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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. Monoadducts 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.

N

can forge DNA−DNA interstrand cross-links in some sequences via reaction of the Ap aldehyde residue with the exocyclic amino groups of nucleobases such as adenine and guanine on the opposing strand of the DNA duplex (Scheme 2).32−40 The cross-linking reactions considered here, involving “true” Ap sites, are distinct from those involving oxidized abasic sites.41,42 Here, we show that Ap sites generated by the nitrogen mustard mechlorethamine (HN2) give rise to interstrand crosslinks of a type not previously associated with this drug. We present gel electrophoretic data that are consistent with an early evolution of the expected drug-bridged cross-links 3 followed by the appearance of Ap-derived cross-links. The evidence is 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

itrogen mustards such as mechlorethamine (HN2) were the first synthetic anticancer drugs1,2 and remain in widespread clinical use.3−6 These bifunctional agents generate aziridinium ions that react with DNA at a variety of locations, including N7-guanine, N3-adenine, N3-cytidine, and the phosphodiester linkages of the backbone (Scheme 1).4,7−21 Monoadducts (1 and 2) at guanine residues are the major DNA alkylation products formed by these drugs (Scheme 1).8,10,11 Interstrand cross-links (3) are generated in much lower yields (1−10% of total adducts) but are generally believed to be the critical lesions responsible for medicinal activity of the nitrogen mustards.4,22,23 Cross-link formation by nitrogen mustards can occur via reactions with two guanine residues in 5′-GNC sequences [3 (Scheme 1)];14,24−28 however, there is also evidence of G-G cross-link formation at 5′-GC sequences as well as G-A and A-A cross-linking at as-yet-undefined sequences.12,26,29 The alkylation of guanine and adenine residues by nitrogen mustards induces the formation of abasic (Ap) sites in genomic DNA (Scheme 1).9,10,30,31 We recently showed that Ap sites © XXXX American Chemical Society

Received: October 21, 2016 Revised: November 22, 2016 Published: November 29, 2016 A

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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.

Scheme 1

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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 as a stock solution in DMF, to give a mixture containing HN2 (1 mM) and labeled DNA in HEPES buffer (50 mM, pH 7) containing 100 mM NaCl and 10% (v/v) DMF that was incubated at 37 °C for 96 h unless otherwise specified. The DNA was ethanol-precipitated from the reaction mixture,44 resuspended in formamide loading buffer,44 and loaded onto a 20% denaturing polyacrylamide gel, and the gel was electrophoresed for 5 h at 1600 V. The amount of radiolabeled DNA

These results provide the first characterization of Ap-derived cross-link formation at sequences in which a cytosine residue directly opposes the Ap site. The ability of such sites to generate interstrand cross-links was by no means certain, because the nature of the directly opposing base can exert significant effects on the structures of Ap-containing duplexes.43 Cross-linking reactions of this type could be important in biology and medicine because Ap sites with directly opposing B

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containing double-stranded DNA (∼400000 cpm) was incubated in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C for 120 h. The DNA was ethanol precipitated, suspended in formamide loading buffer, and resolved on a 2 mm thick 20% denaturing polyacrylamide gel. The late-forming cross-linked duplex band was visualized using X-ray film, the band cut out of the gel, and the gel slice crushed, and the gel pieces were vortex-mixed in elution buffer (200 mM NaCl and 1 mM EDTA) at room temperature for at least 1 h. The mixture was filtered through a Poly-Prep column to remove gel fragments, and the residue was ethanol precipitated, redissolved in water, and mixed with 2× oxidation buffer [10 μL of a solution composed of 20 mM sodium phosphate (pH 7.2), 20 mM NaCl, 2 mM sodium ascorbate, and 1 mM H2O2]. To this mixture was added a solution of iron-EDTA [2 μL of 70 mM EDTA and 70 mM (NH4)2Fe(SO4)2·6H2O] to start the reaction, and the mixture was vortexed briefly and incubated at room temperature for 5 min before addition of a thiourea stop solution (10 μL of a 100 mM solution in water). Hydroxyl radical footprinting reactions, Maxam−Gilbert G reactions, and Maxam−Gilbert A+G reactions were performed on the labeled duplex to generate marker lanes.46 The resulting DNA fragments were analyzed using gel electrophoresis as described above.

in each band on the gel was measured by phosphorimager analysis. The time course for the formation of the late-forming cross-link was determined by incubating a solution containing labeled DNA (approximately 100000 cpm) and HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) and HN2 (1 mM) at 37 °C. At specified time points, aliquots (3 μL) were removed and formamide loading dye was added followed by freezing at −20 °C, and gel electrophoretic analysis as described above. For cross-link formation by LNM, all conditions were identical, except LNM was introduced as a stock solution in acetonitrile, the final concentration of LNM was either 100 or 500 μM, and β-mercaptoethanol (0.5 or 2.5 mM) was added to initiate the DNA alkylation reaction.45 Representative Procedure for Preparation of Duplexes Containing Authentic Ap-Derived Cross-Links. A single-stranded, uracil-containing 2′-deoxyoligonucleotide was 5′-labeled using standard procedures,44 annealed with its complementary strand, and treated with the enzyme UDG (50 units/mL, final concentration) to generate the Ap site. The UDG enzyme was removed by phenol/chloroform extraction and the DNA ethanol precipitated. The Ap-containing duplexes were incubated in a buffer composed of HEPES (50 mM, pH 7) containing NaCl (100 mM) at 37 °C for 120 h unless otherwise specified. The DNA was ethanol precipitated, resuspended in formamide loading buffer, and loaded onto a 20% denaturing polyacrylamide gel that was electrophoresed for 5 h at 1600 V. The amount of radiolabeled DNA in each band on the gel was measured by phosphorimager analysis. The time course for the formation of the dA-Ap cross-link was determined by incubating a solution containing labeled DNA (approximately 100000 cpm) and HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) at 37 °C. At specified time points, aliquots (3 μL) were removed, formamide loading dye was added, and the sample was frozen at −20 °C and analyzed by gel electrophoresis as described above. Sequence Specific DNA Alkylation by HN2, 2(Chloroethyl)ethylsulfide (6), and LNM. Typical alkylation reaction mixtures contained HN2 (1 mM) and a 32P-labeled DNA duplex (Figure 1) in a buffer composed of HEPES (50 mM, pH 7), NaCl (100 mM), and DMF [10% (v/v)] and incubated at 37 °C for 2 h. The DNA was ethanol precipitated, redissolved in aqueous piperidine (50 μL of a 1 M solution), and incubated at 90 °C for 25 min (Maxam−Gilbert workup).46 The solution was frozen on dry ice, lyophilized for 40 min in a SpeedVac Concentrator at 37 °C, redissolved in 20 μL of water, and evaporated again. The dried DNA fragments were dissolved in formamide loading buffer, loaded onto a 20% polyacrylamide denaturing gel, and electrophoresed at 1400 V for 5 h. The amount of radioactivity in the resolved DNA fragments was quantitatively analyzed with a phosphorimager. For alkylation by LNM, all conditions were identical except that LNM was introduced as a stock solution of acetonitrile (instead of DMF) and 50 μM LNM and 500 μM βmercaptoethanol were employed. For alkylation by the sulfur mustard 6, a concentration of 500 μM was used. Hydroxyl Radical Footprinting of a Duplex CContaining Authentic Ap-Derived Cross-Link. We followed literature protocols for the footprinting of cross-link duplex C.35,37,42,47 In this experiment, the strand opposing the Ap-containing oligonucleotide was 5′-labeled using the standard procedure.44 Labeled DNA was annealed with the uracilcontaining complementary strand and the duplex treated with UDG to generate the abasic site as described above. The Ap-

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RESULTS AND DISCUSSION Treatment of Duplex DNA with HN2 Leads to Generation of Distinct Early-Forming and Late-Forming Cross-Links. It is well established that treatment of duplex DNA with HN2 can lead to the generation of interstrand crosslinks that appear as slow-migrating bands on denaturing polyacrylamide gels (located above the full-length singlestranded DNA in the denaturing gels presented here).14,26−28 Indeed, we found that treatment of duplex A (Figure 1) with HN2 (1 mM) in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) and DMF [10% (v/v)] at 37 °C, followed by electrophoretic analysis of the 32P-labeled DNA fragments on a denaturing 20% polyacrylamide gel, revealed several distinct slowly migrating bands (Figure 2). The formation of multiple cross-linked species has previously been observed in duplexes containing both 5′-GNC and 5′-GC sites treated with HN2.14,26,28 Most intriguingly, we found that the two major cross-link bands generated by treatment of duplex A with HN2 displayed very different formation time courses and gel mobilities (Figure 2). A major “early-forming” cross-link band was immediately evident. The yield of this band peaked at the earliest time point (2 h, 5% yield), and then the intensity decreased over the remainder of the experiment (Figure 2B). This behavior was consistent with that expected for the typical drug-linked G-G cross-link in two key regards: (i) Interstrand DNA cross-link formation by HN2 is rapid,30,48−50 and (ii) depurination of HN2 adducts, occurring with a half-life of ∼9 h at 37 °C, was expected to cause spontaneous “unhooking” of the cross-link (with a corresponding disappearance of the crosslink band).10,30,31 We also observed a “late-forming” cross-link band that became visible after 12 h and predominant after 48 h [4% yield (Figure 2)]. It seemed unlikely that this could be a drug-linked cross-link because the chloroethyl groups of HN2 in both DNA monoadducts [1 (Scheme 1)] and free HN2 hydrolyze rapidly (t1/2 = 1−30 min).29,48−51 On the other hand, in light of our recent studies,32−40 it was reasonable to consider that a buildup of Ap sites resulting from depurination of HN2 adducts (1−3) C

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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 crosslinked 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 32Plabeled 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 fulllength 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′deoxyoligonucleotides and the upper bands cross-linked DNA. Lanes 1 and 2 show 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 48 and 96 h, respectively, prior to sequencing gel analysis. Lanes 3−6 show authentic dA-Ap cross-links in duplexes C, B, E, and D, respectively. The 32P-labeled 2′-deoxyoligonucleotides were resolved on a 20% polyacrylamide denaturing gel, and the radioactivity in each band was quantitatively measured by phosphorimager analysis.

might permit the generation of Ap-derived cross-links. In the following sections, we provide evidence that the late-forming cross-link band seen in Figure 2A is a dA-Ap cross-link resulting from alkylation-induced generation of an Ap site at position 2 of duplex A (Figure 1). A DNA Duplex Containing an Authentic dA-Ap CrossLink at Position 2 Co-Migrates with the Late-Forming Cross-Link Band Generated by Treatment of Duplex A with HN2. The formation of Ap-derived cross-links has been observed in two different sequence motifs: 5′-CAp/5′-AG and 5′-ApT/5′-AA (the cross-linked base is underlined; see Figure 1 for images of the cross-link sequence motifs).35,37,38 In the dG-Ap cross-link, the cross-linking guanine residue is offset one base to the 5′-side of the Ap site, while in the dA-Ap cross-link, the cross-linking adenine residue is offset one base to the 3′side of the Ap site.35,37,38 In all of the Ap-derived cross-links described to date, an adenine residue has been located directly opposing the Ap site and the dA-Ap cross-linking motif generates cross-link yields substantially greater than that of the dG-Ap motif.35,37,38 Recognition that alkylation-induced Ap sites have the potential to generate interstrand DNA−DNA cross-links, combined with the knowledge that guanine residues are the primary alkylation sites for HN2, drew our attention to 5′-GT sequences in duplex A as potential progenitors of HN2-induced dA-Ap cross-links. Specifically, alkylation at these sites, followed by depurination of the resulting alkylguanine residue, could allow dA-Ap cross-link formation at the resulting 5′-ApT site.35 In duplex A, there are four such sites involving the guanine residues at positions 1, 2, 6, and 8 (Figure 1). As an initial test of whether the late-forming cross-link band seen in Figure 2

these duplexes varied substantially, with values of 5, 30, 14, and 2%, for duplexes B−E, respectively (Figure S1). Interestingly, cross-linked duplexes B−E displayed distinct gel mobilities (Figure 3). It has previously been observed that the location of cross-links, with respect to the end of the DNA duplex, can dramatically alter mobility in denaturing gels.54−56 In general, our observations follow the trends reported in two earlier studies involving drug-derived cross-links, in which duplexes containing cross-links near their ends were shown to migrate faster in denaturing gels than those containing cross-links near their center.54,55 More important in the context of the current studies is the fact that cross-linked duplexes B, D, and E derived from Ap sites at positions 1, 6, and 8, respectively, did not comigrate with the major late-forming cross-link band produced by treatment of duplex A with HN2. On the other hand, the cross-link derived from the Ap site at position 2 (duplex C) did co-migrate with the major late-forming band from the HN2 reaction (Figure 3). We carefully characterized cross-link formation in the authentic Ap-containing duplex C. We found that this crosslink was generated in an equilibrium yield of 30%, with an apparent formation half-time of 24 h in HEPES buffer (50 mM, D

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Biochemistry pH 7) containing NaCl (100 mM) at 37 °C (Figure S1). IronEDTA 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 crosslink 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 lateforming 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. Electrophoretic analysis of the 32P-labeled DNA fragments on a denaturing 20% polyacrylamide gel showed that guanine residues at positions 2−4 and 6−8 were all alkylated by HN2 [alkylation at position 1 could not be accurately measured because the band was not resolved from full-length DNA, and position 5 could not be measured because the band migrated off the end of the gel (Figure 4 and Figure S4)]. Nonetheless, the results confirmed that position 2 was alkylated in substantial yield by HN2. To provide additional evidence that the late-forming crosslink band generated by treatment of duplex A with HN2 was derived from an Ap site in the duplex, we examined the effects exerted upon the cross-linking reaction by two different treatments that modify Ap sites in duplex DNA. Specifically, we examined the effects of apurinic endonuclease (APE), an enzyme that cleaves on the 5′-side of Ap sites,57 and methoxyamine (MX), a reagent that forms a stable oxime derivative at Ap sites and inhibits the formation of Ap-derived cross-links.35,58 In separate experiments, these reagents were added after 24 h to reaction mixtures containing HN2 and duplex A. These Ap-modifying reagents inhibited generation of the late-forming cross-link band, consistent with the notion that HN2-induced generation of Ap sites was central to the production of the late-forming band (Figure 5). As expected, MX prevented both cross-linking and spontaneous strand cleavage by capping the Ap aldehyde residues as an inert oxime (lane 2, Figure 5).35 APE inhibited cross-link formation while generating the anticipated strand cleavage at Ap sites resulting from spontaneous depurination of HN2−guanine adducts at G2−G4 (lane 3, Figure 5). The multiple bands generated by APE may reflect both cleavage at Ap sites generated by depurination of alkylated adenine residues and the result of the enzyme’s 3′-exonuclease activity on the initial cleavage products. Previous work by Osborne et al. showed that N3alkyladenine residues are spontaneously released from DNA treated with HN2, presumably with concomitant generation of abasic sites.29

Figure 4. Alkylation of guanine residues in duplex A by HN2. 32Plabeled oligonucleotide duplex A was incubated with HN2 in HEPES buffer (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 the labeled fragments on a 20% denaturing polyacrylamide gel. Labeled DNA was visualized by phosphorimager analysis. Lane 1 shows an untreated duplex A. Lane 2 shows a Maxam−Gilbert G reaction on the labeled strand of duplex A. Lane 3 shows an A+G reaction on the labeled strand of duplex A. Lane 4 shows DNA treated with HN2 (1 mM) followed by Maxam−Gilbert workup. The 32Plabeled 2′-deoxyoligonucleotides were resolved on a sequencing gel and visualized by phosphorimager analysis.

Generation of the Abasic-Derived Cross-Link by Monoalkylating Agents 2-Chloro-N,N-diethylethanamine (5), 2-(Chloroethyl)ethylsulfide (6), and Leinamycin (7). In the reaction pathway proposed here, formation of the dA-Ap cross-link following treatment of duplex A with HN2 is dependent upon the alkylating properties of HN2, but not dependent upon the cross-linking properties of the drug. Accordingly, the monofunctional nitrogen mustard 2-chloroN,N-diethylethanamine 5 and other monofunctional DNAalkylating agents that target guanine residues should be competent to induce the late-forming cross-link band in duplex A. With this in mind, we examined the ability of two wellknown21,59 monofunctional DNA-alkylating agents, 2-chloroN,N-diethylethanamine (5) and (2-chloroethyl)ethylsulfide (6), to induce formation of the dA-Ap cross-link band in duplex A. N7-Alkylguanine residues are major DNA adducts generated by both of these agents (Figure S4).21,59,60 We found that treatment of duplex A with either 5 (1 mM) or 6 (1 mM) in HEPES buffer (50 mM, pH 7) containing NaCl (100 mM) for 96 h at 37 °C did indeed produce the cross-linked duplex C in yields comparable to that achieved by bifunctional alkylator HN2 (Figure 6 and Figure S5). We also examined the ability of the Streptomyces-derived anticancer natural product leinamycin (LNM) to generate the late-forming dA-Ap cross-link in duplex A. LNM seems wellE

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(Scheme 3).45,61 (ii) LNM selectively alkylates 5′-GT sequences that support the formation of dA-Ap crossScheme 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 (50 mM, pH 7) containing 100 mM NaCl at 37 °C, and after 24 h, CH3ONH2 (MX) was added to a final concentration of 2 mM and the mixture incubated for an additional 72 h. Lane 3 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, and after 24 h, APE was added and the mixtures were incubated for an additional 72 h. The 32P-labeled 2′-deoxyoligonucleotides were resolved on a sequencing gel and visualized by phosphorimager analysis.

links.60,62 (iii) LNM−guanine adducts undergo unusually rapid depurination to yield abasic sites.63 We found that treatment of duplex A with LNM (100 μM) and 2mercaptoethanol (500 μM) generated a band that co-migrated with cross-linked duplex C (Figure S6). Interestingly, treatment of duplex A with higher concentrations of LNM generated a complex mixture of slow-migrating bands that did not necessarily co-migrate with authentic cross-linked duplexes B−E (Figure S7). The identity of these species remains to be determined.

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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 residue, and formation of a dA-Ap cross-link. In our experiments, Ap-derived cross-links were produced in yields comparable to those of the drug-linked cross-links. The results indicate that care must be exercised when assigning the identity of slow-migrating cross-link bands in gel electrophoretic analyses of mustard-treated DNA; not all slow-migrating bands are necessarily drug-linked cross-links. Following treatment of duplex DNA with HN2, the gel electrophoretic data were consistent with early evolution of the expected drugbridged cross-links, followed by the appearance of Ap-derived cross-links. It seems clear that HN2-induced formation of Apderived cross-links does not occur equally well at all 5′-GT sites in duplex A. The major site of cross-link formation involves alkylation and Ap generation at G2 and cross-linking with A9 (Figure 1). The observed yields of Ap-derived cross-link at any

Figure 6. Treatment of duplex A with the monofunctional nitrogen mustard 5 generates the late-forming dA-Ap cross-link. Duplex A was incubated with compound 5 (1 mM) in HEPES buffer (50 mM, pH 7) containing 100 mM NaCl and 10% (v/v) DMF at 37 °C, and at 0, 2, 4, 8, 12, 24, 48, 72, and 96 h, aliquots were removed from the reaction mixture and frozen at −20 °C prior to gel electrophoretic analysis (lanes 1−9, respectively). Lane 10 shows the authentic dA-Ap containing duplex C. Labeled DNA in the gel was quantitatively detected by phosphorimaging analysis. The bottom band in the gel image is the full-length labeled 2′-deoxyoligonucleotide, and the slowmoving upper band corresponds to cross-linked DNA.

suited for the generation of Ap-derived cross-links in DNA for three reasons. (i) The reaction of LNM with thiols generates a DNA-binding episulfonium ion 8 that selectively and efficiently alkylates position N7 of guanine residues in duplex DNA F

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given 5′-GT site must reflect an interplay of HN2 alkylation efficiency, depurination of the alkylguanine residue, and the inherent potential for Ap-derived cross-link formation at that sequence in the duplex. Generation of the dA-Ap cross-link by HN2 relies upon the DNA alkylating properties of the drug and not its bifunctional cross-linking properties. Accordingly, we found that formation of the dA-Ap cross-link was similarly induced by three different monofunctional alkylating agents that target guanine residues in duplex DNA, 2-chloro-N,N-diethylethanamine (5) and (2chloroethyl)ethylsulfide (6), and LNM. Previously characterized Ap-derived cross-links have been located at sequences in which an adenine residue directly opposes the Ap site.32−40 In biological systems, Ap sites with opposing adenine residues arise via the enzymatic removal of misincorporated 2′-deoxyuridine by the base excision repair enzyme UDG.65 This may be a major source of Ap sites in eukaryotic cells.65 The loss of damaged thymine residues also can give rise to Ap sites with a directly opposing adenine residue.66 The work described here explored the properties of Ap sites arising from another important cellular process, the depurination of guanine and alkylguanine residues. These events give rise to Ap sites opposed by a cytosine residue. At the outset of this work, it was uncertain whether Ap sites opposed by a cytosine residue could engage in interstrand cross-linking reactions. This uncertainty was due to the fact that the nature of the base directly opposing an Ap site can exert significant effects on the structure of an Ap-containing duplex.43,67,68 For example, the Ap residue can adopt an extrahelical conformation when opposed by a pyrimidine residue.43,67,68 Nonetheless, the results reported here show for the first time that Ap sites with directly opposing cytosine residues are competent to form dA-Ap cross-links. Indeed, formation of slow-migrating cross-link bands was easily detected in each of the Ap-containing duplexes B−E, with the equilibrium cross-link yields ranging from 2 to 30%. Given that Ap sites with directly opposing cytosine residues occur frequently in genomic DNA via spontaneous or enzymatic depurination of guanine and N7-alkylguanine residues,9,10,63,69−71 the results provide an additional impetus to examine the occurrence and consequences of Ap-derived crosslinks in cellular DNA.

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We thank the National Institute of Environmental Health Sciences of the National Institutes of Health for support of this work (ES 021007). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS Ap, DNA abasic site; dA, 2′-deoxyadenosine; dG, 2′deoxyguanosine; UDG, uracil DNA glycosylase; T4 PNK, T4 DNA polynucleotide kinase; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; Tris, tris(hydroxymethyl)aminomethane; HN2, mechlorethamine; LNM, leinamycin; MX, methoxyamine; APE, apurinic endonuclease; nt, nucleotide.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b01080. Time courses for the formation of cross-linked duplexes B−E; iron-EDTA footprinting of duplex C; generation of a late-forming cross-link in duplex F; sequence specificity of guanine alkylation by HN2, 6, and LNM; generation of the late-forming cross-link following treatment of duplex A with 6 or LNM (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (573) 882-6763. Fax: (573) 882-2754. ORCID

Kent S. Gates: 0000-0002-4218-7411 G

DOI: 10.1021/acs.biochem.6b01080 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

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