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Examination of the Long-range Effects ofAminofluorene-induced Conformational Heterogeneityand Its Relevance to the Mechanism of TranslesionalDNA Synthesis

Srinivasarao Meneni, Fengting Liang and Bongsup P. Cho

Department of Biomedical and

Pharmaceutical Sciences,College of Pharmacy, Universityof Rhode Island, Kingston,RI 02881, USA

Adduct-induced conformational heterogeneity complicates the understand-

ing of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2-deoxyguanosin-8-yl)-2-aminofluorene], themajor adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previouslycharacterized and are dependent on the positioning of the aminofluorenerings: B is in the B-DNA major groove, S is stacked into the helix withbase-displacement, and W is wedged into the minor groove. Here, weconducted 19F NMR, CD, Tm, and modeling experiments at various primerpositions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the secondset it was paired with A. The FAF-G:C oligonucleotides were found topreferentially adopt the B or S-conformers while the FAF-G:A mismatchones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strandlength; the largest differences in conformation were displayed at lowertemperatures. The CD and Tm results are in general agreement with theNMR data. Molecular modeling indicated that the aminofluorene moiety inthe minor groove of the W-conformer would impose a steric clash with thetight-packing amino acid residues on the DNA binding area of the Bacillusfragment (BF), a replicative DNA polymerase. In the case of the B-typeconformer, the carcinogenic moiety resides in the solvent-exposed majorgroove throughout the replication/translocation process. The presentdynamic NMR results, combined with previous primer extension kineticdata by Miller & Grollman, support a model in which adduct-inducedconformational heterogeneities at positions remote from the replication forkaffect polymerase function through a long-range DNAprotein interaction.

2006 Elsevier Ltd. All rights reserved.

*Corresponding authorKeywords: aminofluorene-DNA adducts; conformational heterogeneity;long-range effect; tranlesion synthesis

Introduction

DNA adduct formation is a signature hallmarkof mutation, ultimately leading to the initiation ofchemical carcinogenesis.1,2 Arylamines and theirnitro derivatives are a major group of mutagensand carcinogens.3,4 The environmental carcinogen2-nitrofluorene and its amino derivatives are the

prototype arylamine carcinogens.5,6 Upon activa-tion in vivo, they react with cellular DNA to formtwo major C8-substituted dG adducts: N-(2-deoxyguanosin-8-yl)-2-acetyaminofluorene (AAF)1

Abbreviations used: AF-adduct,N-(2-deoxyguanosin-8-yl)-2-aminofluorene;AAF-adduct, N-(2-deoxyguanosin-8-yl)-2-acetylaminofluorene; BF, Bacillus fragment; CD, circulardichroism; FAF, 7-fluoro-2-aminofluorene; ICD290-360 nm,induced circular dichroism at 290360 nm; SMI, slippedmutagenic intermediates; TLS, translesion synthesis;

NOESY, nuclear Overhauser effect spectroscopy; H/D,hydrogen/deuterium.

E-mail address of the corresponding author:[email protected]

doi:10.1016/j.jmb.2006.12.023 J. Mol. Biol. (2007) 366, 13871400

0022-2836/$ - see front matter 2006 Elsevier Ltd. All rights reserved.

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and N-(2 -deoxyguanosin-8-yl)-2-aminofluorene(AF) (Figure 1(a)), along with the minor 3-(2-deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF).7 These bulky DNA lesions, if notrepaired, can produce mutations during replication

that initiate the process of carcinogenesis. Inbacteria AF-adducts mostly induce GT transver-sions, whereas AAF-adducts cause mostly deletionmutations.3 In mammalian cells, however, bothadducts promote sequence-dependent base substi-tution mutations.8

The bulky AF and AAF adducts have long beenused as model systems for investigation of thestructure/functionion relationship in the arylaminemutagen family.14 However, the molecular under-standing of how they exert mutation in vivo isgenerally poor and is complicated by adduct-induced conformational heterogeneities. The AAF-

adduct exists predominantly in the base-displacedstacked (S) conformation.911 Because it lacksWatsonCrick base-pairs at the lesion site, theAAF-adduct produces a major structural distortionin DNA. In contrast, the N-deacetylated AF-adductpossesses flexiblility around the glycosidyl bond(), enabling it to adopt multiple conformationalmotifs depending upon the location of the amino-fulorene moiety, including a major-groove bindingB-type (B) conformation and a minor-groovebinding wedged (W) conformation in additionto the S conformation (Figure 1(b)).1218 In general,fully base-paired DNA duplexes that have incorpo-rated AF are present in an S/B equilibrium in which

the tendency for the molecules to favor the S or the Bconformation is dependent upon the flanking-sequence context.1719 Consistent with this observa-tion, the mutational specificity and frequency of theAF-adduct in mammalian cells vary dependingupon the sequence context in which it is embedded.8

The W-conformer has only been observed in AF-modified duplexes with dA and dG-mismatches atthe lesion site;2022 these mispairings apparentlyunderlie GT and GC transversions, respectively.

Normally, DNA replication in a replicative poly-merase proceeds with high fidelity and processivityon a natural DNA template.2326 The process, how-

ever, is interupted significantly when a damagedbase is present in the template. Continued replica-tion of the adduct-containing template is termed astranslesion synthesis (TLS), which is a major sourceof point mutations.27,28 The TLS events are modu-lated by various factors including the lesion-inducedconformational changes of the template, its sur-rounding base sequence context, and by the natureof DNA polymerases.28,29 The slowing of replicationin a replicative polymerase is now understood topredominantly produce switch to a lesion bypasspolymerase in vivo, which is frequently error prone.However, many specific details of this paradigmremains unknown, i.e. how does the polymerase

switch at a site of DNA damage really occur?27Mutagenic outcomes in replicative polymerasesmay also contribute to the overall mutagenic burdenin vivo.30

Although the steady-state kinetics of nucleotideinsertion opposite the lesion have been studiedextensively for many DNA adducts,3134 data deal-ing with the long-range effects of a lesion on TLS arelimited.3539 Lindsley & Fuchs36 have shown that

the rate of primer extension by T7 DNA polymeraseboth at (n) and adjacent to (n+1) the lesion site witha DNA template containing an AF-lesion is reducedsignificantly (104) relative to unmodified controlDNA. A greater rate reduction (106) was ob-served in the presence of an AAF-adduct. Miller &Grollman37 subsequently extended the scope of theinvestigation, probing the long-range effects ofAF and other DNA adducts on the exo Klenowfragment of Escherichia coli DNA polymerase I.Polymerase activity was affected as far as fourbases downstream (nn+3)(104106) of the dG[AF]:dA mismatch lesion. The effect was much less

pronounced (101

102

) for extension of dG [AF]:dC. Similar results were obtained with Pol II & III.39

The slowed replication rate even after incorporationof bases opposite the lesion provides sufficient timefor template alignment, a general model to predictdeletion mutations by various bulky DNA adductsincluding AF and AAF-adducts.28,31

In the present study we have used the well-defined AF-induced B/S/W-conformational hetero-geneity model17,40 as a basis for investigating theadduct structures at various positions relative to theprimer terminus during simulated AF-induced TLS.Temperature-dependent 19F NMR spectra wereobtained for two distinct TLS models: extension of

dG [FAF]:dC-match and dG [FAF]:dA mismatch.The dynamic NMR results coupled with the model-ing work with the thermophilic DNA polymerase IBacillus fragment (BF)41 were examined in thecontext of previous kinetic parameters36,37 in orderto gain insight into the long-range effects of theconformationally flexible AF-adduct on DNA poly-merase function.

Results

The synthesis and purification of an FAF-mod-

ified 12-mer oligodeoxynucleotide and time-of-flight-mass spectrometry characterization were car-ried out using the procedures described.19 Themodified template strand was annealed withappropriate primers in order to produce variousTLS models of the primer extension reaction (n1,n, n +1, n + 3, and n +6, n = primer terminus) (Figure1(c)). Figures 2 and 3 show the dynamic 19F NMRresults for the dC-match and dA-mismatch exten-sion models, respectively. We have recently demon-strated the utility of 19F NMR/CD procedure forprobing the AF-induced B-S-W-conformationalheterogeneities.40 The 19F NMR method takesadvantage of the sensitivity of the fluorine nucleus

to the tertiary structure of DNA, thereby requiringfluorine-tagged FAF as a model probe (Figure1(a)).12 Induced circular dichroism in the 290360 nm FAF-absorbing range (ICD290360 nm) serves

1388 Long-range Effect of Aminofluorene-Heterogeneity

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Figure 1. (a) Structures of C8-substituted dG-aminofluorene adducts. (b) Views from the minor-groove of a duplex for the three conformational motifs of the AF-modifiedDNA: B, S, and W-conformers. The modified dG and the complementary base (dC for B and S; dA for W) residues are shown in red and blue lines, respectively and the AF-moietyis highlighted with red CPK. In the B conformer, anti- [AF]dG maintains WatsonCrick hydrogen bonds, thereby placing the aminofluorene ring in the major groove withdisplacement of the modified dG and partner dC. The aminofluorene moiety of the S-conformer stacks into the helix with the modified dG in the syn-glycosidyl conformation(base-displaced). The modified dG of the W-conformer adopts syn-configuration; however, the steric constraint at the lesion site allows the aminofluorene ring wedged into thesurface of the narrow minor groove. (c) Sequence context studied ( G*=FAF-adduct; X=C, FAF-G:C match series; X=A, FAF-G:A mismacth series).

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Long-rangeEffectofAminofluorene-Heterogeneity

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as a sensitive conformational marker for probingAF-induced B-S-W heterogeneities.19,40

General assignment strategy

19F NMR signal assignments were made on the basis of the H/D isotopic shielding effect, nuclearOverhauser effect spectroscopy (NOESY) andadduct-induced CD experiments, as well as con-sideration of the sequence at the lesion site. The

rationale for the H/D isotope effect is that the

19

Fresonance of the exposed FAF residue in a B or W-conformer should be more susceptible to solvent-induced shielding (usually> 0.2 ppm) than theburied FAF in an S-conformer (usually

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at the same temperature exhibited two well-resolved signals at 117.6 and 119.6 ppm in aratio of approximately 6:4 (Figure 2). These signalswere identified as B and S-conformers, respectively,on the basis of the H/D isotope shielding effects(+0.17 and +0.10 ppm, respectively). Figure 2 insetshows the contour plots of NOESY/exchangespectrum recorded at 20 C, in which the presenceof off-diagonal cross-peaks indicates the intercon-verting nature of the two conformers. In contrast, no

such cross-peaks were present when a spectrum wasobtained at 5 C. Furthermore, the two signalsfollowed line-shape changes characteristic of a two-site exchange with coalescence occurring around40 C. Upon raising the temperature further to60 C, the coalescent signal became narrowed,which is a result of a rapidly rotating FAF moietyin a single strand 12-mer.

Complete line-shape analysis yielded an activa-tion energy value G of 14.6 kcal/mol, and a rateof interconversion (k=200 s1) at 30 C which isequivalent to a chemical exchange lifetime of 5 ms.The dynamics of the S/B equilibrium can also begleaned from the rate constant at the coalescence

temperature (kC =2.22), at which rapid chemicalshift averaging between the two conformers occurs(Figure 2). reflects the separation in frequency inHz between the two conformer signals at 5 C, when

dynamic exchange is minimal. The lower limit for kCwas determined to be 1715 s1, which is much fasterthan the rates of spontaneous base pair opening in aregular B-DNA molecule.44,45 The situation resem-bles base flipping by the DNA repair enzyme uracilDNA glycosylase.46

n+6 dA-mismatch

Unlike the n+6 dC-duplex described above, 19F

NMR spectrum of the dA-mismatch duplex revealeda single prominent resonance at 118.9 ppm at 5 C(Figure 3). This signal revealed a +0.25 ppm H/Deffect, indicative of an exposed fluoro atom. Theimino proton spectrum at 5 C showed well-resolvedsignals for a single conformation (i.e. W-conforma-tion see below). The duplex exhibited a strongpositive ICD2903 60 n m (see below), which is acharacteristic marker for W-conformation.40 Theseresults are in agreement with previous findings thatduplexes containing purine bases (A or G) oppositethe AF-lesion adopt a W-conformation.2022

A new signal (118.2 ppm) appeared at 10 C andit gained intensity with increasing temperatures (i.e.

2030 C). The emerging heterogeneity is evi-denced by the appearance of varying intensities ofproton signals at 20 C in the 1H NMR spectrum, asituation which was distinct from that observed at

Figure 3. Dynamic 19F NMR spectra (116 ppm121 ppm) of various FAF-G:A mismatch template/primer modelsat n-1, n, n+3, and n+6 positions (n=primer terminus; G =FAF-adduct). Spectra of all sequences obtained at sevenstandard temperatures (labeled as 5, 10, 20, 30, 40, 50, and 60 C). Additional temperatures were recorded for n-1 (23and 25 C), n (25 C), n+1 (25 C), n+3 (25 C), and n+6 (25 and 35 C). The inset shows NOESY/exchange spectrumof the n+6 dA-mismatch duplex at 20 C. Note the lack of off-diagonal cross-peaks. Tm (10

4 M) of the n+3 template-

primer is 24 C

29 C. Tm (10

4

) of the n+6 dA template-primer is 41.4(0.4) C.

1391Long-range Effect of Aminofluorene-Heterogeneity

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5 C (Supplementary Data, Figure S2). At 20 C, thedownfield and upfield signals exhibited deuterium-induced shielding of 0.10 and 0.41 ppm, respec-tively. The unusually large isotope effect observedfor the upfield signal is consistent with the fullyexposed fluoro at C7 of the FAF-moiety, which issandwiched within the walls of the narrow minorgroove. The NOESY spectrum of the n +6 dA duplexat 20 C (Figure 3, inset) lacked discernable off-

diagonal cross-peaks, indicating that it does notexhibit conformeric exchange. This is in contrastwith the afore-mentioned dC-duplex data (Figure2(a), inset) obtained at the same temperature, whichadopted a well-defined S/B equilibrium (see above).The shape and chemical shifts of the upfield W-conformer signal did not change substantially untilthe temperature reached 30 C, and then it collapsedin the narrow 30 C35 C range. This behaviorappeared more like that of a duplex melting ratherthan a two-site dynamic exchange. Taken together,these results suggest a local denaturation at thelesion site, which represents a transition from a W-

conformer to the thermodynamically less stable S

and B-conformers and eventually to single strands(Tm =41.4 C).

Duplex stability

We were unable to accurately determineTm valuesfor the n1, n, and n+1 template-primers. The Tmvalues for the n + 3 dC or n + 3 dA template-primerswere determined to be in the 24 C29 C range.The fully paired 12-mer duplexes are a mixture ofmultiple conformers and exist as a mixture of singleand double-stranded forms at ambient tempera-tures. As for the n+6 dC-match duplex, the FAF-adduction decreased the thermal (Tm =7.2 C)and thermodynamic (G=1.8 kcal/mol) stabili-ties (Table 1). This is a typical trend for S/B-conformeric duplexes.19 In contrast, the valuesobserved for the n+6 dA duplex were comparable

(Tm =0.9 C, G =

0 kcal/mol) to those of thecontrol duplex (Table 1). Similar duplex stability has been observed for other W-like conformericduplexes.17,18 This is also reminiscent ofanotherexclusively W-conformeric dG-N2-AAF-modified11-mer duplex, whose thermal (Tm =6.3 C) andthermodynamic stability (G =1.8 kcal/mol) isgreater than the control.47 In this case, the acetyla-minofluorene-moiety is sandwiched very tightlywithin the walls of the minor groove and maintainsWastonCrick hydrogen bonds at the lesion site. Theentropic component of the Gibb's free enegy hasbeen shown to be responsible for the stability of thisunique W-conformation. It should be noted, how-

ever, that this is not true for all minorminor groovebinding structures.18 Thus, it appears that theduplex stability of minor groove conformersdepends on the curvature of the helix and thedepth of the minor groove, which in turn areinfluenced by the size, planarity, shape and stereo-chemistry (in the case of PAH-adducts) of thecarcinogenic adduct.

n-1

The n-1 sequence represents a 12/5-mer TLSmodel, in which replication of the modified strand

would have proceeded prior to the lesion. The

19

F

Figure 4. Stack plots of 19F NMR spectra of various(a) dC-match and (b) dA-mismatch FAF-template/

primer series at 10 C. Dotted arrows specify conformerassignments.

Table 1. Effects of FAF-modification on the thermal and thermodynamic stability of the n+6 dC-match and n+6 dA-mismatch 12-mer duplexes

Sequencea Gb (kcal/mol) Hb (kcal/mol) Tmc (C) Gd (kcal/mol) H e (kcal/mol) Tm

f (C)

AG*T 8.4 (10.2) 68.6 (76.8) 45.6 (52.8) 1.8 8.2 7.2TC AAG*T 7.4 (7.4) 66.5 (74.8) 41.4 (40.5) 0 8.3 0.9TA A

a The central trimer portion of the 12-mer duplex (G*=FAF-adduct; see Figure 1).b The results of curve fit and Tm-lnCt dependence were within 15% of each other and therefore these numbers are average of the two

methods.19 The average standard deviations forG,H, and Tm are0.2,6.3, and0.4,respectively. Values in parenthesis arefor thecontrols.c

Tm values at 104

M taken from the 1/Tm lnCt/4 Meltwin plots.d G =G (FAF-modified) G (control).e H=H (FAF-modified) H (control).f Tm =Tm (FAF-modified) Tm (control).


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signals in the 5 C10 C range were very broad,reflecting the extensive conformational mobility atthe lesion site (Figures 2 and 3). Upon increasingtemperature, the duplex portion (5 nt) of the modelmelted quickly (Tm

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B*, S*/W* and B*/W* equilibria presumably existin the 5 C20 C range. The persistence of theW*-conformer at n + 3 confirms the greater ther-mal and thermodynamic stability of W over S andB-conformers.

ICD290360 nm

Figure 5 shows an overlay of the CD spectra of then+3 and n+6 models for both the dC- and dA-series. The n +6 dC duplex exhibited mostlynegative ICD2903 60 nm because of its high B-conformer content (64%).19 In contrast, the highlypositive ICD290360 nm observed for the correspond-ing n+6 dA duplex must be due to the exclusivepresence of the W-conformer.40 The ellipticityintensities of ICD290360 nm in both dC and dA serieswere decreased significantly in the n + 3 TLS models,

reflecting conformational mobility at the lesion site(Figures 2 and 3). The CD results are in goodagreement with the dynamic 19F NMR data.

Discussion

19F NMR probing of the AF-conformationalheterogeneity

The FAF-adduct conformation was found to beinfluenced greatly by the extent of the primerextension (n1n+6) with respect to the template

lesion. We also found that the conformationaldiversity at a given primer position is modulatedby temperature as well as by the nature of the basepair at the lesion site. For example, while the dC-match series adopted an S/B equilibrium (Figure 2),a more complex B/S/W heterogeneity (Figure 3)was observed for the dA-mismatch series. Ourdynamic 19F NMR results indicated that conforma-tional flexibilities (intermediate states) exist forthose sequences containing the AF-adduct at (n) orflanking (n1, n+1) the primer-template junction.However, conformational distinction improved gra-dually as the length of primer sequences increased.This phenomenon was observed for both dC and

dA-series in a specific manner (Figures 2 and 3) andappeared to be driven by the greater thermal andthermodynamic stabilities of the W-conformer rela-tive to the S and B-conformers. Thus, the extension ofdG [FAF]:dA-mismatch underwent a progressive

conformational transition from a B/S- (n1n+1),B/S/W- (n+ 3)-equilibrium to an exclusive W-conformation (n+ 6) (see Figures 3 and 4(b)). Thetransition for the dC-match series consisted of a B-or S- (n-1), S/B- (n), B/B- (n+1), B/B/S-equilibrium (n+ 3) to an S/B-equilibrium (n+ 6)(Figures 2 and 4(a)). Table 2 summarizes the structureof DNA duplex, the conformational snapshots ofFAF-adducts at various primer positions, and kineticsof extension available form previous studies.36,37

We observed the predominant presence of the W-conformer for the n+6 dA-mismatch duplex at 5 C,which was quickly unraveled into single strands in

the 5 C

30 C range (Figure 3). These results implythe existence of a Wsyn/Banti equilibrium, the con-version of which could take place through a two-step process; i.e. an initial Wsyn/Ssyn transitionfollowed by an Ssyn/Banti one. The dynamic energybarriers of the B/S/W equilibrium for duplexes witha purine mismatch at the lesion site are not yetknown. However, the interconversion energy for theSsyn/Banti equilibrim of fully paired duplexes wasfound to be in the range ofG= 1415 kcal/mol (S.M. et al., unpublished results). The Wsyn/Ssynequilibrium is expected to occur readily since bothconformers maintain very close , , and torsionangles.21,22

Long-range conformational distortion effects

Replication via high fidelity replicative DNApolymerases proceeds by threading DNA throughthe active site in a well-orchestrated manner.2326

Using BF, Johnson & Beese51 characterized thecrystal structures of all 12 possible mismatchescaptured at the growing primer terminus in theactive site of the polymerase. They found thatmismatch-induced distortion is not just localized tothe mismatch site, but extends up to 6 bp down-stream through the polymerase DNA binding

region. This so-called short-term memory ofreplication errors is conceptually analogous to thelong-range distortion effects observed previously by AF-adducts on T7 and KF polymerases.36,37

Thus, inhibition of polymerase function is not onlyat the site of the lesion but also positions up to atleast 3 nt past the adduct site. The rates of primerextension for dG [AF]:dA mispair is known to be104-fold slower compared to the dG [AF]:dCmatch (Table 2).

Recent crystal studies have provided insight intohow incorporation rates might be reduced drasti-cally at the n and n+ 1 positions. Hsu et al.52

presented snapshot structures of the AF-adduct

docked in the active site of a BF that undergoesone round of replication both in solution and as acrystal. The syn-AF-adduct occupied the preinser-tion site with minimal perturbation of the active site.

Figure 5. Overlay of normalized CD plots of dC-match and dA-mismatch series at n+3 and n+6 positionsat 15 C.


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With an incoming dCTP, however, the modified dG

switched its conformation to anti-, enabling Wat-sonCrick hydrogen bonds to form. A subsequentchemical reaction allowed the aminofluorene-moi-ety to project into the solvent-exposed major groove,reminiscent of the B*-conformeric n dG [FAF]:dCmatch TLS model described in the present study.The newly formed dG [AF]:dC base-pair, however, blocks the next template base from occupying thetemplate preinsertion site, thereby stalling DNAsynthesis. Dutta et al.53 have studied a crystal fromthe T7 DNA polymerase treated with ddCTP orddATP and found sketchy electron density aroundthe template AF-adduct. The observed disorderreflects the dynamic mobility of the AF-adduct

within the active site of a crystalline polymerase andis consistent with our 19F NMR finding, indicatingthat the n dC-match model adopts an S*/B*equilibrium (Figure 2). Thus, the dG [AF]-adductat the replication fork adopted either an anti-conformation to form WatsonCrick hydrogenbonds (i.e. B*-conformer) or a syn-conformation toproduce an S*-like conformer and the processevolved into a well-defined S/B mixture. Similarly,population of the W-conformer increases withincreasing the length of primers (Figure 3). Con-formational status is well establshed at n+6, yet thenucleotide insertion rates in both cases were found

to be the slowest between n and n+3 and normalactivities resumed at n+5 and beyond.37

To understand the mechanisms of this long-rangeeffect, we modeled several AF-template-primers (n+1, n+3, n+6) with known coordinates of BF, ahigh-fidelity DNA polymerase with extensivesequence and structural homology to the KFpolymerase.41 No suitable crystal structure of aternary complex for KF is currently available. Figure6 shows the BF polymerase complexed with the AF-modified n + 3 TLS model with either a B (dC-match)or a W (dA-mismatch) conformer as illustrativepurposes. In the B conformation the bulky amino-fluorene-moiety (shown in red CPK) remains in the

solvent-exposed major groove; thus much lessdisruption of replication is expected as the DNAbinding region translocates through the polymerase.On the other hand, the AF-moiety in the minor

groove of the W-conformer limits not only the

mobility of the DNA, but also imposes a serioussteric clash with the polymerase throughout thereplication/ translocation process. As a result, thereplication rate is dramatically reduced. This findingis reminiscent of the minor groove binding (+)-trans-anti-BP-DNA adduct, in which the bulky benzo [a]pyrene imposes major disruption in the duplex binding site.54,55 Similar situations were observedfor the n and n+1 models. At the n+6 position(Figure 6), however, the AF-lesion is located near thetip of the duplex binding region of the polymerase-binding region, and therefore exerts less interferenceupon proteinDNA interactions. The adverse stericimpact of the AF-moiety in the S -conformation is

expected to be in between those observed with theW and B-conformations.

These results are consistent with the steady statekinetic data described by Miller & Grollman36,37

(Table 2). The dramatic differences in the rates ofnucleotide insertion (104-fold) observed for dG[FAF]:dC match and dG [FAF]:dA mismatchemphasize the importance of adduct-induced stericconstraints in the DNA binding area, especially theminor groove interaction between the template-primer DNA and the polymerase, for determiningreplication fidelity.5255 Altering the adduct-induced conformational equilibria is important,

but the complex features of enzyme structure andinteraction with the lesion must be taken intoconsideration.

Mutational implication of the long-rangeAF-conformational heterogeneity

The fact that retardation of DNA synthesis persistseven after incorporation of the base immediatelyopposite of the lesion has an important implicationfor the mechanisms of mutagenesis.5662 Such adelay during TLS increases the propensity toproduce a family of misaligned slipped mutagenicintermediates (SMI), a model that is known to lead

to frameshift mutations by a variety of bulky DNAadducts including AF and AAF. The sequencecontext is a critical factor, which can be used topredict the nature of various targeted or semi-

Table 2. Conformational heterogeneities observed at various FAF-modified primer-templates and their relativenucleotide insertion frequencies by KFexo

Position of primer terminuswith respect to lesion

Conformation fordG*:dC matcha

Conformation fordG:dA mismatcha

Relative frequencyb for nucleotideinsertion catalyzed by KFexo (base opposite)

n-1 S*/B*a

S*/B* 0.37n B(30%), S(70%) S* 0.36 (dC);d 1.1105 (dA)n + 1 B*, B** S* 0.032 (dC); 7.46 105 (dA)n + 3 B*, B**(80%), S(20%) B* (80%)/W* (20%) 0.16 (dC); 5.8 104 (dA)

B* (39%)/S* (24%)/W* (37%)c

n + 6 B(60%), S(40%) S (56%)/W (44%) NAe

a dG*=FAF adduct (Figure 1). Conformation population at 20 C otherwise indicated.b Data taken from Miller & Grollman.37 Fext = (kcat/Km)lesion template/(kcat/Km)control template.c At 10 C.d Preferred nucleotide inserted at lesion site is dC.e NA, not available.


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AF-adduct. As described above, elongation issignificantly hampered by several nucleotides 5 ofthe lesion, and the effect is much greater when a dAis incorrectly inserted than when a dC is correctlyinserted opposite of the lesion. This effect can be

attributed to a growing W-conformer whose stericclash with the protein is transmitted to the active sitethrough long-range distortions. No such stericinterference is expected for the B-conformer oncethe polymerase has progressed past the lesion. Thus,primer extension over the dG [AF]:dA-mismatch isexpected to be more mutagenic synergistically bynot just base mutations, but also various deletions inthe appropriate sequence contexts.

The mutagenic consequences of lesion bypass andreplicative polymerases are very different, bothstructurally and functionally. Primer-extension stu-dies indicate that the AF adduct causes partial

blocking both at

1 and n positions with E. coli DinBand human pol c.62 Zhang et al.64 have modelednucleotide incorporation opposite the major adductderived from the food carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) inDpo4, the DinB family polymerase. Like the AF-adduct, the PhIP adduct exhibits an S/B conforma-tional heterogeneity in solution.65 The resultsindicate that the PhIP ring system is pushed intothe minor groove (as in W-type conformer) toaccommodate an incoming dNTP at the active siteof the enzyme, which seriously disturbs its geome-try, regardless of the identity of dNTP. The B-conformeric PhIP ring on the spaceous major groove

in Dpo4 allows the active site to accommodatedCTP, dTTP or dATP reasonaly well. This iscontrasted with the same adduct in the replicativepolymerase RB69, which experiences considerableactive site distortion when partnered by C or A.66

Polymerase stalling in vivo could lead to switch to anerror-prone bypass polymerase.

In conclusion, adduct or sequence-induced con-formational heterogeneity has become a signatureproperty for many bulky carcinogenDNA adducts.The results from the dynamic 19F NMR, CD, andmodeling experiments provide conformationalinsight into how AF-induced heterogeneity at

positions distant from the primer terminus caninfluence the downstream polymerase activity, anovel aspect of the present study. The S/B con-formational equilibria depend on the adduct posi-tion in relation to the single strand/double strand junction. The dramatic differences in the rates ofnucleotide insertion observed for dG [FAF]:dCmatch and dG [FAF]:dA mismatch can be attributedto the differences in the adduct conformation at thelesion site and their long range distortion effects oninducing replication blockage in the active site of thepolymerase. The energy differences among theconformers are small, such that their relativeconformeric populations could be readily altered

in the active sites of an enzyme. Therefore, theenzyme-induced conformational heterogeneitymust be taken into consideration in order toestablish a meaningful conformationfunction

relationship. In addition, the findings in the presentstudies further reinforce the utility of the dynamic19F NMR/CD procedure in investigating adduct-induced conformational heterogeneities.

Materials and Methods

Crude oligodeoxynucleotides (1015 mol scale) indesalted form were purchased from Sigma-Genosys (TheWoodlands, TX) and purified by reverse phase highperformance liquid chromatography (RP-HPLC). AllHPLC solvents were purchased from Fisher Inc (Pitts-

burgh, PA). The HPLC system consisted of a HitachiEZChrom Elite HPLC system with a L2450 diode array asa detector and a Luna column (10 mm150 mm; 5 m)(Phenomenex, Torrance, CA). The mobile phase systeminvolved a 30 min linear gradient profile of 5%15%(v/v)acetonitrile/0.1 M ammonium acetate buffer (pH 7.0) witha flow rate of 2.0 ml/min. A Sorvall Evolution RCultracentifuge (Thermo Electron, Waltham, MA) wasused to filter NMR samples.

Preparation of the FAF-modified template

An FAF-modified 12-mer DNA template (5-CTTCTAG[FAF]TCCTC-3) was prepared according to a publishedprocedure.12,19 Briefly, an unmodified oligo was treatedwith N-acetoxy-N-trifluoroacetyl-7-fluoro-2-aminofluor-ene, an activated derivative of 7-fluoro-2-aminofluorene.The modified strand was purified by reverse HPLC andcharacterized by mass spectrometric procedure describedpreviously (calculated, 3724.70; found, 3724.82).19

NMR experiments

Approximately 60 optical density units of pure mod-ified oligos were annealed with primer sequences toproduce appropriate template-primers (see Figure 1(c) forsequences). The samples were filtered by ultracentrifuga-tion using a Pall Microsep MF centrifugal device (Yellow,

Mr cutoff= 1000). The centrifuged samples were dissolvedin 300 l of a neutral buffer (10% 2H2O/90% H2Ocontaining 100 mM NaCl, 10 mM sodium phosphate,and 100 M tetrasodium EDTA (pH 7.0)), filtered througha 0.2 m membrane filter, and placed in a Shigemi tube forNMR experiments.

All 1H and 1H-decoupled 19F NMR results were

recorded using a dedicated 5 mm 19F/1H dual probe ona Bruker DPX400 Avance spectrometer operating at 400.0and 376.5 MHz, respectively. Imino proton spectra wereobtained using phase sensitive jump-return sequences at5 C and referenced relative to DSS. 19F NMR spectra werereferenced to CFCl3 by assigning external hexafluoroben-zene in C6D6 at 164.90 ppm. One-dimensional

19F NMRspectra at 5 C60 C were obtained by collecting 65,536points using a 37,664 Hz sweep width and a recycle delayof 1.0 s between acquisitions. A total of 1600 scans wereacquired for each spectrum. The spectra were processed

by zero-filling, exponential multiplication using 20 Hz linebroadening and Fourier transformation. The peak areaswere base-line corrected and integrated using XWINNMR software (Bruker, Billerica, MA). Two-dimensionalNOESY/exchange 19F NMR spectra were carried out inthe phase-sensitive mode using a NOESY pulse sequence:sweep width 4529 Hz, number of complex data points in t21024, number of complex free induction decays in t1 256,


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number of scans 96, dummy scanes 16, recycle delays 1.0 s,and mixing time 400 ms. The data were subjected to sine

bell apodization using 2 Hz line broadening in bothdimensions and then zero-filled before Fourier transfor-mation of the 1024256 data matrix. The data were not

symmetrized. Complete line-shape analysis43

was carriedout using WINDNMR-Pro.

Circular dichroism (CD) spectra

CD measurements were conducted on a Jasco J-810spectropolarimeter equipped with a variable Peltiertemperature controller. Typically, 2 optical density unitsof a FAF-modified template strand were annealed with 1equivalent of primer strands. The samples were dissolvedin 400 l of a neutral buffer (0.2 M NaCl, 10 mM sodiumphosphate, 0.2 mM EDTA (pH 7.0)) and placed in a 1 mmpath-length cell. The sample was heated at 85 C for 5 minand then cooled to 15 C over a 10 min period to ensureduplex formation. Spectra were measured from 200 nm to

400 nm at a rate of 50 nm/min; the final data wereaveraged from ten accumulations. Data points wereacquired every 0.2 nm with a 2 s response time.

UV-thermomelting experiments

Melting experiments were conducted on a Beckman DU800 UV/Vis spectrophotometer equipped with a sixchamber, 1 cm path-length Tm cell. Sample cell tempera-tures were controlled by a built-in Peltier temperaturecontroller. Duplex solutions with a total concentration inthe range of 0.2 M14 M were prepared in solutionscontaining 0.2 M NaCl, 10 mM sodium phosphate, and0.2 mM EDTA (pH 7.0). The concentration of each oligo

sample was estimated based on UV absorbance at awavelength of 260 nm. Thermomelting curves wereconstructed by varying the temperature of the samplecell (1 C/min) and monitoring the absorbance of thesample at 260 nm. A typical melting experiment consistedof forward/reverse scans and was repeated three times.Thermodynamic parameters for bimolecular meltingreactions of the duplexes were obtained from meltingcurve data using the program MELTWIN version 3.5.The margins of the parameters derived from the curve fitdata and from Tm

1-lnCt plots were found to be within15%, therefore average values from the triplicatedexperiments were used as described.19

Modeling of the BFDNA complex

The Biopolymer module of Insight II (Accelrys Inc.) wasused to construct models. The molecular models of theternary FAF-DNA/dNTP/BF structures were generatedusing the coordinates obtained from the Protein Data Bank(PDB ID: 1UA0 (n-1)52 and the unit A of the crystalstructure 1LV5 within the DNA polymerase BF.41 TheDNA sequence in the crystal structures was adjusted asneeded to match those in the present work. For the n-1model, an anti-dG [AF] model was also constructed inaddition to the syn-dG [AF] observed in the crystalstructure. This was achieved by rotating the dG [AF]glycosidic bond by180. Representative anti- and syn-dG[AF] structures were selected by varying the the linkage

site torsion angles and over their 360 range andevaluating steric collisions. For the n+3 and n+6 W-con-former models, the linkage site geometry observed byNorman et al.20 in the NMR solution structure of the dG[AF]:dA mismatch was used. The models were subjected

to minimization, equilibration, and 700 ps dynamicssimulation with AMBER8.The structures in Figure 6 were prepared using

PyMOL.

Acknowledgements

We thank Dr Paul Chiarelli for providing TOF-MS spectra of the FAF-modified 12-mer template.We are grateful to the NIH (R01CA098296) for theirfinancial support of this work. This research was

also made possible in part by the use of theResearch Core Facility supported by the NCRR/NIH (P20 RR016457).

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2006.12.023

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Edited by J. Karn

(Received 6 October 2006; received in revised form 6 December 2006; accepted 12 December 2006)Available online 15 December 2006


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