Letter pubs.acs.org/JPCL
UV-Induced Strand Breaks in Double-Stranded DNA Labeled with 5‑Bromouracil: Frank or Secondary? Justyna Wiczk, Justyna Miloch, and Janusz Rak* Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland S Supporting Information *
ABSTRACT: Some literature reports suggest that in DNA labeled with 5-bromouracil (5BrU), near-UV photons lead to strand breaks that are formed due to the formation of a reactive uracil-5-yl radical capable of abstracting a hydrogen atom from its own or adjacent sugar moiety, which results in a direct strand break. However, other reports propose the formation of 2′-deoxyribonolactone rather than a strand break during the photodamage of 5BrU-substituted DNA. In order to resolve these contradictions, we carried out a series of experiments where 25 nucleotides-long DNA duplexes labeled with 5BrU were irradiated with 300 nm light. Two experimental methods were used to detect and separate the degradation products generated under experimental conditions, DHPLC (completely denaturing high-performance liquid chromatography) and denaturing PAGE electrophoresis. In addition, the identity of the particular products was confirmed with negative ion mass spectrometry. Our studies demonstrate that direct strand breaks reported in the past for 5BrU-labeled oligonucleotides are rather secondary breaks. SECTION: Biophysical Chemistry and Biomolecules
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lmost 60 years ago, Greer and Zamenhof1 and Greer2 demonstrated that the E. coli cells become photosensitive due to pretreatment with 5-bromouridine (BrdU). Later on, it was demonstrated that BrdU is an excellent substrate for thymidine kinase,3 while bromouridine triphosphate is an exceptionally good reactant for DNA replication,4 which suggests that the compound is incorporated into the biopolymer prior to cell sensitization. In the cells pretreated with BrdU, UV radiation enhances the activation of DNA damage signaling with the creation of DNA double strand breaks (DBSs) and cell cycle arrest, particularly in the G1-S phase.5 As a result, the enhanced apoptosis and autophagy-mediated tumor cell death is observed.6 These two features of BrdU, namely, its ability to be incorporated into the genomic DNA of proliferating cells and its capability of photosensitizing the biopolymer, make this bromoderivative, and other modified nucleosides of equivalent properties, a great promise for potential photodynamic anticancer therapies. This explains why the photosensitizing properties of BrdU in the DNA context have gathered particular interest.7−14 Attempts to comprehend the mechanism behind the experimentally observed damage were undertaken at the beginning of the 1990s by the Saito group.7 The investigators suggested 2′-deoxyribonolactone and free adenine to be the primary photoproducts generated in short duplexes that were irradiated with 302 nm at 0 °C. The problem was subsequently analyzed by the Greenberg8−10 and Sanche11 groups for the singly 5-bromouracil (5BrU)-labeled sequences of 20−40 base pairs (bp), as well as by the groups led by Sugiyama12 and Rak14 for long (400−500bp) PCR-labeled oligonucleotides. Interestingly, the groups8−11,14 that employed denaturing © 2013 American Chemical Society
electrophoresis detected the formation of direct (frank) strand breaks (SBs) due to UV irradiation, whereas those7,12,13 using the HPLC method mainly observed depurination coupled to 2′-deoxyribonolactone formation. Because SBs in general and DSBs in particular belong to the most cytotoxic DNA damage15 whose amount directly correlates with cell killing, the propensity of a given photosensitizer to induce DSBs is an important factor determining its potential efficacy in a photodynamic therapy. Considering the paramount importance of SBs in cell killing and contradictory literature reports concerning their UVinduced formation in 5BrU-labeled oligonucleotides, we decided to reassess a system for which direct SBs were reported in the past. We chose DHPLC as our primary analytical method because it appears to have a better resolution and to be a less destructive tool than denaturing PAGE electrophoresis, which is commonly used to assay SBs in DNA.16 The latter approach indeed involves heating of a sample above 90 °C for several minutes before its loading.17 Moreover, the electrophoresis itself lasts several hours, during which the sample is maintained at a temperature above 50 °C. Under these conditions, heat-labile sites can be detected as SBs. In this Letter, we shall demonstrate that SBs shown in the past by denaturing PAGE electrophoresis for oligonucleotides labeled with 5BrU are actually secondary SBs formed out of the heat-labile sites. In order to prove the statement mentioned previously, we analyzed the irradiated solutions containing Received: October 12, 2013 Accepted: November 11, 2013 Published: November 11, 2013 4014
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labeled DNA with the use of DHPLC and high-resolution mass spectrometry. We also showed that denaturing polyacrylamide electrophoresis of UV-damaged DNA seemingly indicates the formation of frank SBs. We chose chemically synthesized oligonucleotide to be the focal subject of our experiment (25 bp long), whose one strand was a point labeled with 5BrU, 5′- CGA GTA CTG CAA Br UAA CGT GTA CAG C-3′ (ss_BrU). This double-stranded oligomer (ds_BrU) was photodamaged in the past, and efficient single SB (SSB) formation was detected with denaturing PAGE electrophoresis.11 Both the labeled strand (see above) and the complementary one (5′- GCT GTA CAC GTT ATT GCA GTA CTC G-3′ (ss_comp)) were purchased from Genomed (Poland) and purified with ion pair reverse-phase HPLC (IPRP-HPLC) prior to usage. DNA hybridization was carried out by adding an equal amount of complementary oligonucleotides in K/PO4 and NaCl buffer (20 mM, pH 7), heating to 90 °C, incubating for 1 min, and then allowing to cool slowly to 50 °C for 10 min.17 The quality of hybridization has been checked by employing nondenaturing PAGE electrophoresis (see Figure S1 in the Supporting Information), and the hybridized samples were kept at 4 °C. Prior to the irradiation, a DNA solution was freshly prepared. Photolysis was carried out in quartz capillaries (3 × 3 mm) filled with the DNA solution at a concentration of 20 μM and a total volume of 50 μL with a 500 W high-pressure mercury lamp for 20 min. The 300 nm wavelength of incident light (half-width of 2.5 nm) was selected using a prismatic monochromator (SPM-2 Carl Zeiss, Jena). First, the photolytes were analyzed by DHPLC. All separations were performed by using a Dionex UltiMate 3000 apparatus with a diode array detector, which was set at 260 nm for monitoring the effluents. An Aeris Widepore reverse-phase XB-C8 column (250 × 4.6 mm; 3.6 μm in particle size) was used. A linear gradient from 0 to 10.4% ACN in the buffer containing TEA acetate (50 mM, pH 7) was maintained over an interval of 40 min, at the flow rate of 1.55 mL/min and column temperature of 70 °C. The irradiated samples were also analyzed with the help of mass spectrometry. Prior to the analysis, the samples had been desalted with Oasis HLB (Waters) for reversed-phase SPE and diluted (1:1) with a 50% water solution of acetonitrile (ACN). Finally, triethylamine (TEA) was added to its final concentration of 1%. The prepared solution was injected into a nanoESI ion source of a QStar XL MS-MS mass spectrometer equipped with a Q-TOF detector (Applied Biosystems). The spectra were recorded in negative polarization, and the ions were monitored within the 400−2000 m/z range. Figure 1 depicts a set of DHPLC chromatograms with regard to the studied system. The comparison of Figure 1C to D and E indicates that under DHPLC conditions, the particular oligonucleotide strands move separately because the two wellseparated signals in Figure 1C match the signals in Figure 1D and E pretty well (note that chromatograms in D and E were obtained by an injection of solutions containing single-stranded oligonucleotides). The chromatogram in Figure 1B, which corresponds to the UV-irradiated ds_BrU, indicates that after 20 min of irradiation, approximately 18.4% of the initial amount of the 5BrU-labeled strand was converted into a product with the retention time of ∼28.5 min. The occurrence of a single product peak strongly suggests that the photochemical process leads to a damage preserving the strand integrity rather than to a SSB, which should produce two fragments of different
Figure 1. DHPLC of (A) ds_BrU irradiated for 20 min and heated at 90 °C for 20 min; (B) ds_BrU irradiated for 20 min; (C) ds_BrU nonirradiated; (D) ss_comp; and (E) ss_BrU. Elution was performed with 50 mM TEA acetate (pH 7.0) containing 0−10.4% ACN over a linear gradient for 40 min at a flow rate of 1.55 mL/min at 70 °C.
mobility. Indeed, if SSB was generated due to irradiation, the two fragments should occur as two separated signals in DHPLC. Moreover, their retention times should be substantially shorter than that of the original strand because the SB would lead to two fragments, 11 bases long (5′- CGA GTA CTG CA-3′; Frag1) and 13 bases long (5′-UAA CGT GTA CAG C-3′; Frag2) (see the sequence of ss_BrU), whereas the retention time of the product peak in the chromatogram in Figure 1B is only ∼1 min shorter than that of the 5BrU-labeled substrate strand (see Figure 1). The most convincing proof of the secondary nature of the studied SB is the chromatogram in Figure 1A. In order to obtain it, the irradiated sample was heated at 90 °C for 20 min before injection. The striking feature of Figure 1A is a disappearance of the product peak with the retention time of 28.5 min (cf. the chromatogram in Figure 1A with B) 4015
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Figure 2. Raw ESI mass spectrum of irradiated ds_BrU oligonucleotide: (A) Frag1, (B) Frag2, and (C) ss_comp.
Scheme 1. Mechanism of SB Formation
namely, 3421.18 (Frag1) and 4024.55 (Frag2), are in complete agreement with the mechanism of SB formation proposed by Sugiyama et al.13 and is depicted in Scheme 1. Hence, electronic excitation of 5BrU induces interstrand electron transfer (ET) from a distant guanine that leads to the formation of a BrU•− radical anion. The latter efficiently releases the bromide anion, leaving the uracil-5-yl radical in the DNA fragment.18−21 Subsequently, the hydrogen atom abstraction (H. abs) from the C1′ of the 5′-side adenosine (the C1′ molecular center of the 5′-side neighbor adenosine seems to be the preferable site of H-abstraction in dsDNA22,23) by the uracil-5-yl radical produces uridine, and back ET (b. ET) to the guanine cation leads to a carbocation on the C1′ of the 5′-side sugar moiety (see Scheme 1). The attack of the water molecule releases adenine, while the 2′-deoxyribose moiety is transformed into 2′-deoxyribonolactone.13 The release of adenine was indeed observed in our DHPLC separations (see the signal with the retention time of ∼2.5 min in the chromatograms in Figure 1A and B). In order to further confirm the presence of adenine, the irradiated sample was extracted with chloroform. Then, the chloroform layer was dried off, dissolved in a small amount of water, and separated using the column and a mobile phase designed for nucleobase/
accompanied by a formation of two new signals with retention times of 21.0 and 22.8 min. The respective MS analysis was carried out in order to determine the identity of these products. Specifically, the solution containing ds_BrU was irradiated for ∼4 hours, which completely converted the labeled oligo into the product. Then, the photolyte was desalted and injected into a nanoESI ion source of the mass spectrometer (the raw mass spectrum of the nonirradiated sample is shown in Figure S2 in the Supporting Information). The raw mass spectrum is shown in Figure 2. It consists of signals corresponding to a series of multiply charged (multiply deprotonated) anions. With the help of reconstruction software (BioAnalyst. 1.1 (Applied Biosystems)), the origin as well as the charge state of the particular signals has been recognized (see Figure 2). The MS spectrum indicates the presence of the original ss_comp oligonucleotide and two fragments that originated from the decomposition of the primary photolysis product (cf. Figure 1A and B). The absence of signals corresponding to the ss_BrU strand results from the fact that the sample was irradiated for 4 h, which completely converted ss_BrU into the product (a complete conversion of ss_BrU warranted the maximum concentration of the studied product, which facilitated the MS analysis). The masses of the two fragments, 4016
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The employment of completely denaturing HPLC instead of commonly used denaturing PAGE electrophoresis for DNA analysis explained a controversy regarding the 5BrU-labeled DNA photodamage mechanism. Our research revealed that photodamage to the labeled double-stranded oligonucleotide, which had been considered to be a direct SB, is actually an abasic site comprising 2′-deoxyribonolactone that does not breach the integrity of the DNA strand. This abasic site gives rise to a SB only at elevated temperatures, and mass spectrometry confirms the formation of two smaller oligonucleotide fragments of size that fits the mechanism pretty well assuming the formation of ribonolactone as a transient species on the path to the secondary SB. Because denaturing PAGE electrophoresis is inseparably related to the elevated temperature, observation of the strand breaks pattern is unavoidable with this methodology, even if such damage is not a primary event. Our results unequivocally demonstrate that photoinduced SBs reported in the past for 5BrU-labeled oligonucleotides are in fact secondary breaks.
nucleoside separation. The comparison of the obtained chromatograms with the chromatogram of an adenine reference solution unequivocally demonstrated that adenine did form in the UV-irradiated solution. The stability of ribonolactone is rather small. The deoxyribolactone is thermally unstable with a half-life at 37 °C of 20 h.24 At elevated temperatures (90 °C), it decomposes immediately, leaving two smaller oligonucleotides, as indicated by Scheme 1. In the case of ss_BrU, Frag1 and Frag2, characterized by masses of 3421.24 and 4024.68, respectively, are produced. The MS measured masses of the Frag1 and Frag2 fragments (see Figure 2) remain in accordance with their theoretical values (3421.18 and 4024.55, respectively), which strongly supports the assumed damage mechanism (Scheme 1). Finally, a picture of denaturing PAGE electrophoresis is shown in Figure 3. As one may expect, the samples containing
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ASSOCIATED CONTENT
S Supporting Information *
Nondenaturing PAGE electrophoresis of the studied oligonucleotides and raw ESI MS spectrum of the nonirradiated sample. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Polish National Science Center (NCN) under Grant No. UMO- 2012/05/B/ST5/ 00368 (J.R.)
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Figure 3. The 20% denaturing polyacrylamide gel electrophoresis in TBE buffer. Most samples were incubated at 95 °C for 20 min before being loaded on the gel. Lane 1: ss_BrU; lane 2: ss_comp; lane 3: ds_BrU nonirradiated; lane 4: ds_BrU irradiated and heated; lane 5: ds_BrU irradiated, no heat pretreatment.
REFERENCES
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