The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Deri

Article

pubs.acs.org/biochemistry

A New Cross-Link for an Old Cross-Linking Drug: The Nitrogen Mustard Anticancer Agent Mechlorethamine Generates Cross-Links Derived from Abasic Sites in Addition to the Expected Drug-Bridged Cross-Links Maryam Imani Nejad,† Kevin M. Johnson,† Nathan E. Price,† and Kent S. Gates*,†,‡

† Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States ‡ Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States

*S Supporting Information

ABSTRACT: Nitrogen mustard anticancer drugs generate highly reactive aziridinium ions that alkylate DNA. Mono- adducts arising from reaction with position N7 of guanine residues are the major DNA adducts generated by these agents. Interstrand cross-links in which the drug bridges position N7 of two guanine residues are formed in low yields relative to those of the monoadducts but are generally thought to be central to medicinal activity. The N7-alkylguanine residues generated by nitrogen mustards are depurinated to yield abasic (Ap) sites in duplex DNA. Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine lead to interstrand cross-links of a type not previously associated with this drug. Gel electrophoretic data were consistent with early evolution of the expected drug-bridged cross-links, followed by the appearance of Ap-derived cross-links. The evidence is further consistent with a reaction pathway involving alkylation of a guanine residue in a 5′-GT sequence, followed by depurination to generate the Ap site, and cross-link formation via reaction of the Ap aldehyde residue with the opposing adenine residue at this site [Price, N. E., Johnson, K. M., Wang, J., Fekry, M. I., Wang, Y., and Gates, K. S. (2014) J. Am. Chem. Soc. 136, 3483−3490]. The monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulﬁde 6, and natural product leinamycin similarly were found to induce the formation of Ap-derived cross-links in duplex DNA. This work provides the ﬁrst characterization of Ap-derived cross-links at sequences in which a cytosine residue is located directly opposing the Ap site. Cross-linking processes of this type could be relevant in medicine and biology because Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues.

itrogen mustards such as mechlorethamine (HN2) were can forge DNA−DNA interstrand cross-links in some the ﬁrst synthetic anticancer drugs1,2 and remain in sequences via reaction of the Ap aldehyde residue with the N − widespread clinical use.3 6 These bifunctional agents generate exocyclic amino groups of nucleobases such as adenine and aziridinium ions that react with DNA at a variety of locations, guanine on the opposing strand of the DNA duplex (Scheme − including N7-guanine, N3-adenine, N3-cytidine, and the 2).32 40 The cross-linking reactions considered here, involving − phosphodiester linkages of the backbone (Scheme 1).4,7 21 “true” Ap sites, are distinct from those involving oxidized abasic Monoadducts (1 and 2) at guanine residues are the major DNA sites.41,42 Here, we show that Ap sites generated by the nitrogen alkylation products formed by these drugs (Scheme 1).8,10,11 mustard mechlorethamine (HN2) give rise to interstrand cross- Interstrand cross-links (3) are generated in much lower yields links of a type not previously associated with this drug. We (1−10% of total adducts) but are generally believed to be the present gel electrophoretic data that are consistent with an early critical lesions responsible for medicinal activity of the nitrogen evolution of the expected drug-bridged cross-links 3 followed mustards.4,22,23 Cross-link formation by nitrogen mustards can by the appearance of Ap-derived cross-links. The evidence is occur via reactions with two guanine residues in 5′-GNC consistent with a reaction pathway involving alkylation of a − sequences [3 (Scheme 1)];14,24 28 however, there is also guanine residue in a 5′-GT sequence, followed by depurination evidence of G-G cross-link formation at 5′-GC sequences as to generate the Ap site, and cross-link formation via reaction of well as G-A and A-A cross-linking at as-yet-undeﬁned the Ap aldehyde residue with the opposing adenine residue at sequences.12,26,29 The alkylation of guanine and adenine residues by nitrogen Received: October 21, 2016 mustards induces the formation of abasic (Ap) sites in genomic Revised: November 22, 2016 DNA (Scheme 1).9,10,30,31 We recently showed that Ap sites Published: November 29, 2016

Scheme 1 cytosine residues occur readily in genomic DNA via spontaneous depurination of guanine and alkylguanine residues. The processes described here expand the list of mechanisms by which nitrogen mustards and other DNA-alkylating drugs can generate cytotoxic interstrand cross-links. ■ EXPERIMENTAL SECTION Materials and General Procedures. Reagents were purchased from the following suppliers and were of the highest purity available: oligonucleotides from Integrated DNA Technologies (Coralville, IA), uracil DNA glycosylase (UDG) and T4 DNA polynucleotide kinase (T4 PNK) from New England Biolabs (Ipswich, MA), [γ-32P]ATP (6000 Ci/mmol) from PerkinElmer, 19:1 acrylamide/bis-acrylamide (40% solution/electrophoresis) from Fisher Scientiﬁc (Waltham, MA), and mechlorethamine hydrochloride and alkylating agents from Sigma-Aldrich (St. Louis, MO). LNM was a gift from Kyowa Hakko Kogyo, Ltd. C-18 Sep-Pak cartridges were purchased from Waters (Milford, MA), and BS Poly Prep columns were obtained from Bio-Rad (Hercules, CA). Quantiﬁcation of radioactivity in polyacrylamide gels was conducted using a Personal Molecular Imager (Bio-Rad) with Quantity One (version 4.6.5). Representative Procedure for Cross-Link Formation Time Courses by HN2, 5, 6, and LNM. Single-stranded 2′- deoxyoligonucleotides were 5′-labeled using standard procedures.44 Labeled DNA was annealed44 with its complementary strand to give the duplexes shown in Figure 1. In a typical cross- Scheme 2

this site (Scheme 2).35 We further showed that the monofunctional DNA-alkylating agents 2-chloro-N,N-diethylethanamine 5, (2-chloroethyl)ethylsulﬁde 6, and natural product leinamycin (LNM) similarly induce Ap-derived cross-links in duplex DNA via alkylation and depurination at 5′-GT sequences. Figure 1. DNA sequences used in these studies. Ap-containing duplexes were generated by the action of UDG on the corresponding dU-containing duplex. Cross-link locations are indicated with a red connection.

linking reaction, HN2 was introduced into the reaction mixture These results provide the first characterization of Ap-derived as a stock solution in DMF, to give a mixture containing HN2 cross-link formation at sequences in which a cytosine residue (1 mM) and labeled DNA in HEPES buffer (50 mM, pH 7) directly opposes the Ap site. The ability of such sites to containing 100 mM NaCl and 10% (v/v) DMF that was generate interstrand cross-links was by no means certain, incubated at 37 °C for 96 h unless otherwise specified. The because the nature of the directly opposing base can exert DNA was ethanol-precipitated from the reaction mixture,44 significant effects on the structures of Ap-containing duplexes.43 resuspended in formamide loading buffer,44 and loaded onto a Cross-linking reactions of this type could be important in 20% denaturing polyacrylamide gel, and the gel was electro- biology and medicine because Ap sites with directly opposing phoresed for 5 h at 1600 V. The amount of radiolabeled DNA

7034 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article in each band on the gel was measured by phosphorimager containing double-stranded DNA (∼400000 cpm) was analysis. The time course for the formation of the late-forming incubated in HEPES buffer (50 mM, pH 7) containing NaCl cross-link was determined by incubating a solution containing (100 mM) at 37 °C for 120 h. The DNA was ethanol labeled DNA (approximately 100000 cpm) and HEPES buffer precipitated, suspended in formamide loading buffer, and (50 mM, pH 7) containing NaCl (100 mM) and HN2 (1 mM) resolved on a 2 mm thick 20% denaturing polyacrylamide gel. at 37 °C. At specified time points, aliquots (3 μL) were The late-forming cross-linked duplex band was visualized using removed and formamide loading dye was added followed by X-ray film, the band cut out of the gel, and the gel slice crushed, freezing at −20 °C, and gel electrophoretic analysis as described and the gel pieces were vortex-mixed in elution buffer (200 mM above. For cross-link formation by LNM, all conditions were NaCl and 1 mM EDTA) at room temperature for at least 1 h. identical, except LNM was introduced as a stock solution in The mixture was filtered through a Poly-Prep column to acetonitrile, the final concentration of LNM was either 100 or remove gel fragments, and the residue was ethanol precipitated, 500 μM, and β-mercaptoethanol (0.5 or 2.5 mM) was added to redissolved in water, and mixed with 2× oxidation buffer [10 initiate the DNA alkylation reaction.45 μL of a solution composed of 20 mM sodium phosphate (pH Representative Procedure for Preparation of Du- 7.2), 20 mM NaCl, 2 mM sodium ascorbate, and 1 mM H2O2]. plexes Containing Authentic Ap-Derived Cross-Links. A To this mixture was added a solution of iron-EDTA [2 μLof70 · single-stranded, uracil-containing 2′-deoxyoligonucleotide was mM EDTA and 70 mM (NH4)2Fe(SO4)2 6H2O] to start the 5′-labeled using standard procedures,44 annealed with its reaction, and the mixture was vortexed briefly and incubated at complementary strand, and treated with the enzyme UDG room temperature for 5 min before addition of a thiourea stop (50 units/mL, final concentration) to generate the Ap site. The solution (10 μL of a 100 mM solution in water). Hydroxyl UDG enzyme was removed by phenol/chloroform extraction radical footprinting reactions, Maxam−Gilbert G reactions, and and the DNA ethanol precipitated. The Ap-containing duplexes Maxam−Gilbert A+G reactions were performed on the labeled were incubated in a buffer composed of HEPES (50 mM, pH duplex to generate marker lanes.46 The resulting DNA 7) containing NaCl (100 mM) at 37 °C for 120 h unless fragments were analyzed using gel electrophoresis as described otherwise specified. The DNA was ethanol precipitated, above. resuspended in formamide loading buffer, and loaded onto a 20% denaturing polyacrylamide gel that was electrophoresed ■ RESULTS AND DISCUSSION for 5 h at 1600 V. The amount of radiolabeled DNA in each Treatment of Duplex DNA with HN2 Leads to band on the gel was measured by phosphorimager analysis. The Generation of Distinct Early-Forming and Late-Forming time course for the formation of the dA-Ap cross-link was Cross-Links. It is well established that treatment of duplex determined by incubating a solution containing labeled DNA DNA with HN2 can lead to the generation of interstrand cross- (approximately 100000 cpm) and HEPES buffer (50 mM, pH links that appear as slow-migrating bands on denaturing 7) containing NaCl (100 mM) at 37 °C. At specified time polyacrylamide gels (located above the full-length single- − points, aliquots (3 μL) were removed, formamide loading dye stranded DNA in the denaturing gels presented here).14,26 28 was added, and the sample was frozen at −20 °C and analyzed Indeed, we found that treatment of duplex A (Figure 1) with by gel electrophoresis as described above. HN2 (1 mM) in HEPES buffer (50 mM, pH 7) containing Sequence Specific DNA Alkylation by HN2, 2- NaCl (100 mM) and DMF [10% (v/v)] at 37 °C, followed by (Chloroethyl)ethylsulfide (6), and LNM. Typical alkylation electrophoretic analysis of the 32P-labeled DNA fragments on a reaction mixtures contained HN2 (1 mM) and a 32P-labeled denaturing 20% polyacrylamide gel, revealed several distinct DNA duplex (Figure 1)inabuffer composed of HEPES (50 slowly migrating bands (Figure 2). The formation of multiple mM, pH 7), NaCl (100 mM), and DMF [10% (v/v)] and cross-linked species has previously been observed in duplexes incubated at 37 °C for 2 h. The DNA was ethanol precipitated, containing both 5′-GNC and 5′-GC sites treated with redissolved in aqueous piperidine (50 μL of a 1 M solution), HN2.14,26,28 Most intriguingly, we found that the two major and incubated at 90 °C for 25 min (Maxam−Gilbert cross-link bands generated by treatment of duplex A with HN2 workup).46 The solution was frozen on dry ice, lyophilized displayed very different formation time courses and gel for 40 min in a SpeedVac Concentrator at 37 °C, redissolved in mobilities (Figure 2). A major “early-forming” cross-link band 20 μL of water, and evaporated again. The dried DNA was immediately evident. The yield of this band peaked at the fragments were dissolved in formamide loading buffer, loaded earliest time point (2 h, 5% yield), and then the intensity onto a 20% polyacrylamide denaturing gel, and electrophoresed decreased over the remainder of the experiment (Figure 2B). at 1400 V for 5 h. The amount of radioactivity in the resolved This behavior was consistent with that expected for the typical DNA fragments was quantitatively analyzed with a phosphor- drug-linked G-G cross-link in two key regards: (i) Interstrand − imager. For alkylation by LNM, all conditions were identical DNA cross-link formation by HN2 is rapid,30,48 50 and (ii) except that LNM was introduced as a stock solution of depurination of HN2 adducts, occurring with a half-life of ∼9h acetonitrile (instead of DMF) and 50 μM LNM and 500 μM β- at 37 °C, was expected to cause spontaneous “unhooking” of mercaptoethanol were employed. For alkylation by the sulfur the cross-link (with a corresponding disappearance of the cross- mustard 6, a concentration of 500 μM was used. link band).10,30,31 Hydroxyl Radical Footprinting of a Duplex C- We also observed a “late-forming” cross-link band that Containing Authentic Ap-Derived Cross-Link. We fol- became visible after 12 h and predominant after 48 h [4% yield lowed literature protocols for the footprinting of cross-link (Figure 2)]. It seemed unlikely that this could be a drug-linked duplex C.35,37,42,47 In this experiment, the strand opposing the cross-link because the chloroethyl groups of HN2 in both DNA Ap-containing oligonucleotide was 5′-labeled using the stand- monoadducts [1 (Scheme 1)] and free HN2 hydrolyze rapidly − ard procedure.44 Labeled DNA was annealed with the uracil- (t =1−30 min).29,48 51 On the other hand, in light of our 1/2 − containing complementary strand and the duplex treated with recent studies,32 40 it was reasonable to consider that a buildup UDG to generate the abasic site as described above. The Ap- of Ap sites resulting from depurination of HN2 adducts (1−3)

7035 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article

could be an Ap-derived cross-link, we simultaneously explored two questions: (i) Does generation of an Ap site at position 1, 2, 6, or 8 in duplex A lead to dA-Ap cross-link formation, and (ii) if so, does the gel mobility of any of the resulting cross- linked duplexes match with that of the late-forming cross-link band produced following treatment of duplex A with HN2? Toward this end, we conducted a series of experiments in which an authentic Ap site was introduced at position 1, 2, 6, or 8 in duplex A. The 5′-32P-labeled Ap-containing duplexes B−E − were prepared as described in our previous work32 40 by the action of uracil DNA glycosylase (UDG) on the corresponding 2′-deoxyuridine-containing oligodeoxynucleotide duplexes.52,53 The formation of slowly migrating, Ap-derived cross-link bands was easily detected in each of the Ap-containing duplexes B−E (Figure 3). The equilibrium yields of the cross-link generated in

Figure 2. Treatment of duplex A with HN2 leads to the generation of distinct early-forming and late-forming cross-links. Duplex A was incubated with HN2 (1 mM) in HEPES buffer (50 mM, pH 7) containing 100 mM NaCl and 10% (v/v) DMF at 37 °C. Aliquots were removed at 0, 2, 4, 8, 24, 48, 72, and 96 h and stored frozen prior to gel electrophoretic analysis (lanes 1−8, respectively). The 32P- labeled 2′-deoxyoligonucleotides were resolved on a sequencing gel, and the radioactivity in each band was quantitatively measured by phosphorimager analysis. The bottom band corresponds to the full- length labeled 2′-deoxyoligonucleotides, and the slowly migrating upper bands correspond to cross-linked DNA. Figure 3. Late-forming cross-link generated by treatment of duplex A with HN2 co-migrates with authentic dA-Ap cross-linked duplex C. The lower bands correspond to the 32P-labeled full-length labeled 2′- might permit the generation of Ap-derived cross-links. In the deoxyoligonucleotides and the upper bands cross-linked DNA. Lanes 1 following sections, we provide evidence that the late-forming and 2 show duplex A incubated with HN2 (1 mM) in HEPES buffer cross-link band seen in Figure 2A is a dA-Ap cross-link resulting (50 mM, pH 7) containing 100 mM NaCl and 10% (v/v) DMF at 37 from alkylation-induced generation of an Ap site at position 2 °C for 48 and 96 h, respectively, prior to sequencing gel analysis. Lanes of duplex A (Figure 1). 3−6 show authentic dA-Ap cross-links in duplexes C, B, E, and D, 32 A DNA Duplex Containing an Authentic dA-Ap Cross- respectively. The P-labeled 2′-deoxyoligonucleotides were resolved Link at Position 2 Co-Migrates with the Late-Forming on a 20% polyacrylamide denaturing gel, and the radioactivity in each Cross-Link Band Generated by Treatment of Duplex A band was quantitatively measured by phosphorimager analysis. with HN2. The formation of Ap-derived cross-links has been observed in two different sequence motifs: 5′-CAp/5′-AG and these duplexes varied substantially, with values of 5, 30, 14, and 5′-ApT/5′-AA (the cross-linked base is underlined; see Figure 2%, for duplexes B−E, respectively (Figure S1). Interestingly, 1 for images of the cross-link sequence motifs).35,37,38 In the cross-linked duplexes B−E displayed distinct gel mobilities dG-Ap cross-link, the cross-linking guanine residue is offset one (Figure 3). It has previously been observed that the location of base to the 5′-side of the Ap site, while in the dA-Ap cross-link, cross-links, with respect to the end of the DNA duplex, can − the cross-linking adenine residue is offset one base to the 3′- dramatically alter mobility in denaturing gels.54 56 In general, side of the Ap site.35,37,38 In all of the Ap-derived cross-links our observations follow the trends reported in two earlier described to date, an adenine residue has been located directly studies involving drug-derived cross-links, in which duplexes opposing the Ap site and the dA-Ap cross-linking motif containing cross-links near their ends were shown to migrate generates cross-link yields substantially greater than that of the faster in denaturing gels than those containing cross-links near dG-Ap motif.35,37,38 their center.54,55 More important in the context of the current Recognition that alkylation-induced Ap sites have the studies is the fact that cross-linked duplexes B, D, and E derived potential to generate interstrand DNA−DNA cross-links, from Ap sites at positions 1, 6, and 8, respectively, did not co- combined with the knowledge that guanine residues are the migrate with the major late-forming cross-link band produced primary alkylation sites for HN2, drew our attention to 5′-GT by treatment of duplex A with HN2. On the other hand, the sequences in duplex A as potential progenitors of HN2-induced cross-link derived from the Ap site at position 2 (duplex C) did dA-Ap cross-links. Specifically, alkylation at these sites, followed co-migrate with the major late-forming band from the HN2 by depurination of the resulting alkylguanine residue, could reaction (Figure 3). allow dA-Ap cross-link formation at the resulting 5′-ApT site.35 We carefully characterized cross-link formation in the In duplex A, there are four such sites involving the guanine authentic Ap-containing duplex C. We found that this cross- residues at positions 1, 2, 6, and 8 (Figure 1). As an initial test link was generated in an equilibrium yield of 30%, with an of whether the late-forming cross-link band seen in Figure 2 apparent formation half-time of 24 h in HEPES buffer (50 mM,

7036 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article pH 7) containing NaCl (100 mM) at 37 °C(Figure S1). Iron- EDTA footprinting47 of the cross-linked DNA provided evidence that the Ap site was cross-linked to the adenine residue at position 9 (Figure 1 and Figure S2). To further probe the involvement of the adenine residue at position 9 in the generation of the late-forming dA-Ap cross- link induced by treatment of duplex A with HN2, we investigated the properties of duplex F, lacking this critical residue (Figure 1). We found that treatment of duplex F with HN2 generated the early-forming cross-link, but not the late- forming cross-link band (Figure S3). This result is consistent with the assignments of the early-forming cross-link band as a drug-linked cross-link and the late-forming cross-link band as a dA-Ap cross-link involving residue A9 of duplex A. Evidence That the Late-Forming Cross-Link Involves Generation of Ap Sites in Duplex A by H2N. Results described above provided evidence that a dA-Ap cross-link was forged between an Ap site at position 2 and the adenine residue at position 9 in duplex A. HN2-induced formation of the requisite Ap site at position 2 must be preceded by alkylation of the corresponding guanine residue at this position. To explore this issue, we treated the 32P-labeled duplex A with HN2 (1 mM) in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) for 2 h at 37 °C, followed by piperidine workup to induce strand cleavage at the alkylated guanine residues. Electro- phoretic analysis of the 32P-labeled DNA fragments on a Figure 4. Alkylation of guanine residues in duplex A by HN2. 32P- labeled oligonucleotide duplex A was incubated with HN2 in HEPES denaturing 20% polyacrylamide gel showed that guanine ff residues at positions 2−4 and 6−8 were all alkylated by HN2 bu er (50 mM, pH 7) containing 100 mM NaCl and 10% (v/v) DMF at 37 °C for 2 h followed by Maxam−Gilbert workup and separation of [alkylation at position 1 could not be accurately measured the labeled fragments on a 20% denaturing polyacrylamide gel. because the band was not resolved from full-length DNA, and Labeled DNA was visualized by phosphorimager analysis. Lane 1 position 5 could not be measured because the band migrated shows an untreated duplex A. Lane 2 shows a Maxam−Gilbert G off the end of the gel (Figure 4 and Figure S4)]. Nonetheless, reaction on the labeled strand of duplex A. Lane 3 shows an A+G the results confirmed that position 2 was alkylated in substantial reaction on the labeled strand of duplex A. Lane 4 shows DNA treated yield by HN2. with HN2 (1 mM) followed by Maxam−Gilbert workup. The 32P- To provide additional evidence that the late-forming cross- labeled 2′-deoxyoligonucleotides were resolved on a sequencing gel link band generated by treatment of duplex A with HN2 was and visualized by phosphorimager analysis. derived from an Ap site in the duplex, we examined the effects exerted upon the cross-linking reaction by two different fi Generation of the Abasic-Derived Cross-Link by treatments that modify Ap sites in duplex DNA. Speci cally, N N ff Monoalkylating Agents 2-Chloro- , -diethylethan- we examined the e ects of apurinic endonuclease (APE), an fi ′ 57 amine (5), 2-(Chloroethyl)ethylsul de (6), and Leinamy- enzyme that cleaves on the 5 -side of Ap sites, and cin (7). In the reaction pathway proposed here, formation of methoxyamine (MX), a reagent that forms a stable oxime the dA-Ap cross-link following treatment of duplex A with HN2 derivative at Ap sites and inhibits the formation of Ap-derived 35,58 is dependent upon the alkylating properties of HN2, but not cross-links. In separate experiments, these reagents were dependent upon the cross-linking properties of the drug. added after 24 h to reaction mixtures containing HN2 and Accordingly, the monofunctional nitrogen mustard 2-chloro- duplex A. These Ap-modifying reagents inhibited generation of N,N-diethylethanamine 5 and other monofunctional DNA- the late-forming cross-link band, consistent with the notion that alkylating agents that target guanine residues should be HN2-induced generation of Ap sites was central to the competent to induce the late-forming cross-link band in duplex production of the late-forming band (Figure 5). As expected, A. With this in mind, we examined the ability of two well- MX prevented both cross-linking and spontaneous strand known21,59 monofunctional DNA-alkylating agents, 2-chloro- cleavage by capping the Ap aldehyde residues as an inert oxime 5 fi 35 N,N-diethylethanamine ( ) and (2-chloroethyl)ethylsul de (lane 2, Figure 5). APE inhibited cross-link formation while (6), to induce formation of the dA-Ap cross-link band in generating the anticipated strand cleavage at Ap sites resulting duplex A. N7-Alkylguanine residues are major DNA adducts from spontaneous depurination of HN2−guanine adducts at generated by both of these agents (Figure S4).21,59,60 We found G2−G4 (lane 3, Figure 5). The multiple bands generated by that treatment of duplex A with either 5 (1 mM) or 6 (1 mM) APE may reflect both cleavage at Ap sites generated by in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) depurination of alkylated adenine residues and the result of the for 96 h at 37 °C did indeed produce the cross-linked duplex C enzyme’s3′-exonuclease activity on the initial cleavage in yields comparable to that achieved by bifunctional alkylator products. Previous work by Osborne et al. showed that N3- HN2 (Figure 6 and Figure S5). alkyladenine residues are spontaneously released from DNA We also examined the ability of the Streptomyces-derived treated with HN2, presumably with concomitant generation of anticancer natural product leinamycin (LNM) to generate the abasic sites.29 late-forming dA-Ap cross-link in duplex A. LNM seems well-

7037 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article

(Scheme 3).45,61 (ii) LNM selectively alkylates 5′-GT sequences that support the formation of dA-Ap cross-

Scheme 3

Figure 5. Treatments that modify Ap sites inhibit generation of the late-forming cross-link in duplex A. Evidence that the late-forming cross-link is derived from an Ap site in duplex A. Lane 1 shows duplex A incubated with HN2 (1 mM) in HEPES buffer (50 mM, pH 7) containing 100 mM NaCl and 10% (v/v) DMF at 37 °C for 96 h. Lane 2 shows duplex A incubated with HN2 (1 mM) in HEPES buffer 60,62 − (50 mM, pH 7) containing 100 mM NaCl at 37 °C, and after 24 h, links. (iii) LNM guanine adducts undergo unusually fi 63 CH3ONH2 (MX) was added to a nal concentration of 2 mM and the rapid depurination to yield abasic sites. We found that mixture incubated for an additional 72 h. Lane 3 shows duplex A treatment of duplex A with LNM (100 μM) and 2- incubated with HN2 (1 mM) in HEPES buffer (50 mM, pH 7) mercaptoethanol (500 μM) generated a band that co-migrated containing 100 mM NaCl and 10% (v/v) DMF at 37 °C, and after 24 with cross-linked duplex C (Figure S6). Interestingly, treatment h, APE was added and the mixtures were incubated for an additional of duplex A with higher concentrations of LNM generated a 32 ′ 72 h. The P-labeled 2 -deoxyoligonucleotides were resolved on a complex mixture of slow-migrating bands that did not sequencing gel and visualized by phosphorimager analysis. necessarily co-migrate with authentic cross-linked duplexes B−E (Figure S7). The identity of these species remains to be determined. ■ CONCLUSIONS Interstrand DNA cross-links are the critical cytotoxic lesions generated by a variety of anticancer drugs, including nitrogen mustards, busulfan, mitomycin C, and cisplatin.64 Nitrogen mustards have the potential to generate a variety of cross-links wherein the drug connects G-G, G-A, and A-A residues.12,26,27,29 Our work provides evidence of the generation of a new type of interstrand cross-link not previously associated with nitrogen mustards, involving alkylation of guanine residues at 5′-GT sites, depurination of the resulting alkylguanine Figure 6. Treatment of duplex A with the monofunctional nitrogen residue, and formation of a dA-Ap cross-link. In our mustard 5 generates the late-forming dA-Ap cross-link. Duplex A was experiments, Ap-derived cross-links were produced in yields incubated with compound 5 (1 mM) in HEPES buffer (50 mM, pH 7) comparable to those of the drug-linked cross-links. The results ° containing 100 mM NaCl and 10% (v/v) DMF at 37 C, and at 0, 2, 4, indicate that care must be exercised when assigning the identity 8, 12, 24, 48, 72, and 96 h, aliquots were removed from the reaction of slow-migrating cross-link bands in gel electrophoretic mixture and frozen at −20 °C prior to gel electrophoretic analysis (lanes 1−9, respectively). Lane 10 shows the authentic dA-Ap analyses of mustard-treated DNA; not all slow-migrating containing duplex C. Labeled DNA in the gel was quantitatively bands are necessarily drug-linked cross-links. Following treat- detected by phosphorimaging analysis. The bottom band in the gel ment of duplex DNA with HN2, the gel electrophoretic data image is the full-length labeled 2′-deoxyoligonucleotide, and the slow- were consistent with early evolution of the expected drug- moving upper band corresponds to cross-linked DNA. bridged cross-links, followed by the appearance of Ap-derived cross-links. It seems clear that HN2-induced formation of Ap- suited for the generation of Ap-derived cross-links in DNA for derived cross-links does not occur equally well at all 5′-GT sites three reasons. (i) The reaction of LNM with thiols generates a in duplex A. The major site of cross-link formation involves DNA-binding episulfonium ion 8 that selectively and efficiently alkylation and Ap generation at G2 and cross-linking with A9 alkylates position N7 of guanine residues in duplex DNA (Figure 1). The observed yields of Ap-derived cross-link at any

7038 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article given 5′-GT site must reflect an interplay of HN2 alkylation Funding efficiency, depurination of the alkylguanine residue, and the We thank the National Institute of Environmental Health inherent potential for Ap-derived cross-link formation at that Sciences of the National Institutes of Health for support of this sequence in the duplex. work (ES 021007). Generation of the dA-Ap cross-link by HN2 relies upon the Notes DNA alkylating properties of the drug and not its bifunctional The authors declare no competing financial interest. cross-linking properties. Accordingly, we found that formation of the dA-Ap cross-link was similarly induced by three different ■ ABBREVIATIONS monofunctional alkylating agents that target guanine residues in Ap, DNA abasic site; dA, 2′-deoxyadenosine; dG, 2′- duplex DNA, 2-chloro-N,N-diethylethanamine (5) and (2- deoxyguanosine; UDG, uracil DNA glycosylase; T4 PNK, T4 chloroethyl)ethylsulfide (6), and LNM. DNA polynucleotide kinase; HEPES, 4-(2-hydroxyethyl)-1- Previously characterized Ap-derived cross-links have been piperazineethanesulfonic acid; EDTA, ethylenediaminetetra- located at sequences in which an adenine residue directly 32−40 acetic acid; Tris, tris(hydroxymethyl)aminomethane; HN2, opposes the Ap site. In biological systems, Ap sites with mechlorethamine; LNM, leinamycin; MX, methoxyamine; opposing adenine residues arise via the enzymatic removal of APE, apurinic endonuclease; nt, nucleotide. misincorporated 2′-deoxyuridine by the base excision repair enzyme UDG.65 This may be a major source of Ap sites in 65 ■ REFERENCES eukaryotic cells. The loss of damaged thymine residues also (1) De Vita, V. T. J., and Chu, E. (2008) A history of cancer can give rise to Ap sites with a directly opposing adenine − residue.66 The work described here explored the properties of chemotherapy. Cancer Res. 68, 8643 8653. Ap sites arising from another important cellular process, the (2) Goodman, L. S., Wintrobe, M. M., Dameshek, W., Goodman, M. J., Gilman, A., and McClennan, M. T. (1946) Nitrogen mustard depurination of guanine and alkylguanine residues. These therapy: use of methyl-bis(beta-chloroethyl)amine hydrochloride and events give rise to Ap sites opposed by a cytosine residue. At tris(beta-chloroethyl)amine hydrochloride for Hodgkin’s disease, the outset of this work, it was uncertain whether Ap sites lymphosarcoma, leukemia, and certain allied and miscellaneous opposed by a cytosine residue could engage in interstrand disorders. JAMA 132, 126−132. cross-linking reactions. This uncertainty was due to the fact that (3) Cheson, B. D., and Rummel, M. J. (2009) Bendamustine: rebirth the nature of the base directly opposing an Ap site can exert of an old drug. J. Clin. Oncol. 27, 1492−1501. significant effects on the structure of an Ap-containing (4) Povirk, L. F., and Shuker, D. E. (1994) DNA damage and duplex.43,67,68 For example, the Ap residue can adopt an mutagenesis induced by nitrogen mustards. Mutat. Res., Rev. Genet. Toxicol. 318, 205−226. extrahelical conformation when opposed by a pyrimidine ’ residue.43,67,68 Nonetheless, the results reported here show (5) Physicians desk reference, 65th ed. (2011) Thomsom PDR, fi Montvale, NJ. for the rst time that Ap sites with directly opposing cytosine (6) WHO Model List of Essential Medicines, 19th List (2015) World residues are competent to form dA-Ap cross-links. Indeed, Health Organization, Geneva. formation of slow-migrating cross-link bands was easily (7) Cullis, P. M., Green, R. E., and Malone, M. E. (1995) Mechanism detected in each of the Ap-containing duplexes B−E, with and reactivity of chlorambucil and chlorambucil-spermidine conjugate. the equilibrium cross-link yields ranging from 2 to 30%. Given J. Chem. Soc., Perkin Trans. 2 2, 1503−1511. that Ap sites with directly opposing cytosine residues occur (8) Mohamed, D., Mowaka, S., Thomale, J., and Linscheid, M. W. frequently in genomic DNA via spontaneous or enzymatic (2009) Chlorambucil-adducts in DNA analyzed at the oligonucleotide − depurination of guanine and N7-alkylguanine resi- level using HPLC-ESI MS. Chem. Res. Toxicol. 22, 1435 1446. − dues,9,10,63,69 71 the results provide an additional impetus to (9) Gates, K. S. (2009) An overview of chemical processes that examine the occurrence and consequences of Ap-derived cross- damage cellular DNA: spontaneous hydrolysis, alkylation, and reactions with radicals. Chem. Res. Toxicol. 22, 1747−1760. links in cellular DNA. (10) Gates, K. S., Nooner, T., and Dutta, S. (2004) Biologically relevant chemical reactions of N7-alkyl-2′-deoxyguanosine adducts in ■ ASSOCIATED CONTENT DNA. Chem. Res. Toxicol. 17, 839−856. *S Supporting Information (11) Haapala, E., Hakala, K., Jokipelto, E., Vilpo, J., and Hovinen, J. (2001) N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chloram- The Supporting Information is available free of charge on the bucil) with 2′-deoxyguanosine. Chem. Res. Toxicol. 14, 988−995. ACS Publications website at DOI: 10.1021/acs.bio- (12) Balcome, S., Park, S., Quirk Dorr, D. R., Hafner, L., Phillips, L., chem.6b01080. and Tretyakova, N. (2004) Adenine-containing DNA-DNA cross-links Time courses for the formation of cross-linked duplexes of antitumor nitrogen mustards. Chem. Res. Toxicol. 17, 950−962. − (13) Dutta, S., Abe, H., Aoyagi, S., Kibayashi, C., and Gates, K. S. B E; iron-EDTA footprinting of duplex C; generation of − a late-forming cross-link in duplex F; sequence specificity (2005) DNA damage by fasicularin. J. Am. Chem. Soc. 127, 15004 15005. of guanine alkylation by HN2, 6, and LNM; generation (14) Millard, J. T., Raucher, S., and Hopkins, P. B. (1990) of the late-forming cross-link following treatment of Mechlorethamine cross-links deoxyguanosine residues at 5′-GNC duplex A with 6 or LNM (PDF) sequences in duplex DNA fragments. J. Am. Chem. Soc. 112, 2459− 2460. ■ AUTHOR INFORMATION (15) Maccubbin, A. F., Caballes, L., Riordan, J. M., Huang, D. H., and Gurtoo, H. L. (1991) A cyclophosphamide/DNA phosphodiester Corresponding Author adduct formed in vitro and in vivo. Cancer Res. 51, 886−892. *E-mail: [email protected]. Phone: (573) 882-6763. Fax: (16) Kohn, K. W., Hartley, J. A., and Mattes, W. B. (1987) (573) 882-2754. Mechanisms of DNA sequence selective alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res. 15, 10531−10549. ORCID (17) Florea-Wang, D., Haapala, E., Mattinen, J., Hakala, K., Vilpo, J., Kent S. Gates: 0000-0002-4218-7411 and Hovinen, J. (2004) Reactions of N,N-Bis(2-chloroethyl)-p-

7039 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article aminophenylbutyric Acid (Chlorambucil) with 2′-Deoxycytidine, 2′- Between Adenine Residues and Abasic Sites in Duplex DNA. J. Am. Deoxy-5-methylcytidine, and Thymidine. Chem. Res. Toxicol. 17, 383− Chem. Soc. 136, 3483−3490. 391. (36) Yang, Z., Johnson, K. M., Price, N. E., and Gates, K. S. (2015) (18) Florea-Wang, D., Haapala, E., Mattinen, J., Hakala, K., Vilpo, J., Characterization of interstrand DNA-DNA cross-links derived from and Hovinen, J. (2003) Reactions of N,N-Bis(2-chloroethyl)-p- abasic sites using bacteriophage φ29 DNA polymerase. Biochemistry aminophenylbutyric Acid (Chlorambucil) with 2′-Deoxyadenosine. 54, 4259−4266. Chem. Res. Toxicol. 16, 403−408. (37) Johnson, K. M., Price, N. E., Wang, J., Fekry, M. I., Dutta, S., (19) Osborne, M. R., and Lawley, P. D. (1993) Alkylation of DNA by Seiner, D. R., Wang, Y., and Gates, K. S. (2013) On the Formation and melphalan with special reference to adenine derivatives and adenine- Properties of Interstrand DNA-DNA Cross-links Forged by Reaction guanine cross-linking. Chem.-Biol. Interact. 89,49−60. of an Abasic Site With the Opposing Guanine Residue of 5′-CAp (20) Bank, B. B. (1992) Studies of chlorambucil-DNA adducts. Sequences in Duplex DNA. J. Am. Chem. Soc. 135, 1015−1025. Biochem. Pharmacol. 44, 571−575. (38) Dutta, S., Chowdhury, G., and Gates, K. S. (2007) Interstrand (21) Price, C. C., Gaucher, M., Koneru, P., Shibakawa, R., Sowa, J. R., crosslinks generated by abasic sites in duplex DNA. J. Am. Chem. Soc. and Yamaguchi, M. (1968) Relative reactivities for monofunctional 129, 1852−1853. nitrogen mustard alkylation of nucleic acid components. Biochim. (39) Gamboa Varela, J., and Gates, K. S. (2015) A Simple, High-Yield − Biophys. Acta, Nucleic Acids Protein Synth. 166, 327 359. Synthesis of DNA Duplexes Containing a Covalent, Thermally- (22) Wijen, J. P. H., Nivard, M. J. M., and Vogel, E. W. (2000) The in Reversible Interstrand Cross-link At a Defined Location. Angew. Chem., vivo genetic activity profile of the monofunctional nitrogen mustard 2- Int. Ed. 54, 7666−7669. chloroethylamine differs drastically from its bifunctional counterpart (40) Gamboa Varela, J., and Gates, K. S. (2016) Simple, high-yield − mechlorethamine. Carcinogenesis 21, 1859 1867. syntheses of DNA duplexes containing interstrand DNA-DNA cross- (23) Yaghi, B. M., Turner, P. M., Denny, W. A., Turner, P. R., ’ links between an N4-aminocytidine residue and an abasic site. Curr. O Connor, C. J., and Ferguson, L. R. (1998) Comparative mutational Protoc. Nucleic Acid Chem. 65, 5.16.1−5.16.15. spectra of the nitrogen mustard chlorambucil and its half-mustard (41) Sczepanski, J. T., Jacobs, A. C., and Greenberg, M. M. (2008) analogue in Chinese hamster AS52 cells. Mutat. Res., Fundam. Mol. − Self-promoted DNA interstrand cross-link formation by an abasic site. Mech. Mutagen. 401, 153 164. J. Am. Chem. Soc. 130, 9646−9647. (24) Dong, Q., Barsky, D., Colvin, M. E., Melius, C. F., Ludeman, L. (42) Sczepanski, J. T., Jacobs, A. C., Majumdar, A., and Greenberg, M., Moravek, J. F., Colvin, M., Bigner, D. D., Modrich, P., and M. M. (2009) Scope and mechanism of interstrand crosslink formation Friedman, H. S. (1995) A structural basis for a phosphoramide ′ − ′ by the C4 -oxidized abasic site. J. Am. Chem. Soc. 131, 11132 11139. mustard-induced DNA interstrand cross-link at 5 -d(GAC). Proc. Natl. (43) Chen, J., Dupradeau, F.-Y., Case, D. A., Turner, C. J., and Acad. Sci. U. S. A. 92, 12170−12174. Stubbe, J. (2007) DNA oligonucleotides with A, T, G and C opposite (25) Ojwang, J. O., Grueneberg, D. A., and Loechler, E. L. (1989) an abasic site: structure and dynamics. Nucleic Acids Res. 36, 253−262. Synthesis of a duplex oligonucleotide containing a nitrogen mustard (44) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular interstrand DNA-DNA cross-link. Cancer Res. 49, 6529−6537. Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (26) Rink, S. M., Solomon, M. S., Taylor, M. J., Rajur, S. B., Plainview, NY. McLaughlin, L. W., and Hopkins, P. B. (1993) Covalent structure of a (45) Gates, K. S. (2000) Mechanisms of DNA damage by leinamycin. nitrogen mustard-induced DNA interstrand cross-link: an N7-to-N7 Chem. Res. Toxicol. 13, 953−956. linkage of deoxyguanosine residues at the duplex sequence 5′- (46) Maxam, A. M., and Gilbert, W. (1980) Sequencing end-labeled d(GNC). J. Am. Chem. Soc. 115, 2551−2557. DNA with base-specific chemical cleavages. Methods Enzymol. 65, (27) Hopkins, P. B., Millard, J. T., Woo, J., Weidner, M. F., Kirchner, − J. J., Sigurdsson, S. T., and Raucher, S. (1991) Sequence preferences of 499 560. (47) Luce, R. A., and Hopkins, P. B. (2001) Chemical cross-linking of DNA interstrand cross-linking agents: Importance of minimal DNA − structural reorganization in the cross-linking reactions of mechloreth- drugs to DNA. Methods Enzymol. 340, 396 412. amine, cisplatin, and mitomycin C. Tetrahedron 47, 2475−2489. (48) Hartley, J. A., Berardini, M. D., and Souhami, R. L. (1991) An (28) Sawyer, G. A., Frederick, E. D., and Millard, J. T. (2004) agarose gel method for the determination of DNA interstrand cross- linking applicable to the measurement of the rate of total and ″second Flanking sequences modulate diepoxide and mustard cross-linking ″ − efficiencies at the 5′-GNC site. Chem. Res. Toxicol. 17, 1057−1063. arm cross-linking reactions. Anal. Biochem. 193, 131 134. (49) Price, C. C. (1958) Fundamental mechanisms of alkylation. Ann. (29) Osborne, M. R., Wilman, D. E. V., and Lawley, P. D. (1995) − Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl)- N. Y. Acad. Sci. 68, 663 668. methylamine. Chem. Res. Toxicol. 8, 316−320. (50) Rutman, R. J., Chun, E. H., and Jones, J. (1969) Observations (30) Bauer, G. B., and Povirk, L. F. (1997) Specificity and kinetics of on the mechanism of the alkylation reaction between nitrogen mustard interstrand and intrastrand bifunctional alkylation by nitrogen and DNA. Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 174, − mustards at a G-G-C sequence. Nucleic Acids Res. 25, 1211−1218. 663 673. (31) Masta, A., Gray, P. J., and Phillips, D. R. (1994) Molecular basis (51) Ulfjohn, A., Kramer, S. P., Dorfman, H., Witten, B., Williamson, of nitrogen mustard effects on transcription processes: role of C., Sass, S., Miller, J. I., and Seligman, A. M. (1964) Duration of the depurination. Nucleic Acids Res. 22, 3880−3886. myelotoxic effects of circulating alkylating agents. Cancer Res. 24, (32) Catalano, M. J., Liu, S., Andersen, N., Yang, Z., Johnson, K. M., 1659−1665. Price, N. A., Wang, Y., and Gates, K. S. (2015) Chemical structure and (52) Varshney, U., and van de Sande, J. H. (1991) Specificities and properties of the interstrand cross-link formed by the reaction of kinetics of uracil excision from uracil-containing DNA oligomers by guanine residues with abasic sites in duplex DNA. J. Am. Chem. Soc. Escherichia coli uracil DNA glycosylase. Biochemistry 30, 4055−4061. 137, 3933−3945. (53) Lindahl, T., Ljunquist, S., Siegert, W., Nyberg, B., and Sperens, (33) Catalano, M. J., Price, N. E., and Gates, K. S. (2016) Effective B. (1977) DNA N-glycosidases: properties of uracil-DNA glycosidase molarity in a nucleic acid controlled reaction. Bioorg. Med. Chem. Lett. from Escherichia coli. J. Biol. Chem. 252, 3286−3294. 26, 2627−2630. (54) Romero, R. M., Rojsittisak, P., and Haworth, I. S. (2013) (34) Price, N. E., Catalano, M. J., Liu, S., Wang, Y., and Gates, K. S. Electrophoretic mobility of duplex DNA cross-linked by mechloreth- (2015) Chemical and structural characterization of interstrand cross- amine at a cytosine-cytosine mismatch pair. Electrophoresis 34, 917− links formed between abasic sites and adenine residue in duplex DNA. 924. Nucleic Acids Res. 43, 3434−3441. (55) Millard, J. T., Weidner, M. F., Kirchner, J. J., Ribeiro, S., and (35) Price, N. E., Johnson, K. M., Wang, J., Fekry, M., Wang, Y., and Hopkins, P. B. (1991) Sequence preferences of DNA interstrand Gates, K. S. (2014) Interstrand DNA−DNA Cross-Link Formation cross-linking agents: quantitation of interstrand crosslink locations in

7040 DOI: 10.1021/acs.biochem.6b01080 Biochemistry 2016, 55, 7033−7041 Biochemistry Article

DNA duplex fragments containing multiple crosslinkable sites. Nucleic Acids Res. 19, 1885−1891. (56) Kumaresan, K. R., Ramaswamy, M., and Yeung, A. T. (1992) Structure of the DNA interstrand cross-link of 4,5′,8-trimethylpsor- alen. Biochemistry 31, 6774−6783. (57) Wilson, D. M., III, and Barsky, D. (2001) The major human abasic endonuclease: formation, consequences and repair of abasic sites in DNA. Mutat. Res., DNA Repair 485, 283−307. (58) Rosa, S., Fortini, P., Karran, P., Bignami, M., and Dogliotti, E. (1991) Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo. Nucleic Acids Res. 19, 5569−5574. (59) Habraken, Y., and Ludlum, D. B. (1989) Release of chloroethyl ethyl sulfide-modified bases by bacterial 3-methyladenine-DNA glycosylases I and II. Carcinogenesis 10, 489−492. (60) Zang, H., and Gates, K. S. (2003) Sequence specificity of DNA alkylation by the antitumor natural product leinamycin. Chem. Res. Toxicol. 16, 1539−1546. (61) Asai, A., Hara, M., Kakita, S., Kanda, Y., Yoshida, M., Saito, H., and Saitoh, Y. (1996) Thiol-mediated DNA alkylation by the novel antitumor antibiotic leinamycin. J. Am. Chem. Soc. 118, 6802−6803. (62) Fekry, M., Szekely, J., Dutta, S., Breydo, L., Zang, H., and Gates, K. S. (2011) Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin. Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dien- one Moiety of the Natural Product Serves As An Atypical DNA Intercalator. J. Am. Chem. Soc. 133, 17641−17651. (63) Nooner, T., Dutta, S., and Gates, K. S. (2004) Chemical properties of the leinamycin 2′-deoxyguanosine adduct. Chem. Res. Toxicol. 17, 942−949. (64) Scharer,̈ O. D. (2005) DNA interstrand crosslinks: natural and drug-induced DNA adducts that induce unique cellular responses. ChemBioChem 6,27−32. (65) Guillet, M., and Boiteux, S. (2003) Origin of endogenous DNA abasic sites in Saccaromyces cerevisiae. Mol. Cell. Biol. 23, 8386−8394. (66) Jian, Y., Lin, G., Chomicz, L., and Li, L. (2015) Reactivity of damaged pyrimidines: formation of a Schiff base intermediate at the glycosidic bond of saturated dihydrouridine. J. Am. Chem. Soc. 137, 3318−3329. (67) Hoehn, S. T., Turner, C. J., and Stubbe, J. (2001) Solution structure of an oligonucleotide containing an abasic site: evidence for an unusual deoxyribose conformation. Nucleic Acids Res. 29, 3413− 3423. (68) Lhomme, J., Constant, J.-F., and Demeunynck, M. (1999) Abasic DNA structure, reactivity, and recognition. Biopolymers 52,65− 83. (69) Lindahl, T., and Nyberg, B. (1972) Rate of depurination of native deoxyribonucleic acid. Biochemistry 11, 3610−3618. (70) Chakravarti, D., Mailander, P. C., Li, K.-M., Higginbotham, S., Zhang, H. L., Gross, M. L., Meza, J. L., Cavalieri, E. L., and Rogan, E. G. (2001) Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H- ras gene. Oncogene 20, 7945−7953. (71) Rinne, M. L., He, Y., Pachkowski, B. F., Nakamura, J., and Kelley, M. R. (2005) N-methylpurine DNA glycosylase overexpression increases alkylation sensitivity by rapidly removing non-toxic 7- methylguanine adducts. Nucleic Acids Res. 33, 2859−2867.

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