by Moolenar et al. [55]. As demonstrated by Jianget al., UvrCBca incises the damaged strands only on the 5′-side ofthe lesion, UvrCTma incises the damaged strands on both sides [51],and the UvrCCho incisions occur only at the ninth phosphodiester

bond on the 3′-side of the lesion, some four nucleotides furtheraway than the normal 3′-incision sites catalyzed by E. coli UvrC. Itwas suggested that Cho acts as a backup nuclease for NER actingon very bulky substrates that block the 3′ incision site of UvrC [55].

. (PDB ID [74]: 2QSG) [23]. The two flipped-out thymines in the unmodified strandid residues in Rad4. The �-hairpin loop is inserted between the two strands, thusB-DNA co-crystal structure (PDB ID [74]: 2FDC) [25] with the DNA extended and ade: �-hairpin, marine; flipped-out bases, red. The figures were prepared using the

Y. Liu et al. / DNA Repair 10 (2011) 684– 696 687

Fig. 2. (A) Sequence contexts and (B) chemical structures of the DNA adducts studied. Subscripted numbers designate the positions of the modified nucleotides (only onelesion per oligonucleotide). In sequences I, VI and VII the labeled adenine and cytosine bases denote 4-OHEN-dA or -dC adducts, respectively. The labeled guanine residues(G6) in sequence I and II (Del) denote either trans- or cis-BP-dG adducts derived from the reactions of (+)-anti-BPDE with the single-stranded sequence I. In sequences III,I ucts i1 re dee

Fcdtseoi

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V and V, G6 denotes the trans-BP-dG adduct. The abbreviations used for these add35-mers containing the indicated duplexes used in HeLa cell NER experiments axperiments are shown in Supplementary data.

urthermore since the Tma and Bca proteins are thermostable, theyan be used at different temperatures to probe the effects of DNAuplex stability on DNA damage recognition. In order to comparehe relative incision rates of different substrates, we assigned theame value of 100 for one of the substrates incised both in HeLa cellxtracts and in UvrABC incubation experiments. The incision ratesf other substrates are expressed relative to this standard substraten the series of bar graphs described below.

.2. Effects of BP-dG adduct stereochemistry in full duplexes I andeletion duplexes II (Del)

.2.1. Repair efficiencies in HeLa cell extractsThe efficiency of repair of the BP-dG adducts by the human NER

ystem in duplexes I and II (Del) was first reported by Hess et al.56]. The (+)-cis and (+)-trans adducts differ from one another onlyy the absolute configuration of substituents about the C10 car-on atom (Fig. 2), but the NER efficiency of the (+)-cis adduct is5 times greater than that of the (+)-trans adduct [36]. This differ-nce has been attributed to the greater disruption of base pairingn the base-displaced/intercalated (+)-cis adduct, and the smallerxtent of structural disruption caused by the minor groove (+)-rans adduct (Fig. 3) [36]. Removing the cytidine opposite thesetereoisomeric lesions almost completely abolishes the repair of

oth these BP-dG adducts in II (Del) duplexes in HeLa cell extracts[56] and present study). Here, we also examined whether simi-ar effects occur in the case of prokaryotic NER proteins in vitroFig. 4A).

n the text are shown in square brackets. The full sequences of the oligonucleotidefined elsewhere [15], and those of the shorter duplexes used in the UvrABC NER

3.2.2. Repair efficiencies in the UvrABC system relative to humanHeLa cell extracts

The kinetics of dual incisions of the same (+)-cis- and(+)-trans-BP-dG lesions in identical 135-mer DNA sequences(see Supplementary data Scheme 1) catalyzed by prokaryoticUvrABcaBBcaCTma proteins at 55 ◦C are depicted in Fig. 4. A typicalgel is shown in Fig. 4A, and the densitometric analyses of these gelautoradiograms yields the kinetic incision curves shown in Fig. 4B.The initial rates, deduced from the initial linear portions of thesekinetic curves, are compared in Fig. 5A. In this bar graph, the ini-tial incision rate of the cis-BP-dG adduct in duplex I was assigned avalue of 100. The relative incision rate of the stereoisomeric transadduct is 20 ± 5% on this scale in HeLa cell extracts [36], and 54 ± 4%in the case of UvrABcaBBcaCTma. Thus, the ratios of initial incisionrates are larger in the case of the cis-BP-dG than the trans-BP-dGadduct in duplex I in both cases, but the cis-BP-dG/trans-BP-dGratios are different: ∼5 in HeLa cell extracts and ∼1.8 in the caseof UvrABC. We note that Zou et al. reported a cis-BP-dG/trans-BP-dG ratio of ∼2 for the same lesions embedded in 50-mer duplexeswith NER assays carried out at 37 ◦C catalyzed by UvrABC proteinsentirely from E. coli [10]. Jiang et al. [51] also studied the repairof the same lesions in 50-mer duplexes, but catalyzed by UvrABca,UvrBBca, and UvrCTma, and reported ratios of 1.4–1.6. All of thesevalues are close to the value for the cis-BP-dG/trans-BP-dG ratio

reported here (Fig. 5A).

In the II (Del) duplexes, the human NER activity is completelyabolished, while with UvrABcaBBcaCTma, it is significantly reducedrelative to the respective values observed for the cis- and trans-BP-

688 Y. Liu et al. / DNA Repair 1

Fig. 3. NMR solution structures of the DNA adducts studied. The trans-BP-dG adductin the full duplex (Duplex I) has the pyrenyl ring system positioned in the B-DNAminor groove directed 5′-along the modified strand with only minor perturbationsof Watson-Crick base pairing [61]. In the cis-BP-dG full duplex I [57] and the dele-tion duplex [58], the conformation is base-displaced/intercalated, with the pyrenylring system inserted into the duplex. In the full duplex I, the modified G and itspartner C are displaced into the minor groove and major groove, respectively, andthe puckered benzylic ring is directed toward the minor groove. In the (+)-cis-BP-dGduplex II (Del), the structure is very similar except that the partner C to the modifiedG is missing. However, the conformation of the trans-BP-dG duplex II (Del) differsfrom that of the full duplex in that it is base-displaced/intercalated, the modifiedG is displaced into the major groove, and the benzylic ring is directed toward themajor groove [76]. In the (−)-SRR-4-OHEN-dC and the (+)-RSS-4-OHEN-dC adducts(Duplexes I and VI), the modified dC is in the anti-glycosidic bond conformation, theequilenin rings are in the minor groove with the distal rings protruding through thehca

daab

3

tma(

elix to the major groove; the rings are directed 5′ along the modified strand in thease of (−)-SRR-4-OHEN-dC and 3′-oriented in the case of the (+)-RSS-4-OHEN-dCdduct [70]. All views are looking into the minor groove of the B-DNA duplex.

G adducts in full duplexes I. Overall, in these examples, the relativectivities of the human and prokaryotic NER systems in the full (I)nd deletion duplexes II (Del) resemble one another qualitatively,ut without being directly proportional.

.2.3. Impact of adduct conformationThe (+)-cis-BP-dG adducts with the base-displaced intercala-

ive conformation are better substrates than the stereoisomericinor groove (+)-trans-adducts in both NER systems [10,36,56],

lthough the difference is larger in HeLa cell extracts. For the IIDel) duplexes, the incision efficiencies relative to the same lesions

0 (2011) 684– 696

in the full duplex I are reduced by a factor of ∼100 in the (+)-cis-adduct, and by a factor of ∼10 in the (+)-trans-BP-dG adduct [56].In contrast, in UvrABcaBBcaCTma experiments, significant levels ofincisions of the cis- and trans-BP-dG adducts in II (Del) duplexesare observed although at lower levels than in the full cis- andtrans-BP-dG (I) duplexes (Fig. 5A). The UvrABcaBBcaCTma experi-ments were conducted at 55 ◦C, an optimal temperature for thethermophilic proteins [51]. This incubation temperature is belowthe melting points of the modified duplexes; spectroscopic experi-ments indicate that the overall adduct conformations are preservedin oligonucleotide duplexes ∼43 nucleotide base pairs in lengthbelow their melting points of 75–80 ◦C [9].

3.2.4. Comparison of incision efficiencies with thermal stabilitiesof different DNA duplex substrates

Insights into the repair-resistance of the deletion duplexes havebeen obtained from thermal melting points (Tm), the temperaturethat corresponds to the dissociation of 50% of the DNA duplexesinto separate strands (Table 1), and NMR and computational molec-ular dynamics studies. The Tm of the (+)-trans-BP-dG II (Del) duplex(Fig. 2) is +6 ◦C higher than the Tm of the modified full duplex I, eventhough the II (Del) duplex is missing the nucleotide C opposite thelesion. However, the analogous increase is strikingly larger, +19 ◦C,in the case of the (+)-cis-BP-dG II (Del) relative to the unmodified II(Del) duplex. Structurally, the (+)-cis-BP-dG-modified full duplex Iand the deletion duplex II (Del) adopt base-displaced/intercalatedconformations [57,58], although with opposite orientations of thepyrenyl ring system as it protrudes through the helix (Fig. 3). In theII (Del) duplex the stacking interactions between the two guaninessurrounding the deletion site and the BP pyrenyl ring system aremore favorable than in the full duplex I. According to recent molec-ular dynamics analyses [16], these enhanced stacking interactionsresult from the collapse of the two guanines upon each other, dueto the absence of the nucleotide between them (Figs. 2A and 3)[58]; consequently, the separation of the two strands at the site ofmodification should be energetically more costly and thus affectthe overall incision efficiency if recognition is a key step, as weassume here. Furthermore, two bases opposite the lesion in thecomplementary strand probably assist in stabilizing the modifiedDNA–XPC complex [23] and the absence of one of these nucleotidesmay diminish the stability of this complex and contribute to theobserved lack of incisions observed in the cis-BP-dG II (Del) duplex.The absence of the partner base opposite the modified nucleotidealso may diminish the binding of the XPC-RAD23B recognition fac-tor to the lesion site. In the case of the (+)-trans-BP-dG adduct induplex II (Del), there is a major structural rearrangement com-pared to the full duplex, since the minor groove-alignment of theBP moiety (with Watson-Crick base pairing being intact in the fullduplex) changes to a base-displaced/intercalated conformation inthe II (Del) duplex (Fig. 3). Therefore, similar structural effectsmay account for the lower incision efficiencies observed in thecase of the trans-BP-dG II (Del) duplex as for the cis II (Del) case(Figs. 4 and 5).

3.3. Effects of flanking bases

3.3.1. (+)-trans-BP-dG lesions in full duplexes III (CG6C) and IV(TG6T)

The relative NER efficiencies in HeLa cell extracts of identicaltrans-BP-dG lesions flanked either by C or by T on both sides, withall other bases in the 135-mer duplexes remaining unchanged,were reported earlier [34]. The initial rates of incisions in HeLa cell

extracts were found to be 1.5 ± 0.2 greater in the sequence contextof duplex IV than in duplex III. In NER experiments using prokary-otic UvrABcaBBcaCTma proteins, the ratio was reported to be 2.3 ± 0.3at 37 ◦C and 1.7 ± 0.4 at 55 ◦C. This loss of discrimination at the

Y. Liu et al. / DNA Repair 10 (2011) 684– 696 689

Fig. 4. (A) Autoradiogram of typical incisions catalyzed by UvrABcaBBcaCTma at 55 ◦C using the cis- and trans-BP-dG lesions embedded in the sequence contexts of duplexesI or II (Del) positioned in the middle of otherwise identical 135-mer DNA duplexes; these duplexes were 32P-endlabeled at the 6th phosphodiester bond on the 5′-side oft 0, 12,2 se of ta produe

hcg

TS

he adduct. Lane M shows the migration distances of oligonucleotide markers 8, 10-mer). The dual incision products migration distance is intermediate between thos a function of incubation time. Time dependence of formation of dual incision

xperiments of this type.

igher temperature is believed be due to the increased dynamiconformational flexibility at the lesion site at 55 ◦C; this has areater impact in the more rigid CG6C sequence context of duplex

able 1ummary of thermal melting data for adducts and sequences investigated.

Sequencea Adduct Tm (Tm Unmod) (◦C) �Tm (◦C)h

I (+)-trans-BP-dG6 41 (51)b −10I (+)-cis-BP-dG6 40 (51)c −11II (Del) (+)-trans-BP-dG6 30 (24)b +6II (Del) (+)-cis-BP-dG6 46 (27)c +19III (+)-trans-BP-dG6 46 (55)d −9IV (+)-trans-BP-dG6 30 (41)d −11V (+)-trans-BP-dG6 55e (60) −5V (+)-trans-BP-dG7 57e (60) −3I (−)-SRR-4-OHEN-dC5 27 (51)f −24I (+)-RSS-4-OHEN-dC7 27 (51)f −24I (−)-SRR-4-OHEN-dC7 26 (51)f −25VI (−)-SRR-4-OHEN-dC4 28 (53)g −25VI (+)-RSS-4-OHEN-dC6 23 (53)g −30VI (−)-SRR-4-OHEN-dA4 43 (53)g −10VI (+)-RSS-4-OHEN-dA8 24 (53)g −29VII (+)-RSS-4-OHE N-dA4 20 (45)g −25VII (−)-SRR-4-OHEN-dA4 30 (45)g −15VII (−)-SRR-4-OHEN-dA6 20 (45)g −25VII (+)-RSS-4-OHEN-dA8 17 (45)g −28VII (−)-SRR-4-OHEN-dA8 19 (45)g −26

a Sequences shown in Fig. 2.b [73].c [16].d [9,13].e [60].f [39].g [42], Table 2.2, Figs. 2.30 and 2.31.h �Tm (◦C) = Tm (Lesion-containing duplex) − Tm (Unmod).

14, . . ., 28, 32 nucleotides in length (the dark band near the middle represents ahe 12 and 14-nucleotide markers. (B) Kinetics of accumulation of incision productscts. The average values and standard deviations are based on seven independent

III than on the more flexible TG6T sequence context of duplexIV [59]. In the case of E. coli UvrAE.coliUvrBE.coliUvrCE.coli, however,the ratio at 37 ◦C was smaller (1.7 ± 0.1) in E. coli UvrABC incisionexperiments [9]. The relative incision rates catalyzed by humanand prokaryotic repair factors are compared in Fig. 5B. The TG6T(IV) duplex rates were assigned a value of 100. The relative inci-sion rates of the CG6C-IV duplexes are 65 ± 9% and 44 ± 6% in HeLacell extracts and UvrABcaBBcaCBca NER experiments, respectively.Again, there is a similar pattern in the human and prokaryotic rela-tive incision rates even though the relative values for the CG6C(III)duplexes catalyzed by UvrCBca are lower than in human cellextracts.

3.3.2. (+)-trans-BP-dG lesions in full duplexes V (CG6G vs. GG7C)and I (CG6C)

The relative rates of incisions catalyzed by human or UvrABca

BBcaCBca NER proteins (55 ◦C) of the trans-BP-dG adducts in thesequence contexts CG6G (V), GG7C (V), and CG6C (I) in duplexes Vand I, respectively, are compared in Fig. 5C. In human cell extracts,the ratio of initial repair rates for trans-BP-dG6 (V)/trans-BP-dG7 (V)is 2.4 ± 0.3 [15], and for the trans-BP-dG6 (V)/trans-BP-dG6 (I) case,it is 4.1 ± 0.3. In Fig. 5C, the value of 100 was assigned to the CG6G(V) substrate. The relative incision rates for the three duplexes,CG6G (V):GG7C (V):CG6C (I) are 100:42:24 in HeLa cell extracts and100:58:37 in the case of UvrABcaBBcaCBca (55 ◦C). Again, the trendsare similar in both systems, but the incision efficiencies appear tobe somewhat more sensitive to sequence context in the case of

the human than the prokaryotic repair systems. The differences inrepair efficiencies in these three duplexes can be rationalized interms of their structural features. An in depth NMR analysis of thesequence-dependence of the imino proton linewidths of the 11-

690 Y. Liu et al. / DNA Repair 10 (2011) 684– 696

Fig. 5. Comparisons of relative initial incision rates measured in human HeLa cell extracts and catalyzed by UvrABC proteins. In all cases, one of the substrates was assigneda value of 100, and the initial incision rates of the other substrates are expressed relative to the value of 100. The sources of the data are cited below in each case. (A)Effects of adduct stereochemistry and conformation: cis- and trans-BP-dG in the full duplexes I and deletion duplexes II (Del); NER data obtained using HeLa cell extractsand UvrABcaBBcaCTma at 55 ◦C. The HeLa data is from [36,56], and the prokaryotic data is from Fig. 4. (B) Effects of base sequence context, . . .TG6T. . . vs. . . .CG6C. . . with G6

denoting the trans-BP-dG adducts in duplexes III and IV (UvrABcaBBcaCBca at 37 ◦C, 43-mer 5′-endlabeled duplexes). The HeLa data is from [59] and the UvrABC data is from[ .CG6CT Fig.

e tion S

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9]. (C) Effects of base sequence context (G6 or G7) in . . .CG6G. . ., . . .GG7C. . ., or . .he HeLa data are from [15]. The UvrABC data for UvrABcaBBcaCBca at 55 ◦C are fromxperiments using 54-mer duplexes; the sequence is shown in Supporting Informa

er CG6G (V) duplex as a function of temperature, demonstrateshat the Watson-Crick hydrogen bonding of the 5′-base pair adja-ent to G6 in the 11-mer CG6G (V) duplex is strongly destabilized;owever, in the GG7C (V) duplex, Watson-Crick hydrogen bonding

s much less disturbed even though a flexible kink at the site of theesion of this duplex has been identified [15,60]. The greater NERfficiency of the CG6G (V) duplex relative to the GG7C (V) duplexas therefore been attributed to its greater local thermodynamicestabilization, stemming from the dynamic, episodic rupturingf the 5′-side base pair adjacent to the lesion at G6 [13,15]. TheG6C (I) duplex appears to be the least structurally distorted [61]:ll Watson-Crick hydrogen bonds are intact in this minor groovedduct (Fig. 3) and the bend associated with the lesion is rigid ratherhan flexible [34].

.4. Incision efficiencies associated with trans-BP-dG lesions arenfluenced by UvrC downstream effects

.4.1. Lesion-recognition versus verificationThe binding of a variety of proteins to double-stranded DNA

ften leads to significant local distortions of the B-DNA double helix

e.g., [62]). These distortions are frequently determined by intrin-ic sequence-governed structural parameters of the DNA duplexes63–65]. The UvrB protein-DNA interaction falls into this patternince complex formation involves the separation of base pairs near

. . . in sequence contexts V, V, and I, respectively, containing trans-BP-dG adducts.S1, Supplementary data (average and standard deviations from four independentcheme 2).

the site of the lesion which is sequence-dependent [66]. Once the�-hairpin has been inserted and the protein-DNA complex hasbeen stabilized as part of the recognition step [28], the DNA is nolonger double stranded and the thermodynamic properties of theduplex should be no longer relevant to the further processing of thelesion. Our assumption is therefore that any correlation betweenthe structural features of lesions in double-stranded DNA and NERefficiencies most likely occur at the recognition step, involving XPC-RAD23B in human and UvrA and UvrB in prokaryotic NER. Thepredominance of the recognition step in determining incision effi-ciencies is illustrated by the example of the cis-BP-dG adduct in thebase-displaced intercalated full and deletion duplexes I and II(Del),respectively. In the full duplex I, the NER efficiency is robust whencatalyzed by either the human [56] or by UvrABC [10] NER sys-tems. While the overall adduct conformation is similar in the II (Del)duplex, the incision efficiency in human cell extracts is abolished[56] and considerably diminished in the UvrABC system (Fig. 4).Since the lesion is identical in both the full duplex I and the II(Del) duplex, it is unlikely that the post-recognition verificationstep, perhaps involving helicase activity, could be very different.Instead, the striking difference in NER efficiencies points to the ini-

tial recognition step. As discussed, the enhanced BP-DNA Van derWaals base stacking interactions in the cis-BP-dG II (Del) may pre-vent the �-hairpin from invading the helix at the site of the lesion[16].

Y. Liu et al. / DNA Repair 10 (2011) 684– 696 691

Fig. 6. Impact of different UvrC endonucleases on relative initial incision rates, catalyzed by UvrABcaUvrBBcaUvrCBca , UvrABcaUvrBBcaUvrCTma , or UvrABcaUvrBBcaUvrCCho proteins,o e origi CTma d

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f trans-BP-dG adducts in duplex V embedded in 5′-endlabeled 54-mer duplexes. Thn Figs. S2 and S3, respectively (Supplementary data), while the UvrABcaUvrBBcaUvr

.4.2. The effects of different UvrC proteins on incision efficienciesatalyzed by UvrABcaUvrBBcaUvrC protein system

While initial damage recognition is critically important, heree find that the nature of the prokaryotic endonuclease UvrC can

lso influence the overall efficiencies of incisions. In Fig. 6, weompare the effects of temperature and different UvrC proteinsUvrCBca, UvrCTma and UvrCCho) on the incision ratios of the trans-P-dG adduct in the CG6G (V) and GG7C (V) duplexes. The HeLaell extract experiments are again shown for reference and, as inig. 6, the incision rates observed for the CG6G (V) duplex weressigned a value of 100 with the HeLa extract and the UvrCBca

xperiments. However, the value of 100 was assigned to the GG7CV) duplex in the UvrCTma and UvrCCho experiments because thisuplex is incised with greater efficiency than the CG6G (V) duplex.he relative incision ratios of the CG6G (V) and GG7C (V) duplexesatalyzed by UvrABcaBBcaCBca at 37 ◦C and 55 ◦C are similar, withinxperimental error (Fig. 6). However, for UvrABcaBBcaCTma at 37 ◦C,he trans-BP-dG6/trans-BP-dG7 ratio is close to unity (experimentst 55 ◦C were not conducted in this case). Interestingly, when theame substrates are incubated with UvrABcaBBcaCCho at 37 ◦C, therans-BP-dG6-V/trans-BP-dG7-V ratio is reversed and is 0.46 ± 0.05.his set of experiments clearly indicates that the bacterial speciesnd therefore the nature of UvrC contributes to the recognition androcessing of lesions that are identical, but positioned in differentequence contexts. Thus, the combination of UvrA and UvrB pro-eins binding to the sites of the lesions, does not alone determinehe overall incision efficiencies. The stable binding of UvrB to dam-ged DNA stimulates the activity of its ATPase, which is essentialor the recruitment of the endonuclease UvrC by this pre-incisionomplex [28]; we speculate that the positioning of UvrC on thevrB-DNA complex depends on the chemical and/or stereochemi-al features of the lesion itself – and its base sequence context; theseactors may govern the dynamic aspects of the UvrB-DNA complexnteractions with UvrC. Thus, a downstream effect involving UvrC,

hen the damaged DNA sequence is in the strand-separated formn the UvrB complex, appears also to be important in processinghe lesions and governing incision efficiencies.

.5. NER efficiencies of 4-hydroxyequilenin – dC and dA adductsn duplexes I, VI and VII

.5.1. Adduct origins and importanceThe reactions of 4-OHEN with nucleotides or DNA generates two

inor and two major stereoisomeric dC, dA and minor proportionsf dG adducts in vitro [38,39]. Such adducts have been found also

inal data for UvrABcaBBcaCBca and UvrABcaUvrBBcaUvrCCho experiments are presentedata is from [77]. The HeLa cell extract results shown are from Fig. 5C.

in rodent [67], and human tissues [68]. Recently, the existence of4-OHEN-dC/dA adducts in human cells exposed to 4-OHEN [69] hasbeen reported.

3.5.2. Structural and thermodynamic propertiesWe investigated the set of 4-OHEN-dC and -dA adducts (Fig. 2)

because they thermodynamically destabilize double-stranded DNAmuch more extensively than the cis- and trans-BP-dG lesions(Table 1). A common feature of 4-OHEN-dC and -dA adducts is theirobstructed Watson-Crick hydrogen bond edge, so base pairing atthe lesion site is impossible. The duplexes with 4-OHEN-derivedadducts therefore exhibit significantly lower Tm values than theBP-dG adducts (Table 1). Another feature of these equine estrogen-derived adducts is that (−)-SRR and (+)-RSS stereoisomers areoppositely directed along the modified strand [40]. For the dCadducts in duplex VI (Fig. 2A), the NMR solution structures [70](Fig. 3) show that the equilenin rings protrude through the helixfrom the minor to the major groove [40]. Here we synthesizedthe two major 4-OHEN-dC/dA adducts at the indicated positionsin sequences I, VI and VII with either (−)-SRR or (+)-RSS absoluteconfigurations (Fig. 2). The (+) and (−)-signs indicate the signs ofthe CD signals measured at 262 nm [40].

3.5.3. 4-OHEN-dC adducts in duplex ITypical gel autoradiograms showing the incisions in duplexes

I containing stereoisomeric 4-OHEN-dC lesions (5′-end labeled)at either positions 5 or 7 catalyzed by E. coli UvrABC proteins at37 ◦C are shown in Fig. 7A. The course of the incision reactions aredepicted in Fig. 7B. The initial rates of incision are determined fromthe initial linear portions of these plots, and are presented in termsof the bar graphs shown in Fig. 7C. These results, after normaliza-tion with respect to the NER efficiencies of the (+)-RSS-dC5 sample(assigned a value of 100) in each type of NER experiment, show thatthe +/−dC5 and +/−dC7 adducts are incised with the same relativeefficiencies in HeLa cell extracts and by the E. coli UvrABC NER appa-ratus. Identical results were obtained when the same samples wereincubated with UvrABcaBBcaCTma at 55 ◦C [41]. The incision efficien-cies are thus the same in TC6G (I) and GC6T (I) sequence contexts,and also do not depend on the (−)-SRR or (+)-RSS stereochemistry.Similar results were also obtained when the same samples were

incised by the UvrABcaBBcaCTma proteins at either 37 ◦C or at 55 ◦C,even though the rate of incisions increased by a factor >10 as thetemperature was raised from the lower to the higher temperature[41].

692 Y. Liu et al. / DNA Repair 10 (2011) 684– 696

Fig. 7. Incisions of stereoisomeric 4-OHEN-dC adducts at either position C5 or C6 in sequence I embedded in 5′-end labeled 43-mer duplexes by E.coli UvrABC proteins at37 ◦C (the full sequence is depicted in Scheme 2, Supplementary data). (A) Typical gel autoradiogram. (B) Incision kinetics. The average values are based on three independentexperiments (data from Chen [41]). The minus and plus signs correspond, respectively, to the (−)-SRR- or the (+)-RSS-4-OHEN-dC adducts (Fig. 2). (C) Comparison of relativei the di bond o( taine

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nitial rates of incisions of stereoisomeric 4-OHEN-dC6 and 4-OHEN-dC7 adducts inncubated in HeLa cell extracts and 32P-internally labeled at the 6th phosphodiester

see Supplementary data Scheme 2) catalyzed by E. coli UvrABC (these data were ob

.5.4. 4-OHEN-dC and -dA adducts in duplexes VI and VIINine sequences with different adducts derived from the reac-

ions of 4-OHEN with either sequence VI or VII and (−)-SRR or+)-RSS absolute configurations were synthesized as described39,42]. The adducts were positioned either at positions A4, C6, or8 in duplex VI, or at A4, A6, or A8 of duplex VII. The sequence of

hese duplexes is identical except for the C or A at the central posi-ions (Fig. 2). In designing these experiments, we hypothesized thathe duplex VII (top strand) contains a sequence of five consecutiveurines which might render this duplex less flexible than duplex VIhich has runs of only two purines, and that this difference might

ffect the NER efficiencies. The initial rates of incisions were mea-ured using UvrABcaBBcaCTma at 55 ◦C, and typical autoradiogramsre shown in Supplementary data. These results are summarized inhe bar graphs shown in Fig. 8. In this figure, the (+)-C6-VI adducterved as the reference sample (assigned a value of 100) in eachf the two sets of studies using human cell extracts and UvrABCroteins.

In duplex VI, the relative initial rates obtained with all fourdducts are similar in value, within experimental error, exceptor the (−)-dA4-VI sample; the latter exhibits a relative incisionate of ∼65% in the human cell extract and ∼104% in the UvrABC

xperiments relative to the standard (the (+)-dC6-VI adduct, Fig. 8And B). In duplex VII, relative to the same standard, the initialvrABcaBBcaCTma-catalyzed incision rates are significantly smaller

by up to a factor of ∼2) than in human cell extracts in the

uplex I sequence context embedded in either (1) the middle of 135-mer duplexesn the 5′-side of the adduct, and (2) in the middle of 5′-endlabeled 43-mer duplexes

d from the initial linear portions of the plots in B).

(+)-dA8-VII and (−)-dA8-VII samples, about ∼ 30% smaller in the(+)-dA4-VII sample, and close to one another for the (−)-dA4-VIIand (−)-dA6-VII adducts (Fig. 8C and D). Thus, there are about three4-OHEN-derived samples out of nine that deviate more markedlyin the two repair systems.

In duplex VI and VII in HeLa cell extracts, all of the nine sub-strates exhibit relative incision values that lie in the range of ∼60–110% (Fig. 8A and B). In the UvrABC incision experiments,six adducts exhibit incision rates relative to the same (+)-dC6-VIadduct standard in the ∼60–100% range, and the other three inthe ∼ 40–50% range (Fig. 8C and D). We suggest that the effectsof sequence context and stereochemistry may play a lesser rolewhen the lesions themselves are extremely destabilizing as in the4-OHEN-derived adducts (Table 1).

3.6. Overall comparisons of prokaryotic and eukaryotic NERincision efficiencies

In order to obtain an overview of eukaryotic and prokaryotic NERincision efficiencies of the 21 different substrates collected here,we constructed the plot shown in Fig. 9, based on data reported inFigs. 5–8 (the single UvrCCho result was omitted). In this plot, the

relative incision efficiencies observed in HeLa cell extracts are plot-ted on the horizontal axis, and the relative efficiencies observed inthe various UvrABC incision experiments are given on the verticalaxis for the same adduct. Of course, the incision efficiency at a par-

Y. Liu et al. / DNA Repair 10 (2011) 684– 696 693

Fig. 8. Comparisons of relative NER incision efficiencies of different stereoisomeric 4-OHEN-dC or -dA adducts in sequence contexts VI or VII embedded in the middle ofinternally labeled 135-mer DNA duplexes (see Supplementary data Scheme 1), in human HeLa cell extracts and prokaryotic UvrABcaBBcaCTma proteins at 55 ◦C. The value of100 was assigned to the (+)-4-OHEN-dC6 adducts in sequence context VI (abbreviated as the (+)-dC6-VI adduct in the figure), and all other incision efficiencies are expressedr Supplt positi

tvdl

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elative to this value. The HeLa and UvrABC data (typical gels are shown in Fig. S4,he stereochemical properties of the adducts (Fig. 2), and the subscripts denote the

icular data point is relative to the same adduct that was assigned aalue of 100 in the eukaryotic and prokaryotic NER experiments, asescribed in each of the figures. Any data point lying on the straight

ine shown in Fig. 9 would indicate a perfect correlation.The relative incision efficiencies in HeLa cell extracts and in

vrABC experiments obtained with 12 4-OHEN-derived lesions inuplexes I, VI, and VII, taking into account the error bars, show thatnly three of the 12 data points (L, P, and G in Fig. 9) deviate signif-cantly from the ideal correlation line. In duplex VII, the pattern ofncision rates is, overall, much less well correlated with the relativencision rates observed in HeLa cell extracts than in duplex VI.

The BP-dG adducts show reasonable qualitative correlations forll adducts except for point E; this point represents the relative inci-ion efficiency catalyzed by UvrCTma, and is an outlier, showing noifference in relative incision rates of . . .CG6G. . . (V) and . . .GG7C. . .V) duplexes (Fig. 9). The trans-BP-dG/cis-BP-dG ratios and the

elative incision ratios observed with the trans-BP-dG adduct inifferent sequence contexts do resemble one another in the eukary-tic and prokaryotic NER incision experiments, although there aren some cases better quantitative correlations than in others. For

ementary data), are excerpted from Reeves [42]. The minus and plus signs denoteons of the modified nucleotides in sequences VI or VII.

example, with human HeLa cell extracts, in contrast to compara-tively robust incision efficiencies in full duplexes I [36,56], the NERactivity is practically abolished in the deletion II (Del) duplexes; sig-nificantly reduced incision activities with the deletion duplexes arealso observed in prokaryotic protein incision experiments. How-ever, the reduction in the II (Del) duplex is not complete, and theresemblance is therefore qualitative (points A and B in Fig. 9). Inthe prokaryotic experiments, the incision rates can also dependremarkably on the nature of the UvrC endonuclease (Fig. 6). In gen-eral, the prokaryotic repair system seems to be less sensitive thanthe human HeLa NER system to changes in adduct stereochem-istry, adduct conformation, and base sequence context effects. Inconsidering all of the adducts studied, we compute a correlationcoefficient of 0.595 for this full data set shown in Fig. 9.

A lack of correlation was observed between incision efficien-cies catalyzed by human and E.coli NER systems in the case of

acetylaminofluorene-dG adducts at different positions in the NarIsequence context [8]. The observations of several outliers in ourincision experiments are not surprising. Incision efficiencies aredependent not only on the structural features of the lesions and the

694 Y. Liu et al. / DNA Repair 10 (2011) 684– 696

Fig. 9. Comparisons of all relative incision efficiencies in human HeLa cell extracts and UvrABC experiments shown in the individual bar graphs in Figs. 5–8. The origins ofeach of the points are also shown. Incision efficiencies for each figure are expressed relative to a designated adduct assigned a value of 100. The straight line corresponds toa e incisw

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hypothetical ideal correlation; any point on this line would signify that the relativas calculated for all the data points.

ase sequence context, but also on downstream damage processingvents after recognition, such as the nature of the bacterial speciesrom which UvrC was purified. The structural and functional down-tream damage processing steps leading up to efficient incision inukaryotes are not understood.

. Conclusions

On the basis of the 21 substrates investigated here (Fig. 9),n overall qualitative correlation between NER incision efficien-ies in human HeLa cell extracts and UvrABC NER experiments isbservable. We find a qualitatively similar pattern in the relativencision efficiencies in human HeLa cell extracts and UvrABC-atalyzed incision experiments in vitro, within ±30% for two-thirdsf the different substrates studied. For the BP-dG adducts, the rela-ive incision efficiencies exhibit similar trends for stereoisomericis-and trans-BP-dG adducts. The resemblance of NER efficien-ies in human and prokaryotic repair systems is consistent withhe hypothesis that the recognition step of bulky DNA lesions

ay involve a common �-hairpin insertion in the eukaryoticPC-RAD23B and in the prokaryotic UvrB systems. This stephould depend on the ease of separation of the two strands atr near the lesion site, determined by local thermodynamic sta-ilization/destabilization. The thermodynamic properties of theouble-stranded region containing the lesion depend on the chem-

cal nature of the lesion, including stereochemistry, topology andhysical size, and its sequence context; the impact of these variableactors on the local DNA structure provide the thermodynamic sig-al. In this concept, the ease of insertion of the �-hairpin betweenhe two strands is facilitated by lesions that destabilize the local

egion of the duplex, and is hindered by lesions that stabilize theocal structure of the duplex by van der Waals stacking and otherypes of interactions. Hairpin insertion could be facilitated by bend-ng and concomitant unwinding [20,32], either dynamically due

ion efficiencies are identical in both NER systems. A correlation coefficient of 0.595

to the lesion or through protein-DNA interactions. As shown in arecent crystal structure [20], UvrA bends and unwinds the DNA,which would facilitate strand opening and recognition by UvrB.The stabilization of the opened DNA helix by the repair protein alsoneeds to be considered. Furthermore, UvrB is believed to interactwith the damaged substrate through base stacking with a tyrosineresidue at the base of the beta-hairpin and through a base flippingevent [25], and the yeast homologue of human XPC-RAD23B [23]also utilizes base-flipping interactions with the protein. Base flip-ping free energy profiles and repair susceptibility of damaged DNAare currently under investigation [71].

Finally, it is important to realize that, while it is apparent thatcertain protein motifs such as a �-hairpin are common recognitionelements in NER proteins from prokaryotes and eukaryotes, the twosystems show remarkable differences. Considering the three ordersof magnitude difference between the genomes, the huge degreeof genome compaction of the eukaryotic genome into chromatinand the different structural organization of chromatin (transcrip-tionally active or inactive) within the nucleus of eukaryotes, it isnot surprising that eukaryotes have evolved a more complex NERprocessing system [72] which involves more than 30 proteins ascompared to just six in bacterial species. It is likely that eukaryoticchromatin organization of DNA, which is wound around histonesin nucleosomes, may provide additional helical stress in repair-susceptible, destabilizing lesions to promote damage recognition,while stabilizing repair-resistant lesions can diminish nucleosomedynamics [78]. Future work on the structure, function, and dynam-ics of NER proteins, as well as, the role of chromatin reorganizationduring repair will give clues to both the similarities and differencesbetween these two systems. For example, a co-crystal structure of

UvrB bound to a damage site might show structural similaritiesof damage verification provided by XPD as part of TFIIH-DNA co-crystal [17]. Current work in our laboratories is focused on theseexciting and fundamental topics of NER.

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cknowledgements

This work was supported by NIH grants CA112412 andA099194 to NEG, CA75449 and CA28038 to SB, and ES019566o BVH. We also gratefully acknowledge TeraGrid resources pro-ided by the Texas Advanced Computing Center supported by theational Science Foundation. The content is solely the responsibil-

ty of the authors and does not necessarily represent the officialiews of the National Cancer Institute or the National Institutes ofealth. Components of this work were conducted in the Shared

nstrumentation Facility at NYU that was constructed with supportrom the Research Facilities Improvement Program (C06RR-16572)rom the National Center for Research Resources, National Insti-utes of Health. The acquisition of the Bruker Ultraflex Maldi masspectrometer at this facility was supported by the National Scienceoundation (CHE-0958457).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.dnarep.2011.04.020.

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