DNA damage-site recognition by lysine conjugates Boris Breiner, Jo¨rg C. Schlatterer, Igor V. Alabugin†, Serguei V. Kovalenko, and Nancy L. Greenbaum Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306-4390 Communicated by Michael Kasha, Florida State University, Tallahassee, FL, June 20, 2007 (received for review January 30, 2007)
Simple lysine conjugates are capable of selective DNA damage at sites approximating a variety of naturally occurring DNA-damage patterns. This process transforms single-strand DNA cleavage into double-strand cleavage with a potential impact on gene and cancer therapy or on the design of DNA constructs that require disassembly at a specific location. This study constitutes an example of DNA damage site recognition by molecules that are two orders of magnitude smaller than DNA-processing enzymes and presents a strategy for site-selective cleavage of single-strand nucleotides, which is based on their annealing with two shorter counterstrands designed to recreate the above duplex damage site. damage recognition 兩 double-stranded DNA cleavage 兩 phosphate recognition 兩 sequence-specific DNA modification
A
side from the common double helix, DNA forms a wide range of structural motifs, such as hairpin loops, triplex, tetraplex, and bulged structures, as well as nicks and gaps. The individual structural features of these motifs make them potential candidates for specific targeting (1–6). Among these structural elements, nicks and gaps are promising for the development of sequence-specific DNA cleavage, because they already feature a break in the phosphate backbone of DNA. Cleavage of the phosphate backbone of DNA can be caused by chemical reagents (7) such as radicals (8) and by radiation damage (9). To survive, cells develop enzymatic mechanisms for the repair that work efficiently on single-strand (ss) damage (10). Any further cleavage on the opposite strand at the damage site leads to ds cleavage, which is hard to repair (11). The ds cleavage requires either a bifunctional reagent (12–20) or detection and targeting of the damaged site. The only literature example of the latter is a complex natural antibiotic, bleomycin (21). In this paper, we show that simple lysine conjugates can identify ss damage sites with high selectivity and induce DNA cleavage at the strand opposite to the damage site (Scheme 1). Results and Discussion We used an internally 32P-labeled‡ 54-nt DNA (BW 54s, 5⬘-TAA TAC GAC TCA CTA TAG GCC CAG GGA AAA CTT GTA AAG GTC TAC CTA TCT *ATT; the 32P label is indicated by an asterisk). This sequence incorporates several isolated guanine (G) nucleotides as well as GG diads, GGG triads, and an AT tract, the latter serving as a natural binding site for a family of lysine conjugates. We have shown that a combination of AT selectivity of binding and G selectivity of activation through photoinduced electron transfer (22–29) can be used for selective targeting of guanines flanking the AT tract (30). By annealing BW 54s with a variety of counterstrands, we built a family of constructs (Fig. 1) inspired by a selection of sites that formed either in the process of chemical damage of DNA or, transiently, during enzymatic processing of DNA (31). Nicked DNA (constructs B and C) is involved in DNA topological transitions, DNA repair synthesis, and DNA replication of the lagging strand (32). Single-nucleotide gaps with 3⬘- and 5⬘-phosphorylated ends (constructs D and E) can be formed by AP lyase activity of DNA glycosylases (32) or from gaps with 3⬘-phospholycolate-5⬘phosphate ends generated as the result of 4⬘-H abstraction by natural antibiotics such as bleomycin or by hydroxyl radicals produced by ionizing radiation (9) The structure of the lysine-enediyne
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Scheme 1. Two components are potentially responsible for damage site recognition and subsequent cleavage: (i) formation of a hydrophobic pocket and (ii) electrostatic interaction between the additional negative charges, because of the presence of terminal phosphate moieties at the negatively charged DNA backbone [(⫺) signs] and the protonated amines of lysine moiety [(⫹) charges]. (A) Annealing process that positions recognition site opposite the target at the original ss oligonucleotide. (B) Recognition of the target site by lysine conjugates. (C) Sequence-selective photochemical conversion of ssDNA cleavage into ds cleavage.
conjugate 1 and the lysine-acetylene conjugate 2 are shown in Fig. 2. These simple compounds are equipped with potent photoactivated ‘‘warheads’’§ (33, 34) and satisfy Lipinski’s rule of five (35) for drug design with respect to their molecular weight (⬍500) and H-bonding ability (not more than five donors, not more than 10 acceptors). We envisioned that the hydrophobic ‘‘warheads’’ may occupy a hydrophobic pocket created by the omission of a base, whereas the mono- or bis-protonated hydrophilic lysine residue will additionally experience strong Coulombic attraction toward a terminal phosphate mono- or dianion, as shown in Scheme 1. Four sets of target sites were constructed within BW 54. The first site was chosen to be opposite G26 of the G24G25G26¶ triad, which is a natural (but minor) cleavage site for the reaction of this conjugate with undamaged duplex DNA. The second one was opposite G19 of the G18G19 diad, which is not part of a natural binding site. The third and fourth sites were opposite G34 and A36, respectively. These sites were chosen to ascertain whether it was possible to target a single G site or other nucleotides. We wanted to know whether the damage remains localized or propagates to the nearby G34 and G39G40 by the hole-hopping mechanism (22–29), which may be important when electron transfer from DNA is involved in the ‘‘warhead’’ activation step. We irradiated the constructs in the presence of compound 1 or 2 (10 M) and analyzed the resulting cleavage patterns by Author contributions: B.B., J.C.S., and I.V.A. designed research; B.B., J.C.S., and S.V.K. performed research; S.V.K. contributed new reagents/analytic tools; B.B., J.C.S., I.V.A., S.V.K., and N.L.G. analyzed data; and B.B., J.C.S., and I.V.A. wrote the paper. The authors declare no conflict of interest. Abbreviation: ss, single strand. †To
whom correspondence should be addressed. E-mail:
[email protected].
‡The
internal label is necessary because a terminal label would consist of a terminal phosphate group, which would serve as a competing recognition element.
§The warhead in compound 1 was first investigated by Turro and coworkers (33, 34). It was
later found that DNA damage caused by this warhead is at least in part caused by electron transfer (see ref. 30). ¶The
bold letter indicates the exact location of the target site within a (G)n sequence.
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0705701104/DC1. © 2007 by The National Academy of Sciences of the USA
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Design of control DNA duplex and ss damage sites.
denaturing PAGE, followed by phosphorimaging and quantification with SAFA software (36) (Fig. 3). All constructs showed enhanced cleavage at the respective target sites. Constructs with a ‘‘gap,’’ an unpaired base in the target strand, displayed the greatest effect, especially when the target was within the natural AT-rich binding site (D, E, E⬘, G, G⬘). Gaps outside of the AT tract were less effective (constructs F and F⬘) and showed migration of damage away from the target G19 base not only to the adjacent G18 but also to a more remote G7 site. Interestingly, the migration of damage was unidirectional, and no enhancement is observed at the G24G25G26 triad. In all cases, however, cleavage at the site targeted by the construct was clearly enhanced. In the case of constructs without the ‘‘gap’’ (B, C, C⬘), the enhancement was also evident, albeit to a smaller degree than in the ‘‘gapped’’ constructs. In the cases where the target site was not at a (multiple) G (constructs B and D), the cleavage was distributed over an array of bases terminated by the nearest G. Both the single G34 and the G39G40 doublet showed increased cleavage, but also the whole A/T area between these two sites displayed more damage relative to the duplex DNA. Remarkably, once the target site is moved only two bases to G34 (constructs G and G⬘), all of the damage enhancement is localized at that site, resulting in amplification by a factor of 5–6 on what used to be a minor cleavage site in the ds construct (Fig. 4). Localization of damage at a single G in the presence of nearby (G)n sites illustrates that dissipation of the damage by hole hopping does not interfere (37).储 To compare the different types of constructs [with/without gap, with/without phosphate group(s)], we compared constructs C, C⬘, E, and E⬘ directly, again using the ‘‘undamaged’’ ds construct A as reference (Fig. 5). Enhancement of piperidine-induced cleavage is identical within each pair of constructs with (E and E⬘) and without a gap (C and C⬘), suggesting that, unlike the gap, phosphate groups apparently have no influence at the oxidative piperidine-induced DNA cleavage. On the other hand, frank photocleavage induced by our compounds, which may occur because of direct H abstraction by radical species, is clearly enhanced both by the presence of phosphate groups and by the hydrophobic pocket (Fig. 6).
To understand the role of the phosphate groups in the recognition of lysine conjugates, we also varied the buffer conditions. The directing effect, as well as the reactivity, is much more pronounced in borate buffer than in phosphate buffer (Fig. 7). Although we observe an enhancement by a factor of approximately eight in borate buffer, the directing efficiency of the target site is reduced by ⬇50% in 20 mM phosphate buffer. These findings suggest that recognition between the ammonium groups of lysine and phosphates of the DNA does contribute to the directing effect and that this contribution decreases upon ‘‘dilution’’ with the external phosphate moieties. Nevertheless, even in the presence of a 4,000– 20,000 molar excess of phosphate, the directing effect is still present, suggesting the robustness of these recognition patterns. Both the robustness and the versatility of DNA damage recognition are further illustrated by reactivity of another lysine conjugate (2) over a wider range of conditions. Differences in selectivity are minimal for experimental results for pH 6, 7, and 8, and cleavage at the target site is amplified by a factor of 5–6, even in the presence of phosphate buffer (Fig. 8). These experiments suggest that the effect is not limited to the system and the conditions used here but can be expanded to other DNA-cleaving agents. The directing effect of ss damage sites provides an explanation for the unusually high ratios of ds/ss cleavage of plasmid DNA by enediyne-lysine conjugates (38), where statistical evidence based on the Poisson distribution suggested a dramatic increase in the amount of ds cleavage compared with that expected from a combination of random ss cleavages. Although statistical evidence does not differentiate between the true ds cleavage and this alternative scenario, the possibility of directing a second attack
储The observation that strand cleavage is often faster than hole propagation in these systems
is very interesting. It may result either from a very quick cleavage or from a relatively slow propagation. It is known that the rate of hole migration is sensitive to static and dynamic fluctuations in the local DNA structure. For a particularly relevant example that illustrates the role of counterions, see ref. 37.
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Fig. 2. Structures of lysine conjugates 1 and 2 (shown as unprotonated amines). PNAS 兩 August 7, 2007 兩 vol. 104 兩 no. 32 兩 13017
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Fig. 1.
Fig. 3. Comparison between the intact DNA duplex A and constructs B–E. The constructs (0.2 M) were irradiated in the presence of compound 1 (10 M) in borate buffer (pH 7.6). (Upper) Autoradiogram of PAGE analysis. The arrows show the position of the target site. (Lower) Histograms showing the quantified cleavage data, normalized relative to the largest peak in construct A.
through recognition of the initial ss cleavage points to a new strategy in the design of highly reactive dsDNA cleaving agents. In conclusion, we have shown that it is possible for small molecules (Mr ⬍500) to recognize damage sites in one strand of dsDNA and to use this recognition to direct subsequent damage to a specified location at the counterstrand, thus converting ss cleavage to ds cleavage. The recognition site can be further fine-tuned by creating or omitting a gap and/or adding or excluding phosphates at the f lanks. Although such recognition is known for enzymes and some large natural 13018 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0705701104
products, we have demonstrated that the underlying working pattern can be simulated with compounds that are smaller than enzymes by two orders of magnitude. Moreover, this finding points to a new strategy for a site-selective cleavage of ss nucleotides such as mRNA, because annealing of a target ss with two appropriately designed counterstrand pieces creates the above duplex with a ss break at a position of our choice, which, as we have shown above, serves as the recognition site for the lysine conjugates. This procedure is equivalent to site-specific cleavage at a ssDNA with the advantage that the specificity of Breiner et al.
Fig. 4. Comparison between DNA construct A and constructs F, F⬘, G, and G⬘. The constructs (1 M) were irradiated in the presence of compound 1 (10 M) in borate buffer (pH 7.6). The arrows show the position of the target site. (Upper) Autoradiogram of PAGE analysis. (Lower) Histograms showing the quantified cleavage data, normalized relative to the largest peak in construct A.
Materials and Methods General Information. All reagents used were purchased from Sigma (St. Louis, MO) and Acros Organics (Geel, Belgium) if Breiner et al.
not otherwise noted. All buffers were prepared and pH-adjusted at room temperature (25°C). Irradiation Conditions. Samples were placed on ice at a distance of
20 cm from 200-W Hg–Xe lamp [Spectra-Physics, Laser and Photonics Oriel (Stratford, CT), with long-pass filter with 324 nm cut-on wavelength]. 32P
Labeling of DNA Oligomers for Phototriggered Damage. The
synthesis of BW 54s was described previously (30). PNAS 兩 August 7, 2007 兩 vol. 104 兩 no. 32 兩 13019
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cleavage is easily controlled by the choice or the two counterstrand pieces and the attachment of the phosphate moiety. Thus, the system is modular and flexible and has significant potential in localized control of gene expression, with clear and powerful implications for targeted medical therapy.
Fig. 5. Comparison between DNA construct A and constructs C⬘, E⬘, C, and E. The constructs (1 M) were irradiated in the presence of compound 1 (10 M) in borate buffer (pH 7.6). (Upper) Autoradiogram of PAGE analysis. The arrows show the position of the target site. (Lower) Histograms showing the quantified cleavage data, normalized relative to the largest peak in construct A.
Cleavage Reactions. In a typical reaction, 2.5 l of oligomer solution (2 M in H2O), 1 l of borate buffer (200 mM), and 1.5 l (33 M) of an aqueous solution of compound 1 were
Fig. 6. Histograms showing the amount of frank cleavage at the G24G25G26 triad in various constructs under identical conditions. Intensities are relative to the largest peak in construct A. E and E⬘ correspond to gapped constructs with and without terminal phosphate groups. 13020 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0705701104
irradiated for the indicated amount of time in a microcentrifuge tube. After irradiation, all samples were evaporated to dryness in vacuo. Samples that were not treated with piperidine were immediately dissolved in loading buffer (80% formamide vol/vol/10 mM EDTA/0.1 mg/ml Xylene cyanol/1 mg/ml brom phenol blue/5 mM NaOH). The remaining samples were treated with piperidine (20 l, 90°C, 30 min), evaporated to dryness, and coevaporated with H2O (20 l) twice before dissolving them in loading buffer. The reactions were analyzed by using a 12% denaturing (8 M urea/25% formamide) polyacrylamide gel. Electrophoreses were performed at 2,000 – 2,500 V and were generally complete after 3–3.5 h. The gels were cooled to ⬇4°C and visualized and quantified by using a phosphorimaging screen (Molecular Dynamics, Sunnyvale, CA), and a Storm 860 Scanner (Molecular Dynamics). Financial support was provided in part by Material Research and Technology Center (MARTECH) at Florida State University, the National Science Foundation (I.V.A.), and the National Institutes of Health (N.L.G.). I.V.A. is grateful to the 3M Company for an Untenured Faculty Award. Breiner et al.
Fig. 7. Histograms showing the quantified cleavage data for comparison between DNA constructs A and E in different buffer conditions. The constructs (1 M) were irradiated in the presence of compound 1 (10 M) in borate buffer (pH 7.6, 20 mM) (Left), borate buffer (pH 7.6, 4 mM) (Center), and phosphate buffer (pH 7.6, 20 mM) (Right). The data are normalized relative to the largest peak in construct A. The arrows show the position of the target site. See supporting information (SI) for complete data.
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Fig. 8. Histograms showing the quantified cleavage data for comparison between DNA constructs A and E in different buffer conditions. The constructs (1 M) were irradiated in the presence of compound 2 (10 M) in phosphate buffer. (Left) pH 6. (Center) pH 7. (Right) pH 8. The data are normalized relative to the largest peak in construct A. The arrows show the position of the target site. See SI for complete data.
