Action of Bleomycin on Structural Mimics of Intermediates in DNA

José A da Costa-Nunes , Cláudio Capitão , Jaroslav Kozak , Pedro Costa-Nunes , Gloria M Ducasa , Olga Pontes , Karel J Angelis. BMC Plant Biology 2...
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Chem. Res. Toxicol. 1998, 11, 1580-1585

Action of Bleomycin on Structural Mimics of Intermediates in DNA Double-Strand Cleavage Kwabena Charles and Lawrence F. Povirk* Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 Received June 30, 1998

Bleomycin-induced cleavage was examined in several nicked, gapped, or intact duplex DNA substrates, including a structure designed to mimic a proposed singly nicked intermediate in double-strand cleavage. This nicked structure appeared to correctly target the second cleavage event in the complementary strand, resulting in a blunt-ended double-strand break, similar to that induced directly by bleomycin alone in an intact duplex of the same sequence. A onebase-gapped structure was markedly less efficient in correctly targeting bleomycin attack in the complementary strand. The results are consistent with a model of bleomycin-induced doublestrand cleavage in which the nick formed by the initial bleomycin attack serves to target secondary attack to a specific position in the complementary strand, resulting in a doublestrand break with a defined geometry.

Introduction About 10% of the DNA strand breaks induced by the antitumor antibiotic bleomycin are double-strand breaks (1, 2). Because the kinetics of double-strand cleavage are strictly single-hit (1, 3), it has been inferred that a single bleomycin molecule probably effects cleavage of both strands in turn, without fully dissociating from the DNA between the two cleavage events (3-6). Moreover, because typically only one of the two cleavage sites (designated the “primary” site) follows the canonical G-Py specificity of bleomycin-induced single-strand cleavage, it has been proposed that the position of “secondary” cleavage is determined primarily by the local perturbation of DNA structure resulting from cleavage at the primary site (4, 7). At the chemical level, bleomycin-induced cleavage of each DNA strand involves an oxidative fragmentation of deoxyribose, which results from hydrogen abstraction, followed by O2 addition, at C-4′ (6, 8-10). The base and deoxyribose carbons 1′-3′ are released as a basepropenal (base-CHdCHCHO), leaving a one-base gap with a 5′-phosphate terminus, and with the 4′ and 5′ carbons still attached as a 3′-phosphoglycolate (PO4CH2COO-) terminus (Figure 1). Although the precise order of the events involved in bleomycin-induced doublestrand cleavage has not been unambiguously determined, it is known that release of the base-propenal is much slower than either single- or double-strand cleavage (11, 12). This finding implies that even if strand breakage at the primary site consistently occurs before bleomycin attack at the secondary site, secondary attack (i.e., C-4′ abstraction at the secondary site) almost certainly occurs before base-propenal release at the primary site. More* To whom correspondence should be addressed: Medical College of Virginia, P.O. Box 980230 MCV Station, Richmond, VA 23298. Telephone: (804) 786-9640. Fax: (804) 371-8079. E-mail: LPOVIRK@ gems.vcu.edu. For delivery by means other than U.S. Postal Service, use the following address: Medical College of Virginia, Sanger Hall, Rm. 6-016, 1101 E. Marshall St., Richmond, VA 23298.

over, NMR data strongly suggest that this transitory attachment of the base to the cleaved strand is through the 5′- rather than to the 3′-terminus (12). Thus, the putative singly nicked intermediate that is the substrate for secondary attack may be a duplex having a break at the primary site, with a phosphoglycolate on the 3′-end of the break, and the base of the target nucleotide still attached to the 5′-end via a labile three-carbon sugar fragment (Figure 1b). In this report, we present data on bleomycin-induced cleavage of various synthetic oligomeric substrates, including intact duplex DNA and mimics of the putative singly nicked intermediates. The results are consistent with the proposal that such a singly nicked intermediate targets the second cleavage event that produces a doublestrand break.

Experimental Procedures Blenoxane (Bristol, Syracuse, NY), the clinical mixture containing primarily bleomycins A2 and B2, was dissolved in distilled H2O at 6 mM, and Fe(III)‚bleomycin was prepared by addition of equimolar ferric ammonium sulfate as described previously (13). Oligonucleotides, obtained from Integrated DNA Technologies (Coralville, IA), were 5′-32P-end-labeled with [γ-32P]ATP (6000 Ci/mmol, New England Nuclear, Boston, MA) and T4 polynucleotide kinase according to standard procedures (14), and were purified by denaturing polyacrylamide gel electrophoresis followed by HPLC (15). A 3′-phosphoglycolate-terminated 9-mer was prepared by bleomycin-induced cleavage of a partial duplex (16), and similarly purified. Annealing reaction mixtures in 50 mM 4-(2-hydroxylethyl)1-piperazineethanesulfonic acid/NaOH (pH 8) contained 0.3 µM labeled 18-mer, 0.4 µM unlabeled complementary oligomer(s), 0.1 M NaCl, and 0.1 mM EDTA and were assembled at 22 °C and cooled for 10 min on ice. The annealed DNA was then diluted 5-fold into the same buffer containing 50 µg/mL calf thymus DNA (to ensure that the overall drug/DNA ratio remained approximately constant) and 1 mM H2O2. Samples were placed on ice, and then the reaction was initiated by addition of Fe(III)•bleomycin to a final concentration of 0-15

10.1021/tx980154g CCC: $15.00 © 1998 American Chemical Society Published on Web 11/13/1998

Intermediates in DNA Cleavage by Bleomycin

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1581 µM. Following treatment for 1 h, 5 µL of each sample was added to 5 µL of formamide containing 20 mM EDTA and the mixture immediately loaded onto a 24% denaturing polyacrylamide sequencing gel. The gels (40 cm × 30 cm × 0.8 cm) were run for 6 h at 50 W, covered with cellophane wrap, and subjected to quantitative phosphorimage analysis. The cleavage frequency was calculated as the phosphorimage intensity of each band divided by the total intensity at and above that band on the gel. Exclusion of any faster-migrating radioactivity from the denominator in these calculations takes into account the fact that cleavage at a particular site will not be detected in any individual molecules which have sustained an additional break closer to the labeled end. Because samples treated with 15 µM bleomycin often showed nearly complete depletion of the fulllength fragment, only the 5 and 10 µM samples were used for analysis of cleavage frequency.

Results

Figure 1. Steps in bleomycin-induced DNA double-strand cleavage. Abstraction from C-4′ at the primary site (left strand) is followed by oxygen addition, single-strand cleavage by sugar fragmentation, and regeneration of an activated form of bleomycin, which then effects a similar attack in the complementary strand. Eventually, two base-propenals are released, and a double-strand break with 5′-phosphate and 3′-phosphoglycolate termini is formed. However, since base-propenal release is slow with respect to strand cleavage, the substrate for the second hydrogen abstraction may be a nicked molecule with the base of the primary target nucleotide still attached to the 5′-end of the nick.

Action of Bleomycin on Nicked and Gapped Substrates. Previous work showed that, in bleomycininduced DNA degradation, G in the sequence GGCG is a nearly pure secondary site; that is, cleavage at this site occurs only in the context of primary cleavage at the CGCC site directly opposite (4). This sequence was incorporated into an 18-base duplex containing three primary cleavage sites in the GGCG strand so that these sites could serve as internal controls for assessing cleavage specificity. Bleomycin-induced cleavage was examined in various intact, nicked, and gapped duplexes based on this sequence, as well as in several half-duplexes. To examine the DNA cleavage frequency at a temperature (0 °C) where the stable oligomeric duplexes would form, activated bleomycin was generated by combining Fe(III)‚ bleomycin and H2O2 (6). The phosphorimage of a denaturing gel from a representative experiment is shown in Figure 2, and pooled data from several such experiments are summarized in Figure 3. When the intact 16-mer duplex was treated with bleomycin, cleavage in the GGCG-containing strand was largely restricted to the predicted primary (...AGTGGCGTTC...) and secondary (...AGTGGCGTTC...) sites (Figure 2a). As expected, the cleavage frequency was greatly reduced in the labeled single strand alone (Figure 3a), or in a partial duplex containing only bases to the 5′-side of the putative CGCC primary site (Figure 3b; note that for each site, the relative cleavage frequency is normalized to the cleavage frequency at the corresponding site in the intact duplex). The nicked duplex in Figure 3d was designed to mimic the putative singly nicked intermediate in bleomycininduced double-strand cleavage (see Figure 1b); it differs from that intermediate only in having a 3′-hydroxyl rather than a 3′-phosphoglycolate at the primary break site, and an intact deoxyribose in place of the labile threecarbon species of the intermediate. Although there was no detectable bleomycin attack at the primary (CGCC) site in the nicked strand of this substrate (as assessed using a duplex labeled at the 3′-end of the nicked strand; data not shown), substantial cleavage nevertheless occurred at the secondary (GGCG) site in the unnicked strand. Indeed, the relative cleavage frequency at this secondary site in the nicked duplex was 50% greater than that in the intact unnicked duplex. Thus, there were significant differences in the action of bleomycin on the nicked versus the intact substrate, and those differences are consistent with the nick at the primary site serving

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Figure 2. Sequencing gels showing fragmentation of a DNA duplex (a), half-duplexes (b and c) and nicked (d) and gapped (e) duplexes of the same sequence, induced by 0, 5, 10, and 15 µM bleomycin, as indicated. The label in all cases was at the 5′-end of the top strand. Dotted lines match 3′-phosphate-terminated Maxam-Gilbert markers with the corresponding bleomycin-induced 3′phosphoglycolate fragments, which migrate slightly faster.

to target cleavage to the secondary site in the unnicked strand. However, targeting was not 100% efficient; i.e., there was still substantial cleavage at two of the three primary sites in the unnicked strand. A partial duplex containing only the half-strand 3′ to the nick (Figure 3c) was damaged with a pattern similar to that of the fully nicked duplex (Figure 3d). However, shortening the duplex region by a single base (Figure 3e) dramatically decreased the cleavage frequency at the secondary GGCG site (as well as at the adjacent GGCG site), suggesting that the 5′-terminal deoxycytidine nucleotide plays a major role in directing the putative secondary cleavage. A one-base-gapped substrate (similar to the final structure of a bleomycin-induced singlestrand break after base-propenal release) also showed substantially less cleavage at the GGCG site than did the nicked substrate (compare d and e in Figure 2 and d and f in Figure 3). Substrates with 3′-Phosphoglycolate Termini. The putative singly nicked double-strand cleavage intermediate (Figure 1b) is expected to have a 3′-phosphoglycolate at the nick site, rather than the 3′-hydroxyl of the substrate in Figure 3d. To determine whether this difference would influence the degree or the specificity of apparent targeting of bleomycin attack, the 3′-phosphoglycolate 9-mer pCGAGGAACG was generated by bleomycin-induced cleavage of the partial duplex pCGAGGAACGCGTCCGGC‚GCCGGACGCG (16) and

purified by gel electrophoresis and HPLC. The purified oligomer was then used to prepare a nicked substrate identical to that in Figure 3d, but with a nick bearing a 3′-phosphoglycolate terminus. While there was substantial cleavage at the GGCG putative secondary site in this substrate (Figure 4), the extent of cleavage was no greater than that for the substrate with a 3′-hydroxyl at the nick, suggesting that the additional -2 charge of the phosphoglycolate did not greatly affect the specificity of bleomycin for this substrate. Effect of a Guanosine f Inosine Substitution at the Primary Site. The exocyclic N2-amino group of guanine 5′ to a cleavage site is thought to play a critical role in targeting bleomycin cleavage to GPy sequences (17). Thus, as expected, substitution of inosine for guanosine at the primary site (CGCC f CICC) reduced the cleavage frequency at the target cytosine by approximately 10-fold (data not shown). It also reduced the cleavage frequency at the corresponding secondary site in the complementary strand (GGCG) by approximately 5-fold (Figure 5), confirming the dependence of secondary cleavage on interactions at the primary site. However, in the case of a nicked complex with inosine substitution at the same primary site, the cleavage frequency at the secondary GG site in the nicked duplex was comparable to that seen in the unsubstituted nicked duplex (Figure 2d) or in the intact unsubstituted duplex (Figure 2a). This result provides additional evidence that, in the nicked

Intermediates in DNA Cleavage by Bleomycin

Figure 3. Quantitative analysis of relative cleavage rates of the various nicked and gapped duplexes and half-duplexes. The cleavage frequency was normalized to that seen in an intact duplex, the full sequence of which is shown at the top of the figure. The label in all cases was at the 5′-end of the top strand. Values are the average ( SE of five to seven independent samples (except b, three samples).

complex, targeting to the secondary site was not dependent on prior targeting to the primary site.

Discussion The critical event in bleomycin-induced DNA degradation is the abstraction of hydrogen from C-4′ of deoxyribose, probably by a transient high-valence iron-oxo complex of bleomycin (6, 9, 10). Under normal conditions, about half of the resulting C-4′ free radicals react with O2 to form peroxyl radicals, which in turn decompose to produce backbone cleavage and, eventually, release of the base and carbons 1′-3′ as a base-propenal (baseCHdCHCHO) (Figure 1). Double-strand cleavage is thought to occur when the high-valence complex is regenerated, probably by reaction between the putative peroxyl free radical on C-4′ and the immediate bleomycin product of the initial abstraction (6, 7, 18). Bleomycin presumably then undergoes a conformational rearrangement to effect a chemically identical attack in the complementary strand, resulting in a double-strand break. The position of this secondary cleavage is virtually always either directly opposite the primary cleavage or on a one-base 5′-stagger, with the choice between these two positions being uniquely determined by a hierarchy of sequence-dependent selection rules (4, 7, 18). The dependence of cleavage at the secondary (generally nonGPy) site on prior cleavage at a primary (GPy) site in the complementary strand (4) has been confirmed by the findings that specific suppression of primary cleavage by deuterium incorporation at C-4′ of a primary site (18),

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or a guanine f isobutyrylguanine (13) or guanine f inosine (Figure 5) substitution immediately 5′ to primary site, results in a coordinate inhibition of cleavage at the corresponding secondary site as well. The finding that the position of secondary cleavage is so strictly constrained by the position of primary cleavage raises the possibility that it is the presence of the primary break, and the resulting perturbation in DNA structure, that targets cleavage to a specific secondary site. Support for this view is provided by the apparent targeting of bleomycin-induced cleavage to certain other perturbations in DNA structure, such as single-base bulges (19). Previously, Keller and Oppenheimer (20) examined bleomycin-induced cleavage of various synthetic duplexes containing a one-base gap with 3′- and 5′-phosphate termini, a structure designed to mimic the fully mature bleomycin-induced strand break (from which it differs only in having a 3′-phosphate rather than a 3′-phosphoglycolate). Such a gap, when positioned so it replaces A in the sequence GGACG in a 14-mer duplex, was found to strongly target cleavage to the GT site directly opposite, resulting in a blunt-ended double-strand break. However, in other sequences with similar gaps (at C in the sequence CGCC, for example), bleomycin treatment resulted almost exclusively in double-strand breaks with one-base 3′-overhangs, a break specificity never induced by bleomycin directly (4). Burger et al. (11) showed that the bleomycin-induced decrease in DNA viscosity (presumably reflecting doublestrand cleavage) occurred much faster than base-propenal release. These results imply that the primary break is not fully mature when secondary cleavage occurs, but rather that the base of the nucleotide sustaining primary attack would still be attached to the DNA at that time. NMR studies strongly suggest that this base is attached to the 5′- rather than to the 3′-end of the break, presumably by a labile three-carbon deoxyribose fragment (12), the other two carbons being linked to the 3′-end as a phosphoglycolate (Figure 1b). Thus, the singly cleaved intermediate in double-strand cleavage is expected to be a nicked rather than a gapped duplex. A mimic of such a structure (differing only in having an intact deoxyribose in place of the three-carbon fragment at the primary cleavage site, and a 3′-hydroxyl rather than a 3′ phosphoglycolate) was found to sustain significant cleavage at the G in GGCG, indicating that blunt-ended breaks were formed just as in direct bleomycin-induced doublestrand cleavage. However, whereas in the intact duplex cleavage at GGCG is only detected in the context of directly opposed cleavage at CGCC, there was no damage at the CG∧CC site in the nicked substrate (where ∧ indicates the position of the nick). This result raises the possibility that, both in the bona fide intermediate and in the synthetic mimic, it was the presence of the nick that targeted cleavage to the GGCG site. This proposal is further supported by the finding that for a duplex with a CGCC f CICC substitution (which markedly reduces the cleavage frequency at the CICC site), significant cleavage at the GGCG in the opposite strand was detected only when the inosine-containing strand was nicked. Thus again, cleavage at the secondary site was detected in the absence of damage at the primary site, but only when a nick equivalent to that produced by primary cleavage was present. Replacement of the hydroxyl with a phosphoglycolate did not appreciably alter the cleavage pattern, suggesting that targeting was

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Figure 4. Bleomycin-induced cleavage opposite a nick bearing either a 3′-hydroxyl or 3′-phosphoglycolate (indicated by •) terminus. The top strand was 5′-end-labeled, and the full sequence is shown in Figure 3.

Figure 5. Cleavage of an intact, nicked, or partial duplex containing a guanine f inosine (I) substitution at the 3′-terminus of the nick. The top strand was 5′-end-labeled, and the full sequence is shown in Figure 3.

Intermediates in DNA Cleavage by Bleomycin

primarily a function of the nick rather than of the increase in negative charge. A one-base-gapped structure, more similar to a mature bleomycin-induced singlestrand break, was much less efficient in targeting secondary cleavage. Thus, these results are consistent with the view that it is the primary nick per se that targets cleavage to the secondary site. Although cleavage of the nicked substrates more closely mimics true double-strand cleavage than does cleavage of gapped substrates, even the nicked duplex targets only a fraction of the cleavages to the “correct” secondary site. However, this is not surprising since, as suggested by the single-hit cleavage kinetics, the bleomycin molecule that effects secondary cleavage was probably already DNA-bound at a specific site and with a specific geometry, as required to effect primary cleavage. Perhaps only a fraction of the bleomycin molecules that bind de novo to the nicked structure assume this same binding mode, while the remainder bind to adjacent primary sites just as they would for an intact duplex. On the basis of two-dimensional NMR of Co(III)‚ bleomycin bound to oligomeric DNA duplexes, two research groups (21, 22) have recently proposed structures in which the bleomycin bithiazole rings are intercalated between the base pair containing the target nucleotide and the base pair to its 3′-side (e.g., between the two adjacent C’s in GGCC), while the metal coordination site is hydrogen-bonded to the N2 of the G immediately 5′ of the target. A nick just 5′ to the target C (i.e., at the primary site) could increase DNA flexibility so it could more easily accommodate this bithiazole intercalation. Vanderwall et al. (23) showed with computer-based molecular modeling that, with the bithiazole remaining intercalated at a single site in an intact DNA duplex, bleomycin can undergo a rearrangement such that the metal coordination site can be positioned for both primary and secondary cleavage (23). This result raises the possibility that, in double-strand cleavage, initial free radical attack and hydrogen abstraction at both the primary and the secondary site could occur before actual cleavage at either site. However, if this is the case, it is difficult to explain why deuterium incorporation at the primary site suppresses attack at the secondary site (18); if anything, deuterium at this position would be expected to prevent reaction between the putative high-valence iron-oxo bleomycin and the primary site, and thus increase the chance of it attacking the secondary site. Overall, the data appear to be more compatible with a model wherein nicking at the primary site changes bleomycin binding geometry sufficiently to both promote secondary attack and restrict it to a specific site in the complementary strand.

Acknowledgment. This work was supported by Grant CA40615 from the National Cancer Institute.

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