doi:10.1016/j.jmb.2006.12.023 J. Mol. Biol. (2007) 366, 1387–1400
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 andPharmaceutical Sciences,College of Pharmacy, Universityof Rhode Island, Kingston,RI 02881, USA
Abbreviations used: AF-adduct,N-(2′-deoxyguanosin-8-yl)-2-aminofAAF-adduct, N-(2′-deoxyguanosin-8acetylaminofluorene; BF, Bacillus fradichroism; FAF, 7-fluoro-2-aminofluinduced circular dichroism at 290–3mutagenic intermediates; TLS, transNOESY, nuclear Overhauser effect shydrogen/deuterium.E-mail address of the correspondi
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 ofboth 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 DNA–protein interaction.
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 theprototype 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
1388 Long-range Effect of Aminofluorene-Heterogeneity
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 replicationthat initiate the process of carcinogenesis. Inbacteria AF-adducts mostly induce G→T 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.1–4 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-displaced“stacked” (S) conformation.9–11 Because it lacksWatson–Crick 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(χ), enabling it to adopt multiple conformationalmotifs depending upon the location of the amino-fulorene moiety, including a major-groove binding“B-type” (B) conformation and a minor-groovebinding “wedged” (W) conformation in additionto the S conformation (Figure 1(b)).12–18 In general,fully base-paired DNA duplexes that have incorpo-rated AF are present in an S/B equilibrium in whichthe tendency for the molecules to favor the S or the Bconformation is dependent upon the flanking-sequence context.17–19 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.8The W-conformer has only been observed in AF-modified duplexes with dA and dG-mismatches atthe lesion site;20–22 these mispairings apparentlyunderlie G→Tand G→C transversions, respectively.Normally, DNA replication in a replicative poly-
merase proceeds with high fidelity and processivityon a natural DNA template.23–26 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 polymeraseswitch at a site of DNA damage really occur?27
Mutagenic 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,31–34 data deal-ing with the long-range effects of a lesion on TLS arelimited.35–39 Lindsley & Fuchs36 have shown thatthe 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 (∼10−4) relative to unmodified controlDNA. A greater rate reduction (∼10−6) 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 (n∼n+3)(10−4∼10−6) of the dG[AF]:dA mismatch lesion. The effect was much lesspronounced (10−1∼10−2) 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.28,31In 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 ofdG [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 (n−1,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 nucleusto the tertiary structure of DNA, thereby requiringfluorine-tagged FAF as a model probe (Figure1(a)).12 Induced circular dichroism in the 290–360 nm FAF-absorbing range (ICD290–360 nm) serves
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, andW-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 Watson–Crick 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).
1389Long-range
Effect
ofAminofluorene-H
eterogeneity
Figure 2. Dynamic 19F NMR spectra (−116 ppm∼−121 ppm) of various FAF-G:C match template/primer models atn-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 (23 and25 °C), n (12, 15, 21, 22, 23 and 25 °C), n+1 (15, 22 and 25 °C), n+3 (15 and 18 °C), and n+6 (15, 25, 33 and 35 °C). Theinset shows NOESY/exchange spectra of the n+6 dC-match duplex at 5 °C and 20 °C (dotted red circles indicate theareas for possible off-diagonal cross-peaks). Tm (10−4 M) of the n+3 template-primer is 24∼29 °C. Tm (10−4) of the n+6dC template-primer is 45.6(±0.4) °C.
1390 Long-range Effect of Aminofluorene-Heterogeneity
as a sensitive conformational marker for probingAF-induced B-S-W heterogeneities.19,40
General assignment strategy
19F NMR signal assignments were made on thebasis 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. Therationale for the H/D isotope effect is that the 19Fresonance 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<0.1 ppm)when the deuterium content is increased from 10 to100%.12,42 While the ICD290–360 nm of a B-conformeris mostly negative, W and S-conformers are char-acterized by positive ICD290–360 nm values withmuch greater intensity for the former.40 While thesequence-dependent S/B equilibrium is a commonconformational theme for fully paired DNAduplexes,19 the W-conformer has been observedexclusively in sequences that contain a purinemismatch opposite the lesion.20–22 Strong tempera-ture dependence of off-diagonal NOESY contoursindicates a transfer of magnetization from oneconformer to another.12 In addition, dynamic
coalescence behavior was taken as evidence forexchanging conformers. Lineshape analyses of thedynamic 19F NMR spectra were performed todetermine various thermodynamic and kineticparameters (ΔG≠, kC and τ).43
The FAF-lesions at (n) or adjacent to (n−1, n+1)the primer terminus generally exhibited broad NMRsignals, indicating their conformational flexibility inthe solvent-exposed environment. These duplexesmostly exist in single strand form at an ambienttemperature due to their lower Tm values (<20 °C).Therefore, the adduct conformation at these sitescould not be defined clearly, thus considered asbeing in an “intermediate state” (marked with anasterisk: B*, S*, and W*).
n+6 dC-match
This fully paired n+6 dC-match duplex representsa simulated TLS model, in which the templatewould have undergone six replication/translocationprocesses from the FAF-lesion site (Figure 2). Westudied previously a similar 12-mer duplex with areversed flanking sequence context (-TG [FAF]A-).12
The imino proton spectrum at 5 °C exhibited morethan 12 proton signals with varying intensities,consistent with conformational heterogeneity (Sup-plementary Data, Figure S1). The 19F NMR spectrum
Figure 3. Dynamic 19F NMR spectra (−116 ppm∼−121 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
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, nosuch cross-peaks were present when a spectrumwasobtained at 5 °C. Furthermore, the two signalsfollowed line-shape changes characteristic of a two-site exchange with coalescence occurring around∼40 °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 ΔG≠ of 14.6 kcal/mol, and a rateof interconversion (k=200 s−1) 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 coalescencetemperature (kC=2.22×Δν), at which rapid chemicalshift averaging between the two conformers occurs(Figure 2). Δν 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 s−1, 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, 19FNMR 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 showedwell-resolvedsignals for a single conformation (i.e. W-conforma-tion see below). The duplex exhibited a strongpositive ICD290–360 nm (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.20–22A new signal (−118.2 ppm) appeared at 10 °C and
it gained intensity with increasing temperatures (i.e.20∼30 °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 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.
1392 Long-range Effect of Aminofluorene-Heterogeneity
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 °C–35 °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
Table 1. Effects of FAF-modification on the thermal and thermismatch 12-mer duplexes
a The central trimer portion of the 12-mer duplex (G*=FAF-adductb The results of curve fit and Tm-lnCt dependence were within ±15%
methods.19 The average standard deviations for −ΔG, −ΔH°, and Tm arcontrols.
c Tm values at 10−4 M taken from the 1/Tm − lnCt/4 Meltwin plotsd ΔΔG=ΔG° (FAF-modified) − ΔG° (control).e ΔΔH=ΔH° (FAF-modified) − ΔH° (control).f ΔTm=Tm (FAF-modified) − Tm (control).
and B-conformers and eventually to single strands(Tm=41.4 °C).
Duplex stability
Wewere unable to accurately determine Tm valuesfor the n−1, 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 °C∼29 °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 (ΔTm=−7.2 °C)and thermodynamic (ΔΔG°=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(ΔTm=0.9 °C, ΔΔG=∼0 kcal/mol) to those of thecontrol duplex (Table 1). Similar duplex stability hasbeen observed for other W-like conformericduplexes.17,18 This is also reminiscent of “another”exclusively W-conformeric dG-N2-AAF-modified11-mer duplex, whose thermal (ΔTm=6.3 °C) andthermodynamic stability (ΔΔG°=−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 maintainsWaston–Crick 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 minor–minor groove-binding 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 strandwould have proceeded prior to the lesion. The 19F
modynamic stability of the n+6 dC-match and n+6 dA-
) ΔΔGd (kcal/mol) ΔΔH e (kcal/mol) ΔTmf (°C)
.8) 1.8 8.2 −7.2
.5) 0 8.3 0.9
; see Figure 1).of each other and therefore these numbers are average of the twoe ±0.2, ±6.3, and ±0.4, respectively. Values in parenthesis are for the
.
1393Long-range Effect of Aminofluorene-Heterogeneity
signals in the 5 °C∼10 °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<17 °C) to give rise to a sharpsignal, which shifted gradually downfield. Thenarow signals represent the mobile FAF residue ofthe denatured single strand template. Previous 1HNMR studies have shown that a similar n-1 modelAF-modified 13/9-mer adopts AF-stacked S-likeconformation, reminiscent to the intercalated (+)-anti-trans-N2- [BP]dG adducts in the same sequencecontext.48 In addition, the 1H NMR results revealedthe existence of the AF-rotamers with respect to theβ′ torsion angle. In concordance, further evidence ofmobility of the carcinogen moiety is supported bythe observation of a relatively large (+0.21 ppm at20 °C) H/D isotope effect. Therefore, the signalsbelow 20 °C appear to indicate the presence of S* orS*-like conformers with possible AF-β′-rotamers.The existence of a mixed S*/B* equilibrium, how-ever, cannot be ruled out.
n
In the n model replication would have proceededup to the adduct site with either the normal partnerdC (Figure 2) or the mismatch dA (Figure 3)directly opposite of the lesion. There were notmany differences between the n and n-1 dA-mismatch models in terms of spectral patterns andH/D solvent effect (+0.19 ppm at 20 °C) (Figure 3).Thus, definite conformational assignment for thedA mismatch model was difficult (Tm<20 °C),although it could be considered a mixture of S*/B*equilibrium. On the other hand, the spectrum of then dC-match model (Figure 2) revealed two signalsat −116.8 and −117.8 ppm in a 3:7 ratio at 20 °C,which were coalesced at ∼30 °C (Figure 2). Thedownfield −116.8 ppm signal, which was dominantin the lower temperatures, exhibited an H/Disotope shift (+0.19 ppm at 20 °C). This is contrastedwith the small effect (+0.02 ppm at 20 °C) observedfor the upfield signal. The distinctive resonancepatterns observed for the two n models arepresumably due to the fact that dC, but not dA,can form Watson–Crick base-pairs at the lesion site.These results support the presence of a B*(−116.8 ppm)/S* (−117.8 ppm) equilibrium. The S-conformer is generally more susceptible to a ringcurrent effect, which enables it to be shieldedrelative to the B-conformer.12
The observation that the n dC model adopts an S/B equilibrium is not in line with previous 1H NMRresults,49 which showed the presence of an S-typeconformer for the AF-modified n-model 13/10-merregardless of whether dC or dA was presentopposite of the lesion. The (+)-anti-trans-N2- [BP]dG adducts in the same sequence context exhibitedthe S-type structural feature as well.50 The reason forthe discrepancy is not clear, but could be due to thefact that the 13/10-mer sequence possesses a longerduplex (10 versus 6 bp) segment and a shorter single
strand segment (3 versus 6 nt) as well as differentflanking sequence context (e.g. -CG [AF]C). It is alsoworth mentioning that the authors pointed out thepresence of an envelope of broad imino signalscentered around 11.0 ppm.49 They assigned only oneof those to the imino proton for the modified dG ofthe dG [AF]:dC match sequence. This is contrastedwith the sharp resonance detected for the sameproton of the same 13/10-mer containing the dG[AF]:dA mismatch, thus hinting multiple conforma-tions for the dG [AF]:dC match sequence.
n+1∼n+3 dC-match
The dynamic profile of the n+3 dC-match modelbelow 10 °C was analogous to that observed for then+6 dC duplex (Figure 2). The signals at −117.5 and−119.4 gave H/D isotope effects of +0.38 and+0.01 ppm at 10 °C, as expected for B and S-conformers, respectively. Unlike the fully paired n+6 duplex, however, the downfield B-conformersignal split up into two signals (B* and B**) at 15 °Cand coalesced at 30 °C. The H/D isotope shieldingof the split signals was found to be in the0.34 ppm∼0.38 ppm range, indicating the presenceof a fully solvent-exposed FAF residue (i.e. B-conformer). In contrast, a small shift (+0.01 ppm)was measured for the upfield S-conformer−119.4 ppm signal. Figure 4(a) shows the stackedplots of all dC-extension models at 10 °C, demon-straing a clear progression of conformational dis-tinction with increasing length of the primer. Thepattern observed for the n+1 model is similar to thatof the n+3 model, except for the absence of a well-defined S⁎-conformer signal. The nature of the B-conformer split is not clear, although the conforma-tional flexibility at the lesion site could in principleproduce a number of low-energy structures includ-ing rotamers with respect to α′ and β′ tortionalangles.
n+1∼n+3 dA-mismatch
The dynamic NMR profile of the n+1 dA-mismatch model did not differ substantially fromthose of the n-1 and n models except that thesignals were narrowed significantly at lowertemperatures. The adduct conformation waswell-defined at the n+3 position, in which threedistinctive resonances were observed at −118.1,−118.5, and −118.9 ppm in the 5 °C ∼10 °C range.These signals merged into the most shielded one(−118.9 ppm) at n+6, which was identified as aW-conformer (see above). The two downfieldsignals (−118.1 and −118.5 ppm) were identifiedas B* and S*-conformers, respectively, on the basisof the chemical shift and consideration of the ringcurrent effect.12 Figure 4(b) shows the stack plotof all six dA-mismatch models at 10 °C, whichillustrates the dramatic S*-B*/S*/W*-W conforma-tional transition. A rapid disappearance of the S*-conformer at 20 °C indicated its intermediarynature in the B*/S*/W*-equilibrium. Various S*/
1394 Long-range Effect of Aminofluorene-Heterogeneity
B*, S*/W* and B*/W* equilibria presumably existin the 5 °C∼20 °C range. The persistence of theW*-conformer at n+3 confirms the greater ther-mal and thermodynamic stability of W over S andB-conformers.
ICD290–360 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 ICD290–360 nm because of its high B-conformer content (∼64%).19 In contrast, the highlypositive ICD290–360 nm observed for the correspond-ing n+6 dA duplex must be due to the exclusivepresence of the W-conformer.40 The ellipticityintensities of ICD290–360 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 (n−1∼n+6) with respect to the templatelesion. 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 (n−1, 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
Figure 5. Overlay of normalized CD plots of dC-match and dA-mismatch series at n+3 and n+6 positionsat 15 °C.
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 progressiveconformational transition from a B⁎/S⁎- (n−1∼n+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 inthe 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/Wequilibrium 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 of ΔG≠=14–15 kcal/mol (S.M. et al., unpublished results). The Wsyn/Ssynequilibrium is expected to occur readily since bothconformers maintain very close χ, α′, and β′ 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.23–26Using 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 bindingregion.” This so-called “short-term memory ofreplication errors” is conceptually analogous to the“long-range distortion effects” observed previouslyby AF-adducts on T7 and KF polymerases.36,37Thus, 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 be∼104-fold slower compared to the dG [AF]:dCmatch (Table 2).Recent crystal studies have provided insight into
how incorporation rates might be reduced drasti-cally at the n and n+1 positions. Hsu et al.52
presented snapshot structures of the AF-adductdocked 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.
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)
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.
1395Long-range Effect of Aminofluorene-Heterogeneity
With an incoming dCTP, however, the modified dGswitched its conformation to anti-, enabling Wat-son–Crick 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-adductwithin 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 Watson–Crick 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 foundto 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 thesolvent-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 themobility 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 duplexbinding 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 protein–DNA interactions. The adverse stericimpact of the AF-moiety in the S -conformation isexpected to be in between those observed with theW and B-conformations.These results are consistent with the steady state
kinetic 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.52–55 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.56–62 Such adelay during TLS increases the propensity toproduce a family of misaligned slipped mutagenicintermediates (SMI), a model that is known to leadto 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-
Figure 6. Example n+3 TLS models of BF complexed with AF-modified template/primer DNA. Arrows indicate (a)the n+3 dC-match (B-type conformer) and (b) the n+3 dA-mismatch (W-conformer) template-primers. The carcinogenicaminofluorene moiety is shown in red CPK. Template and primer sequences are colored green and magenta, respectively.For illustrative purpose, the n+1 and n+6 positions relative to the primer terminus n in the template strand are coloredorange (under the surface) and yellow, respectively.
1396 Long-range Effect of Aminofluorene-Heterogeneity
targeted deletions. For example, it has been shownin Escherichia coli that insertion of a correct dCMP(most frequent) opposite of the AAF-lesion canproduce one, two or longer-base deletions with highfrequency when the appropriate complementarybase match is available 5′ to the lesion.59–62Analogous insertion-misalignment mechanismscould also be applied to mammalian cells.57 It hasrecently been reported that the structure of the AAF-adduct in the E. coli NarI hot-spot sequence in the
polymerase active site is very different from that of anon-NarI sequence, highlighting the importance ofproper active site binding.63dAMP is the second most common nucleotide to
be incorporated opposite of the AF-lesion.57 Theresulting dA-mismatch adducts, whose structureshave been studied in both fully paired duplexes20,21
and at primer-template junctions,58 provide themechanistic rational for G→T transversions, whichrepresent the major mutation observed for an
1397Long-range Effect of Aminofluorene-Heterogeneity
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 beattributed 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 and
replicative polymerases are very different, bothstructurally and functionally. Primer-extension stu-dies indicate that the AF adduct causes partialblocking both at −1 and n positions with E. coli DinBand human pol κΔc.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 groovein 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 carcinogen–DNA adducts.The results from the dynamic 19F NMR, CD, andmodeling experiments provide conformationalinsight into how AF-induced heterogeneity atpositions 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 strandjunction. 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 alteredin the active sites of an enzyme. Therefore, the“enzyme-induced conformational heterogeneity”must be taken into consideration in order toestablish a meaningful “conformation–function”
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 (10–15 μmol 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 mm×150 mm; 5 μm)(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 μl of a neutral buffer (10% 2H2O/90% H2Ocontaining 100 mM NaCl, 10 mM sodium phosphate,and 100 μM tetrasodium EDTA (pH 7.0)), filtered througha 0.2 μmmembrane 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 °C–60 °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 processedby 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,
1398 Long-range Effect of Aminofluorene-Heterogeneity
number of scans 96, dummy scanes 16, recycle delays 1.0 s,and mixing time 400 ms. The data were subjected to sinebell apodization using 2 Hz line broadening in bothdimensions and then zero-filled before Fourier transfor-mation of the 1024×256 data matrix. The data were notsymmetrized. 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 μl 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 to400 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 μM–14 μM were prepared in solutionscontaining 0.2 M NaCl, 10 mM sodium phosphate, and0.2 mM EDTA (pH 7.0). The concentration of each oligosample 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 within±15%, therefore average values from the triplicatedexperiments were used as described.19
Modeling of the BF–DNA 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 by∼180°. Representative anti- and syn-dG[AF] structures were selected by varying the the linkage
†version 7.1.6, J. Chem. Educ. Software Series; Reich,H. J., University of Wisconsin, Madison, WI.
site torsion angles α′ and β′ 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 subjectedto 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 wasalso 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
