Mutagenesis of 8-Oxoguanine Adjacent to an Abasic Site in Simian

As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simi...
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AUGUST 2005 VOLUME 18, NUMBER 8 © Copyright 2005 by the American Chemical Society

Communications Mutagenesis of 8-Oxoguanine Adjacent to an Abasic Site in Simian Kidney Cells: Tandem Mutations and Enhancement of GfT Transversions M. Abul Kalam and Ashis K. Basu* Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269 Received May 3, 2005

Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T . C > A > G, the same at the GUC site was T . A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23-24% GfT mutants from a single 8-oxoG in a TG8-oxoT sequence context, the tandem lesions UG8-oxoT and TG8-oxoU generated ∼60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G8-oxofT transversions that included both single and tandem mutations at the TG8-oxoU site was nearly 60% relative to about 30% at the UG8-oxoT site. A higher mutational frequency at the TG8-oxoU sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG8-oxo(AP*) sequence in a 12-mer was lower than the same in a (AP*)G8-oxoT 12-mer with ∆∆G 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G8-oxoT 12-mer duplex with a ∆∆G of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG8-oxo(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG8-oxo(AP*) sequence relative to (AP*)G8-oxoT. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG8-oxo(AP*) sequence context.

Introduction Clustered lesions or multiply damaged sites (MDS)1 in DNA, which include two or more lesions within ∼15 base * To whom correspondence should be addressed. Tel: 860-486-3965. Fax: 860-486-2981. E-mail: [email protected].

pairs, are one of the consequences of ionizing radiation (1, 2). Damage within the MDS may be located in the 1Abbreviations: MDS, multiply damaged sites; AP, apurinic/ apyrimidinic; AP* represents tetrahydrofuran, the model AP site; 8-oxo-dG, 7,8-dihydro-8-oxo-2′-deoxyguanosine; 8-oxoG, 8-oxoguanine; ss, single stranded; NER, nucleotide excision repair.

10.1021/tx050119r CCC: $30.25 © 2005 American Chemical Society Published on Web 07/15/2005

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same or the complementary strands. In addition to strand breaks, MDS has been shown to contain oxidized bases and apurinic/apyrimidinic (AP) sites (3, 4). While ∼20% of MDS induced by γ-irradiation may contain double strand breaks, about 80% contain base damages including tandem lesions (5). A tandem lesion in which a pyrimidine base is degraded to a formylamine and an adjacent guanine is oxidized to 8-oxoguanine (8-oxoG) has been detected in X-irradiated DNA in oxygenated aqueous solutions (6). This type of tandem DNA damages may result from a single initial radical event (7), which implies that a number of thymine and guanine lesions could be involved as part of tandem lesions. In vitro studies on MDS have shown that, unless a very high concentration of the repair enzyme is used, only one of the two lesions can be excised by DNA repair proteins, which may generate a base damage with a closely located AP site (8-10). A hierarchy in the excision of lesions within the MDS, in which 5-hydroxyuracil and 5-formyluracil excisions are much more efficient than 8-oxoG, has been observed in human whole cell extracts (11). Repairability of MDS depends significantly on both the relative location of the lesions and the sequence context (12). Although a limited number of studies on the repair of MDS have been reported (8-12) and most clustered DNA damages are believed to be genotoxic (2-4), few studies have explored the mutagenic consequence of MDS. Even so, one can anticipate that misreplication of one lesion might be influenced by the presence of another lesion in the vicinity. Indeed, of the limited number of studies on replication errors of MDS, one noted that the mutagenicity of 8-oxoG in Escherichia coli increases when another 8-oxoG is located nearby on the complementary strand (13). In another study, the enhanced mutagenic potential of 8-oxoG in E. coli was demonstrated when a uracil was placed in the opposite strand within five bases of the 8-oxoG site (14). For the 8-oxoG-formylamine tandem lesion, increased insertion of adenine opposite the formylamine and increased replication blockage have been detected in mammalian cells, even though in this case mutagenicity of 8-oxoG remains unaltered (15). In the current work, we have evaluated the mutagenicity of 8-oxoG adjacent to a uracil in simian kidney cells with the notion that the uracil would be excised efficiently by the enzyme uracil DNA glycosylase in vivo generating an AP site. We believe that this tandem DNA damage not only represents a model lesion but also reflects a scenario when another γ-radiation-induced lesion adjacent to 8-oxoG might be excised preferentially.

Materials and Methods Materials. [γ-32P]ATP was from Du Pont New England Nuclear (Boston, MA). T4 DNA ligase and T4 polynucleotide kinase were obtained from New England Biolabs (Beverly, MA). E. coli strain DH10B was purchased from Invitrogen (Carlsbad, CA). COS-7 cells were available in our laboratory. Singlestranded (ss) phagemid pMS2 DNA was prepared from E. coli JM109 with the aid of the helper phage M13K07 (NEB) as reported by Moriya (16). Lesion-Containing Oligonucleotides. Uracil-containing, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-Oxo-dG)-containing, AP*containing, and control dodecamers of the sequences d[TGC AGU GTC AGC], d[TGC AGT GUC AGC], d[TGC AGT G8-oxoTC AGC], d[TGC AGU G8-oxoTC AGC], d[TGC AGT G8-oxoUC AGC], d[TGC AG(AP*) G8-oxoTC AGC], d[TGC AGT G8-oxo(AP*)C AGC], and d[TGC AGT GTC AGC] were synthesized by the

Communications Midland Certified Reagent Company, Inc. (Midland, TX). The 8-oxo-dG-containing oligonucleotides were deprotected with concentrated NH4OH for 18 h at 55 °C in the presence of 0.25 M β-mercaptoethanol and purified by C18 reverse phase HPLC followed by denaturing polyacrylamide gel electrophoresis. Mass spectrometric analysis by MALDI-TOF and/or ESI-MS verified the molecular weight of the oligonucleotides. Construction and Characterization of pMS2 Vector Containing a Single 8-Oxo-dG and/or Uracil. The ss pMS2 shuttle vector DNA (58 pmols, 100 µg), which contains a hairpin region, was digested with a large excess of EcoRV (300 pmol, 4.84 µg) for 1 h at 37 °C followed by room temperature overnight. A 58-mer scaffold oligonucleotide was annealed overnight at 9 °C to form the gapped DNA. The control and lesion-containing 12-mers were phosphorylated with T4 polynucleotide kinase, hybridized to the gapped pMS2 DNA, and ligated overnight at 16 °C. Unligated oligonucleotides were removed by passing through Centricon-100, and the DNA was precipitated with ethanol. The scaffold oligonucleotide was digested by treatment with T4 DNA polymerase and exonuclease III, the proteins were extracted with phenol/chloroform, and the DNA was precipitated with ethanol. The final construct was dissolved in 1 mM TrisHCl-0.1 mM EDTA, pH 8, and a portion was subjected to electrophoresis on 1% agarose gel in order to assess the amounts of circular DNA. On the basis of the proportion of circular DNA on the agarose gel, the ligation efficiency for each lesioncontaining dodecamer and the control were estimated to be ∼50%. Transfection and Mutation Frequency Determination. COS-7 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. The cells were seeded at 5 × 105 cells per 60 mm plate. Following overnight incubation, the cells were transfected with 50 ng of ss DNA by electroporation. The culture was incubated for 2 days, and the progeny plasmid was recovered by the method of Hirt (17). It was then used to transform E. coli DH10B, and transformants were analyzed by oligonucleotide hybridization (16, 18, 19). As described (16, 19), left and right probes were used to select phagemids containing the insert. Additional probes were used to determine the wild-type and G-to-T mutants. Transformants that failed to bind with both left and right probes were omitted. DNA sequencing was performed on all transformants that did not hybridize with either the wild-type or the G-to-T probe but were positive to left and right probes. In addition, a fraction of wild-type and G-to-T progeny DNA was sequenced from each batch. In some of the experiments, an A:A mismatch three nucleotides 5′ to the lesion (or a control) and a T opposite 8-oxoG (or G) was introduced in the scaffold to estimate the extent of scaffold removal. On the basis of the number of transformants that contained a T three nucleotides 5′ to the lesion ( dTMP (12%) > dCMP (2%), whereas in the AGTGXC sequence it was dTMP (16%) > dGMP (12%) > dCMP (9%) (where X represents the AP site) (Table 1). Although frameshifts were not common, a low frequency of targeted one-base deletions and insertions occurred in UGT and UG8-oxoT sequences but not in TGU (or TG8-oxoU) sequences (Table 1). Our result differs from several other reports on translesion synthesis of AP sites in COS cells. For example, a similar frequency of incorporation of dAMP, dTMP, and dCMP opposite a natural abasic site was reported in COS cells (21-24). Another study in COS cells reported preferential dAMP incorporation opposite a model AP site (tetrahydrofuran) accompanied by a small number of targeted deletions (25). These differences may reflect the differences among the experimental systems. In E. coli, preferential dAMP incorporation opposite AP sites, a phenomenon known as the “A rule,” has been demonstrated in many studies (for reviews, see refs 26 and 27). A site specific study in E. coli indicated that the frequency of incorporation of dAMP and the other nucleotides may depend on the sequence context (28). In this study, nucleotide incorporation other than dAMP followed the order dTMP > dGMP > dCMP opposite AP site in a GXT sequence and dGMP > dTMP > dCMP in a TXG sequence. Our results in COS cells are similar to this study in E. coli in that dGMP was preferred over dTMP when a purine was located 3′ to the AP site, whereas dTMP was preferred over dGMP when a pyrimidine was located 3′ to the AP site. Both studies also agreed that dCMP was the least preferred nucleotide to be incorporated opposite the AP site. The nucleotide incorporation frequency in the current study, however, changed when the 5′ G in the GXC or the 3′ G in GXG sequence was replaced with 8-oxoG. As shown in Table 1 and Figure 1, at both sites, dTMP incorporation opposite the AP site became more pro-

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nounced than dGMP incorporation. Other changes in the nucleotide incorporations are shown in Figure 1. For the GXG8-oxo sequence, the incorporation opposite the AP site showed dTMP (19%) > dGMP (12%) > dCMP (5%), whereas for the G8-oxoXC sequence it was dTMP (29%) > dGMP (20%) > dCMP (19%). Therefore, the total incorporation of nucleotides other than dAMP remained approximately the same (36% for both G and G8-oxo) in the GXG sequence, even though the relative incorporations of the three nucleotides changed significantly with an adjacent 8-oxoG. By contrast, in the GXC sequence, nucleotide incorporation other then dAMP nearly doubled with a 5′ G8-oxo to the AP site (37 vs 68% for G and G8-oxo, respectively). It is noteworthy that dCMP incorporation opposite the AP site doubled in each site with an adjacent 8-oxoG. The reasons for these changes are not known but may arise from structural effects [e.g., 8-oxoG may rotate to syn orientation and alter the phosphate backbone structure (29, 30)] and/or DNA-protein interactions [which may include the interactions of the modified template with DNA polymerases, accessory proteins, and/ or DNA repair proteins (10-12, 14)]. These changes are unlikely to be the result of excision of 8-oxoG prior to replication, because, unlike uracil DNA glycosylase that excises uracils from either single- or double-stranded DNA, most 8-oxoG repair systems require duplex DNA as a substrate (31). Mutagenicity of 8-Oxo-dG Adjacent to AP Site. Single-stranded vectors such as the pMS2 phagemid are convenient probes to determine the in vivo mutagenicity of DNA damage in the absence of most repair systems that can only operate in duplex DNA. Several earlier site specific studies showed that mutation frequency of 8-oxoG in ss DNA in repair competent E. coli is low ranging from 0.7 to 3%, whereas the same in simian kidney cells is in the range 4-7%, inducing primarily GfT transversions (reviewed in ref 32). It is believed that both E. coli and COS cells have functional MutY protein, which removes adenines from G8-oxo:A pairs after DNA replication is completed, and both are efficient in avoiding a major fraction of 8-oxoG mutagenesis. We serendipitously discovered that 8-oxoG in the TG*T sequence context used in this study generates progeny with 2324% GfT mutations in COS cells (Table 1),2 which is severalfold higher than other sequence contexts studied by us2 and others (16, 19). An explanation for the increased mutagenesis in this sequence has been provided elsewhere.2 When a uracil was placed 5′ to 8-oxoG, the single GfT events remained approximately the same. However, nearly 10% tandem mutations were detected, in which GfT transversions accompanied UfA, UfC, and UfG events, which therefore increased the total GfT substitutions to ∼30%. Similarly, when uracil was 3′ to 8-oxoG, the tandem mutations were nearly 40% and the total GfT frequency climbed to nearly 60% (Table 1 and Figure 2). What could be the reason(s) for this increase of dAMP incorporation opposite 8-oxoG? There are at least two scenarios that can rationalize our result. In the TG8-oxo(AP) sequence, the DNA polymerase must first bypass the AP site, whereas in the (AP)G8-oxoT sequence, the replicating 2Kalam, M. A., Haraguchi, K., Alimchandani, S., Loechler, E. L., Moriya, M., Greenberg, M. M., and Basu, A. K. Genetic effects of oxidative DNA damages: Comparative mutagenesis of the imidazole ring-opened fapy lesions and 8-oxo-purines in simian kidney cells. Manuscript in preparation.

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Table 1. Mutagenicity of a Single U and G8-Oxo Adjacent to U in Simian Kidney Cells tandem mutations

sequence TG8-oxoT UGT UG8-oxoT TGU TG8-oxoU

expt. no.

total colonies

TGT (%)

GfT (%)

1 2 total 1 2 total 1 2 total 1 2 total 1 2 total

326 271 597 131 49 180 110 143 253 114 130 244 78 105 183

248 (76) 208 (77) 456 (76) 81 (62) 33 (67) 114 (63) 36 (33) 66 (46) 102 (40) 79 (69) 73 (56) 152 (62) 12 (15) 11 (11) 23 (13)

78 (24) 62 (23) 140 (24)

28 (26) 25 (18) 53 (21)

16 (21) 21 (20) 37 (20)

UfA (%)

18 (14) 3 (6) 21 (12) 17 (16) 15 (11) 32 (13) 14 (12) 26 (20) 40 (16) 15 (19) 19 (18) 34 (19)

a A single GfC was detected. b U deletion. c GUGTfTTGT. and four C insertions (i.e., UG8-oxofTCG).

d

UfC (%)

30 (23) 10 (20) 40 (22) 10 (9) 15 (11) 25 (10) 12 (11) 17 (13) 29 (12) 4 (5) 8 (8) 12 (7)

UfG (%)

1 (1) 2 (4) 3 (2) 7 (6) 0 (0) 7 (3) 9 (8) 13 (10) 22 (9) 0 (0) 5 (5) 5 (3)

GfT UfA (%)

6 (6) 10 (7) 16 (6) 0 (0) 1 (1) 1 (0.4) 8 (10) 11 (11) 19 (10)

GfT UfC (%)

GfT UfG (%)

other mutations

2 (2) 3 (2) 5 (2)

0 (0) 4 (3) 4 (2)

1 (0.4)a 1 (0.2)a 1 (1)b 1 (2)c 2 (1) 4 (4)d 5 (4)e 9 (4)

6 (8) 17 (16) 23 (13)

17 (22) 13 (12) 30 (16)

Insertion of C between U and G (i.e., UG8-oxofTCG). e One UG8-oxofAC Table 2. Melting Temperature and Thermodynamic Parameters for DNA Duplexes Containing 8-OxoGa base pairing

sequence

G8-oxo:C TG8-oxoT (AP*)G8-oxoT TG8-oxo(AP*) G8-oxo:A TG8-oxoT (AP*)G8-oxoT TG8-oxoT(AP*) a

Figure 1. Frequency of single UfT, UfA, UfC, and UfG events in COS cells are shown as solid bars in UGT (top left), TGU (bottom left), UG8-oxoT (top right), and TG8-oxoU (bottom right) sequence contexts. The gray bar in each case shows the same when G8-oxofT mutations also occurred.

Figure 2. G8-oxofT transversion frequencies are shown as single (solid bar) and part of tandem (gray bar) base changes upon replication of T G8-oxoT, U G8-oxoT, and T G8-oxoU constructs in COS cells.

DNA polymerase first encounters 8-oxoG. At the AP site, the replicative DNA polymerase may dissociate, and recruitment of one or more bypass DNA polymerases may occur. These polymerases may interact differently to these lesions. It is well-known, for example, that pol η, which very efficiently bypasses 8-oxoG incorporating C opposite it, is inefficient in bypassing AP sites (33, 34). By contrast, eukaryotic polymerases R, δ, and  preferentially incorporate dAMP opposite 8-oxoG (33, 35). A difference in DNA polymerases could alter the mutational

Tmb -∆G° -∆H° -∆S° (°C) (kcal/mol) (kcal/mol) (cal/mol K) 59.4 43.2 40.5 56.9 35.6 38.2

16.9 ( 0.3 11.0 ( 0.2 10.2 ( 0.2 14.0 ( 0.5 9.4 ( 0.3 9.8 ( 0.3

95 ( 3 68 ( 3 63 ( 4 71 ( 5 67 ( 7 65 ( 7

263 ( 8 190 ( 9 177 ( 13 192 ( 14 192 ( 21 185 ( 23

Obtained by plotting 1/Tm vs ln CT. b 15 µM DNA.

frequency. An alternative, but not mutually exclusive, hypothesis involves participation (or the lack) of postreplicative mismatch repair proteins such as MYH (the mammalian homologue of mut Y glycosylase) that excises adenines from 8-oxoG:A pairs (36, 37). It is conceivable that the mismatch repair protein binding is less efficient in the TG8-oxo(AP) sequence relative to (AP)G8-oxoT because of a change in three-dimensional structure of the substrate, which in turn may result in a higher frequency of GfT substitutions in the TG8-oxo(AP) sequence. Thermodynamic Stability of 8-OxoG Opposite C vs A. A change in three-dimensional structure of a DNA fragment would likely alter base pairing interactions with the complementary strand. Optical melting curves provide a straightforward approach to determine base pairing strengths of short DNA duplexes (see, for example, ref 20). We compared the 8-oxoG:C pairing with 8-oxoG:A to determine the influence that an AP site might have when it is situated either 5′ or 3′ to the 8-oxoG residue. For these experiments, we used tetrahydrofuran (AP*) as the model AP site, in a dodecamer, since it is significantly more stable than a natural AP site (25). As expected, the presence of AP* destabilized each duplex (Table 2). In each case, C opposite 8-oxoG was more stable than A opposite it, and for the TG8-oxoT dodecamer, the 8-oxoG:C pair exhibited increased stability of ∆∆G ∼ 3 kcal/mol as compared to 8-oxoG:A. Most notable, however, was the result that ∆∆G for 8-oxoG:C relative to 8-oxoG:A pair for AP* 5′ to 8-oxoG was about 1.6 kcal/ mol, whereas the same for AP* 3′ to 8-oxoG was only ∼0.4 kcal/mol. Assuming that thermodynamic stability of a base pair plays a role in nucleotide insertion and extension, this suggests that the discrimination of a polymerase to incorporate dCMP over dAMP would be less

Communications

efficient in the TG8-oxo(AP*) sequence. Moreover, the efficiency of excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG8-oxo(AP*) sequence context, if base pairing stability is a criterion used by the protein for recognition and/or repair. To determine if an adjacent abasic site influences repair of 8-oxoG, in a preliminary investigation, we have analyzed the fpg repair kinetics of 35-mer oligonucleotides of local sequences TG8-oxoT, (AP*)G8-oxoT, and TG8-oxo(AP*) in duplex form (with ...A-C-A... 35-mer complementary strand). As compared to the substrate in which 8-oxoG was the solitary lesion, the excision of 8-oxoG followed by strand cleavage by fpg protein was at least an order of magnitude slower when an AP* site was located 3′ to 8-oxoG (data not shown). By contrast, the effect was modest when the AP* site was 5′ to 8-oxoG. It is conceivable that mismatch repair might also be affected in a similar manner. Irrespective of the mechanism, the mutational data reported here suggest that an AP site adjacent to 8-oxoG would result in an increased frequency of GfT transversions as single mutations (when the nucleotide incorporated opposite the AP site is correct) and as part of tandem mutations (when it is incorrect). How common are the tandem mutations in the ionizing radiation- or oxidative DNA damage-induced mutational spectra? Many studies on mutational specificity of ionizing radiation have been performed in bacteria (see, for example, refs 38-41). However, significant variations in the mutational spectra have been observed, and the results depend greatly on how the experiments were conducted. In an investigation in E. coli, a γ-radiation-induced mutational spectrum includes 19% GfT substitutions and approximately 1% tandem base substitutions (41). γ-Radiation mutagenesis studies in mammalian cells are not as extensive. A small population of mutants was analyzed in most of these studies, and tandem mutations have not been detected (42, 43). We suspect that radiation-induced tandem mutations are less frequent than the same from UV light. It is interesting to note, however, that oxidative DNA damage induced by copper and hydrogen peroxide promotes CGfTT tandem mutations at methylated CpG sites in nucleotide excision repair (NER) deficient human fibroblasts (44). The DNA lesion(s) responsible for this tandem mutation is yet to be identified. In a more recent study, peroxyl radicalmediated oxidative DNA damage-induced mutagenesis in human cells was mapped along the supF gene of the pSP189 shuttle vector (45). Tandem base substitutions have been detected in both repair competent and NER deficient human fibroblast cell lines. In the repair competent cells, 14% of the mutants are tandem mutations, whereas 3-5% mutants are tandem mutations in XPA, XPG, and XPF cell lines (45). Nearly half of the tandem mutations promoted by peroxyl radical occurred at GG sites that are well-known hot spots for oxidative damage (46). It is conceivable that some of these tandem mutations are the results of tandem DNA damages similar to the one investigated in the current work. In conclusion, a uracil has been used to generate an AP site adjacent to 8-oxoG in a phagemid vector, which resulted in an altered mutagenic profile of each lesion in COS cells. Although incorporation of dAMP opposite the AP site could not be quantified accurately, for other nucleotide incorporations opposite the AP site, there was a preference of dTMP incorporation in two different

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sequence contexts with an adjacent 8-oxoG. The presence of an AP site either 5′ or 3′ to 8-oxoG increased the frequency of G8-oxofT transversions, but it was as high as ∼60% when the AP site was located 3′ to 8-oxoG. Thermal melting studies showed that base pairing strengths of 8-oxoG with C and A were also influenced by the adjacent AP site. Even though C formed a more stable pair with 8-oxoG than A in all cases, the discrimination was significantly diminished when the AP site was 3′ to 8-oxoG. Evidently, additional structural and biological studies would be needed to provide further insight.

Acknowledgment. We are grateful to Maasaki Moriya (SUNY, Stony Brook) for providing the pMS2 shuttle vector. This work was supported by NIEHS Grant ES09127. Supporting Information Available: van’t Hoff plots of 1/Tm vs ln CT for helix to coil transitions of dodecamers. This material is available free of charge via the Internet at http:// pubs.acs.org.

Note Added after ASAP Publication. This manuscript was originally published July 15, 2005 with a different heading for the third paragraph of the Materials and Methods section. The corrected version was published July 18, 2005.

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