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Formation Mechanism and Structure of a GuanineUracil DNA Intrastrand Cross-Link Cassandra D. M. Churchill,† Leif A. Eriksson,‡ and Stacey D. Wetmore*,† † ‡
Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada T1K 3M4 School of Chemistry, National University of Ireland—Galway, University Road, Galway, Ireland
bS Supporting Information ABSTRACT: The formation and structure of the 50 G[85]U30 intrastrand crosslink are studied using density functional theory and molecular dynamics simulations due to the potential role of this lesion in the activity of 5-halouracils in antitumor therapies. Upon UV irradiation of 5-halouracil-containing DNA, a guanine radical cation reacts with the uracil radical to form the cross-link, which involves phosphorescence or an intersystem crossing and a rate-determining step of bond formation. Following ionizing radiation, guanine and the uracil radical react, with a rate-limiting step involving hydrogen atom removal. Although cross-link formation from UV radiation is favored, comparison of calculated reaction thermokinetics with that for related experimentally observed purinepyrimidine cross-links suggests this lesion is also likely to form from ionizing radiation. For the first time, the structure of 50 G[85]U30 within DNA is identified by molecular dynamics simulations. Furthermore, three conformations of cross-linked DNA are revealed, which differ in the configuration of the complementary bases. Distortions, such as unwinding, are localized to the cross-linked dinucleotide and complementary nucleotides, with minimal changes to the flanking bases. Global changes to the helix, such as bending and groove alterations, parallel cisplatin-induced distortions, which indicate 50 G[85]U30 , may contribute to the cytotoxicity of halouracils in tumor cell DNA using similar mechanisms.
’ INTRODUCTION DNA damage has been the focus of experimental and computational research in attempts to maintain the integrity of the genetic code.1 DNA damage can also be harnessed as a tool to kill unwanted tumor cells.2 For example, the potent anticancer drug cisplatin forms DNA intrastrand cross-links between adjacent guanine nucleobases and exerts cytotoxic effects through several pathways that ultimately activate cell apoptosis.3,4 Halogenated pyrimidines have similarly been used as antitumor therapies.510 5-Halo-20 -deoxyuridines (chloro, bromo, and iodo) are of particular interest since they are thymidine analogues that are readily incorporated into DNA.7 For example, irradiation of DNA containing 5-bromouracil (BrU) results in the creation of the uracil-5-yl radical (U•),7,11 which can generate cross-links1220 and strand breaks.2126 Several factors led to the administration of 5-halo-20 -deoxyuridines in conjunction with ionizing radiation as an antitumor therapy in clinical trials into the 1990s.57,2731 First, halouracils show relatively low toxicity in nonradiated tissues.19 Second, DNA damage can be controlled both through preferential incorporation of 5-halouracil (XU) into actively dividing (tumor) cells and by limiting the radiated area.6,7 Finally, U• is reactive under the hypoxic conditions found in tumor cells, compared to many other radicals that require reaction with O2 prior to initiating DNA damage.32 However, clinical trials revealed no survival advantage for patients.6 The major obstacle was achieving adequate incorporation of XU in tumor DNA without overexposing normal tissue.6,33 r 2011 American Chemical Society
Interest in the use of XU in antitumor therapy has recently refocused due to new strategies for increased incorporation into DNA.8,9,34 Most notably, administration of 5-chloro-20 -deoxycytidine in conjunction with several enzyme inhibitors exploits the elevated levels of deoxycytidine kinase and cytosine deaminase in tumor cells and therefore uses natural cell machinery to preferentially incorporate 5-chloro-20 -deoxyuridine into tumorous DNA rather than normal rapidly dividing tissues.9 Additionally, a recent study identified mutations to human deoxycytidine kinase that increase specificity toward 5-substituted 20 deoxycytidines,34 which would ultimately result in higher concentrations of 5-halo-20 -deoxyuridines in the nucleotide pool available for incorporation into DNA. Complementing the above advances regarding XU incorporation into tumor cell DNA, recent investigations have focused on revealing the cellular reactions of U•. Experiments have identified inter-1214,16,18,19 and intrastrand15,17,20 cross-links, as well as strand breaks and alkali labile lesions,2126 as products of U• generated by UV or ionizing radiation. The variety of products observed is indicative of the ability of therapeutics based on U• to overcome drug resistance and induce clustered lesions within DNA that are difficult to repair.3537 It is therefore essential to further understand the reactions of U• and use this information to enhance its ability to damage DNA and/or aid the design of alternate treatments with enhanced antitumor capabilities. Received: August 3, 2011 Published: November 07, 2011 2189
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Figure 1. Structure and numbering of the (a) halogenated 50 GXU30 sequence, (b) 50 G•+U•30 and 50 GU•30 reactants, and (c) the 50 G[85]U30 product. Activation of the 50 GXU30 sequence by UV (blue) or ionizing radiation (red) produces the 50 G•+U•30 and 50 GU•30 reactants, respectively, which ultimately yield 50 G[85]U30 . The χG ( — (O40 C10 N9C4)), χU ( — (O40 C10 N1C2)), and θ ( — (N9C8C5C4)) torsion angles describing the nucleobase orientations are highlighted in green.
Among the DNA lesions resulting from U•, single and double strand breaks (SSBs and DSBs, respectively)25 and the alkalilabile 20 -deoxyribonolactone intermediate3841 are the most widely known and studied, including computational examination of certain aspects of their formation mechanisms.42,43 U•, produced from the exposure of BrU-containing DNA to UV13,16,18,21,2426,44 or ionizing12,22,23,25 radiation, leads to strand breaks between U• and an adjacent 50 purine. While strand breaks have been implicated as the primary initiator of cell death from U•,19 these lesions cannot completely account for all cell death observed in survival curves, which suggests cellular processing of the initial damage22,25 or generation of additional forms of damage.23,25 More recent experimental work has detected interstrand cross-links resulting from U• generated by UV or γ-irradiation of BrU in DNA,1214,16,18,19 although the nucleobase composition and connectivity of these lesions have not been determined. In addition, intrastrand cross-links have been detected following UV irradiation of BrU-substituted synthetic and cellular human DNA.15,17,20 Among the four intrastrand cross-links identified in synthetic dinucleoside monophosphates, oligonucleotides,15 and cellular human DNA,17 the most abundant forms between C8 of a 50 -guanine and C5 of a 30 -uracil (denoted as 50 G[85]U30 , Figure 1).17 While the intrastrand lesions have not been reported from exposure of BrU-containing DNA to ionizing radiation, evidence in the literature suggests these lesions may be generated under therapeutically relevant conditions. First, strand breaks and interstrand cross-links form regardless of the type of radiation used to create U•.1214,16,18,19,2126 Additionally, related purinepyrimidine intrastrand cross-links, similarly formed by a pyrimidine C5 (or C5-methyl) radical, result from the exposure of natural DNA to ionizing radiation.4549 Furthermore, a study examining the products of BrU-containing DNA after exposure to ionizing radiation postulated that the observed decrease in the number of SSBs in the 50 GBrU30 sequence was due to the preferential formation of intrastrand cross-links.18 Finally, intrastrand cross-links may have been produced following the exposure of BrU-containing DNA to ionizing radiation but were not discovered due to commonly used digestion and detection procedures that complicate the simultaneous identification of SSBs, DSBs, and inter- and intrastrand cross-links.14,18,19 These studies provide indirect support for the ability of ionizing radiation to generate intrastrand cross-links, which may contribute to the cytotoxicity of XU used in therapeutic treatments, making it important to consider intrastrand cross-link formation under these conditions.
Although the connectivity of intrastrand cross-link products of U• has been unveiled,15,17,20 there is no structural data. Furthermore, the proposed reaction mechanisms15 have not been studied, and no information exists on the orientation of the cross-links in DNA or their effects on the double helix. Structural changes are particularly important since the extent of lesioninduced helical distortion is indicative of the apoptotic potential toward the tumor cells, and related G-pyrimidine intrastrand cross-links have been found to distort the helix46,4951 and affect DNA replication.46,48,49,5254 Since previous computational work has successfully shed light on the initial hydrogen-abstraction step of SSB formation resulting from U•,42,43 computational chemistry is used in the present study to examine the formation mechanism for the most abundant UV-intrastrand cross-link, 50 G[85]U30 (Figure 1), under conditions relevant to both UV and ionizing radiation. In particular, we establish whether 50 G[85]U30 is likely to be generated under therapeutically relevant (ionizing radiation) conditions. For the first time, the cross-link structure is determined, and the conformation(s) of cross-linked DNA studied and compared to that of natural DNA. Overall, this will provide an enhanced understanding of the structure and formation mechanism of 50 G[85]U30 and unveil the potential contributions of this lesion to the mechanism of action of XU in antitumor therapies.
’ COMPUTATIONAL METHODS The 50 G[85]U30 lesion was studied using a dinucleoside monophosphate complexed with a Na+ counterion, which maintained its position between the two anionic phosphate oxygens in all calculations. Optimizations were performed using IEFPCM-M06-2X/ 6-31G(d,p) in water (ε = 78.4).55,56 The structural accuracy of this approach was previously verified by reproducing the conformation of natural B-DNA,57 and the ability of M06-2X/6-31G(d,p) to describe ππ interactions has also been established.58 The unrestricted formalism was used for systems with unpaired electrons (Mulliken atomic spin densities provided in Table S-1, Supporting Information). Frequency calculations confirmed the nature of all stationary points and provided scaled (0.9580)59 zero-point corrected energies (ΔEZPVC), scaled (0.9470)59 thermal corrections to the enthalpy (ΔH), and unscaled thermal corrections to the Gibbs free energy (ΔG) under standard conditions (1 atm and 298.15 K). To obtain ΔG, the solvation energy (including the nonelectrostatic component) was calculated with the SMD solvation model.60 The reaction coordinates were confirmed by following the imaginary mode corresponding to the transition state in both directions to the neighboring minima. Throughout the reaction pathway, the terminal C50 -hydroxyl group was constrained to eliminate 2190
dx.doi.org/10.1021/tx2003239 |Chem. Res. Toxicol. 2011, 24, 2189–2199
Chemical Research in Toxicology interactions non-native to natural B-DNA ( — (HO50 C50 C40 ) = 180). The relative stability of stationary points is discussed using the optimization energy (ΔE), unless otherwise noted. All DFT calculations were performed with Gaussian 09.61 To gain insight into the conformations of the intrastrand cross-link within DNA, as well as helical distortion due to the formed cross-link, 55 ns molecular dynamics (MD) simulations of DNA containing 50 G[85]U30 were performed using YASARA.62 Double-stranded complementary B-DNA was generated with the 50 -d(GCATGGCGTGCTATGC)-30 sequence using the nucgen module of AmberTools.63 This sequence was chosen due to the formation of 50 G[85]U30 in the synthetic 50 d(ATGGCGBrUGCTAT)30 oligonucleotide following UV exposure15 and include additional capping GC bases to reduce unwinding of the helix during the simulation.64 Cross-linked DNA was generated by overlaying the backbone of the M06-2X-optimized dinucleoside monophosphate product onto the central 50 GT30 sequence (bold). Simulations were performed using the AMBER03 forcefield65 for the natural components and GAFF66 (Table S-2, Supporting Information) for the cross-linked dinucleotide (Figure S-2, Supporting Information). For comparison, the corresponding natural strand was simulated for 28 ns. Simulation snapshots were saved every 25 ps, and analysis was performed on all structures following a 3 ns equilibration. DNA strands were analyzed using the Curves+ program.67 Further details of the simulation (page S-7), including the atom types and charges used (Table S-2, Supporting Information), the coordinates of all DFT stationary points, and the coordinates of cross-linked DNA can be found in the Supporting Information.
’ RESULTS We begin by discussing intrastrand cross-link formation upon UV irradiation of a 50 GXU30 sequence, which corresponds to the experimental conditions under which the cross-link has been observed. In this instance, absorption of a photon creates a locally excited XU, followed by intramolecular electron transfer from G to XU to create a guanine radical cation (G•+) and a 5-bromouracil radical anion (BrU•).68 Subsequently, this charge-transfer
Figure 2. Reaction scheme for cross-link formation resulting from (a) UV and (b) ionizing radiation and important stationary points on the lowest-lying triplet state (blue), singlet ground state (green), and lowestlying doublet surface (red).
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state collapses to release X and yield a uracil radical adjacent to G•+ (50 G•+U•30 , Figure 2a).68,69 Intrastrand cross-link formation will also be studied upon exposure to ionizing radiation, which is used in antitumor treatments. In this case, a free electron produced by radiolysis attaches to XU and causes X dissociation to create a uracil radical adjacent to (undamaged) guanine (50 GU•30 , Figure 2b).70,71 This reactant is also relevant to UV irradiation of DNA containing 5-iodoracil since the C5I bond may be cleaved homolytically.72 Thus, the following sections present the pathways starting from the 50 G•+U•30 and 50 GU•30 reactants to understand the formation of 50 G[85]U30 from different halouracils and under different reaction conditions. Subsequently, the structure of the cross-link, as well as induced helical distortions, will be discussed. Cross-Link Formation from UV Radiation. The triplet 50 G•+U•30 biradical reactant formed upon UV exposure of X U-containing DNA (RT, Figure 3) is characterized by an anti orientation of the nucleobases (χG = 255.4 and χU = 277.5, Figure 1), a stacked basebase orientation, and a slightly nonplanar U (RT, Figure 3). Both sugar moieties adopt B-DNA C20 -endo puckering.73 Mulliken spin densities (Table S-1, Supporting Information) reveal an unpaired electron localized at C5 of U and another highly delocalized on G. Cross-link formation from UV radiation proceeds to a singlet product (50 G[85]U30 ) with a spin flip, which can occur via one of the two pathways discussed below (Figure 2). Pathway 1: Bond Formation on the Lowest-Lying Triplet Surface. Prior to bond formation on the triplet surface (Pathway (1), Figure 2a), a conformational change occurs (Figure 3), where U rotates about the glycosidic bond (χU) from the stacked RT (χU = 277.5) to a T-shaped orientation in the intermediate (I1T, χU = 179.7). These minima are connected by a small transition barrier (TS1T, ΔEq = 21.0 kJ mol1 (Figure 4a), ΔGq = 4.3 kJ mol1 (Table 1)). Subsequently, association occurs as the C8C5 distance decreases from I1T (d(C8C5) = 4.162 Å) through the transition state (TS2T, d(C8C5) = 2.231 Å) to the cross-linked intermediate (I2T, d(C8C5) = 1.512 Å). During this process the C8H8 bond lengthens, C8 becomes sp3hybridized, and χG changes from 243.7 (I1T) to 282.7 (I2T). Additionally, a twist (θ, Figure 1) of 207.2 occurs between the bonded nucleobases in I2T. Mulliken spin densities for I2T show that the two unpaired electrons are delocalized over G and U (Table S-1, Supporting Information). The bond formation barrier is 70.5 kJ mol1 (ΔGq = 61.7 kJ mol1), and this step releases energy (ΔE = 40.2 kJ mol1, Table 1).
Figure 3. Stationary points for the formation of 50 G[85]U30 from 50 G•+U•30 . 2191
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Following bond formation, a spin flip from I2T yields the singlet cross-linked intermediate (I2S) accompanied by the emission of phosphorescence (Figure 2a). These minima share
Figure 4. Cross-link formation resulting from (a) 50 G•+U•30 on the triplet (blue) and singlet (green) surfaces or (b) 50 GU•30 on the doublet surface (red). M06-2X energies (kJ mol1) are reported relative to the corresponding reactant.
Table 1. Relative Energies and Thermodynamic Parameters (kJ mol1) for the Formation of 50 G[85]U30 from 50 G•+U•30 a stationary pointb
ΔEc,d
ΔEZPVCd,e
ΔHd,f
ΔGg
TΔSh
RT + H2O
0.0
0.0
0.0
0.0
0.0
TS1T + H2O I1T + H2O
21.0 12.6
19.3 11.5
18.5 12.2
4.3 5.2
14.2 7.0 21.4
TS2T + H2O
83.1
78.5
77.9
56.5
I2T + H2O
27.6
27.0
27.4
63.1
35.7
I2S + H2O
262.9
255.8
257.2
303.3
46.1
I2S 3 3 3 H2O TS3S 3 3 3 H2O PS 3 3 3 H3O+ PS + H3O+
315.4
294.7
300.5
268.9
31.6
279.2
270.7
278.7
237.1
41.6
385.2
369.5
377.3
344.1
33.2
250.7
238.9
241.0
307.9
66.8
Reactant generated from the exposure of 50 GXU30 to UV radiation. Refer to Figure 3 for the structures of corresponding stationary points. c Optimization energy. d Calculated with IEFPCM-M06-2X/6-31G(d,p). e Includes scaled (0.9580) zero-point vibrational energy correction. f Includes scaled (0.9480) thermal correction to the enthalpy. g Calculated using the SMD-M06-2X/6-31G(d,p) energy (including nonelectrostatic component) and IEFPCM-M06-2X/6-31G(d,p) thermal correction to the Gibbs Free Energy. h Calculated as TΔS = (ΔG ΔH). a b
similar backbone conformations and only slight differences in the glycosidic bonds (in I2T, χG = 282.7 and χU = 188.7, while in I2S, χG = 298.2 and χU = 190.8). The I2S intermediate is characterized by d(C8C5) = 1.505 Å and θ = 205.4. Energy is released (ΔE = 235.3 kJ mol1) upon transition to I2S primarily as a 178.9 kJ mol1 photon, which corresponds to a wavelength of light (668.7 nm) in the visible region. A proton is removed from I2S (Figure 2a) to obtain the experimentally observed cross-linked product (PS). To model this step, it was necessary to include a discrete water molecule, I2S 3 3 3 H2O. In the transition state for proton abstraction (TS3S), the C8H8 distance elongates (from 1.098 Å to 1.330 Å) as C8 becomes sp2-hybridized, and a barrier of 36.2 kJ mol1 (ΔGq = 31.8 kJ mol1, Table 1) is overcome to form a complex (PS 3 3 3 H3O+). In the final step of the reaction pathway, a decomplexation step yields the infinitely separated species (PS + H3O+, Figure 3). Pathway 2: Bond Formation via an ISC. Bond formation may be coupled to an intersystem crossing (ISC, Figure 2a), which permits transition from the triplet reactant (RT) to the singlet cross-linked intermediate (I2S). Since an ISC cannot be located using conventional optimization procedures, a coordinate-driven approach (see page S-3 and Figure S-1 in Supporting Information) is used to estimate its location.74 An ISC was estimated at approximately d(C8C5) = 2.09 Å and an energy of 76.4 kJ mol1 relative to RT. This energetic cost is attributed to geometric distortion, which primarily changes χG, χU, and the C8C5 distance. Following an instantaneous spin flip at d(C8C5) = 2.09 Å, the cross-linked intermediate undergoes vibrational relaxation to I2S. Following bond formation, proton removal from C8 occurs on the singlet surface as discussed above. Cross-Link Formation from Ionizing Radiation. The optimized 50 GU•30 doublet reactant (RD, Figure 5) contains planar, stacked nucleobases in the anti orientation. The 50 deoxyribose adopts B-DNA sugar puckering, while the 30 -deoxyribose adopts a C30 -exo pucker. Mulliken spin densities show the unpaired electron is localized on C5 of U (Table S-1, Supporting Information). The reaction proceeds from this reactant along the lowest-lying doublet surface to a cross-linked intermediate (Figure 2b). Cross-link formation is initiated by rotation of U from the stacked reactant (RD, χG = 299.2 and χU = 274.9) to a T-shaped intermediate (I1D, χG = 239.1 and χU = 178.1), which has a barrier of 17.8 kJ mol1 (ΔGq = 6.6 kJ mol1, Table 2). Subsequent bond formation occurs with a barrier of 37.0 kJ mol1 (ΔGq = 23.6 kJ mol1). This step is accompanied by a decrease in d(C8C5) from 3.706 Å in I1D to 2.326 Å in TS2D and 1.517 Å in I2D, as well as a substantial change in the G orientation (χG = 290.1), C8 hybridization to sp3, and a small change in U orientation (χU = 193.1). In I2D, the
Figure 5. Stationary points for the formation of 50 G[85]U30 from 50 GU•30 . 2192
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Table 2. Relative Energies and Thermodynamic Parameters (kJ mol1) for the Formation of 50 G[85]U30 from 50 GU•30 a stationary pointb
ΔEc,d
ΔEZPVCd,e
ΔHd,f
ΔGg
TΔSh
RD
0.0
0.0
0.0
0.0
0.0
TS1D
17.8
13.3
13.2
6.6
6.6
I1D
13.0
9.7
10.9
0.8
10.1
TS2D
50.0
42.0
42.8
24.4
18.4
I2D TS3D
107.0 64.8
109.1 44.0
108.9 43.8
136.7 28.6
27.8 15.5
PS 3 3 3 H• PS + H•
34.3
15.5
16.5
3.6
12.9
43.7
19.1
23.3
14.5
37.8
Reactant generated from the exposure of 50 GXU30 to ionizing radiation. b Refer to Figure 5 for the structure of the corresponding stationary points. c Optimization energy. d Calculated with IEFPCMM06-2X/6-31G(d,p). e Includes scaled (0.9580) zero-point vibrational energy correction. f Includes scaled (0.9480) thermal correction to the enthalpy. g Calculated using the SMD-M06-2X/6-31G(d,p)) energy (including nonelectrostatic component) and IEFPCM-M06-2X/ 6-31G(d,p) thermal correction to the Gibbs free energy. h Calculated as TΔS = (ΔG ΔH). a
unpaired electron is mainly delocalized on G (Table SI-1, Supporting Information). While U rotation results in an increase in energy (ΔE = 13.0 kJ mol1), bond formation releases energy, and I2D lies 107.0 kJ mol1 below the reactant. Following bond formation, a hydrogen atom is removed (Figure 2b) to yield the same ground state singlet product (PS) as the UV pathways. This transition has a larger barrier (ΔEq = 171.8 kJ mol1, ΔGq = 165.3 kJ mol1) than that for proton removal in the UV pathway and is associated with an increase in the C8H8 distance from 1.099 Å (I2D) to 1.837 Å (TS3D) and a localization of the spin density on H8 (0.801 e). Attempts were made to include a mediating molecule (hydroxyl radical, water) in our model, but stationary points could not be obtained. The product complex (PS 3 3 3 H•, Figure 5) connected to TS3 involves a hydrogen atom (0.964 e) bridged between the terminal O50 of the backbone (d(O50 3 3 3 H•) = 2.681 Å) and O4 of U (d(O4 3 3 3 H•) = 2.859 Å). A final decomplexation step (Figures 4b and 5) connects this complex (PS 3 3 3 H•) to the infinitely separated (PS + H•) products. The overall reaction is endothermic (ΔHRxn = 23.3 kJ mol1) and slightly exergonic (ΔGRxn = 14.5 kJ mol1). Cross-Link Structure. Both reaction pathways discussed above yield the same cross-link product (PS). Comparison of the optimized geometries of 50 G[85]U30 and the corresponding natural 50 GT30 dinucleoside monophosphate57 (Figure 6a and Table S-3, Supporting Information) reveals only small changes in the backbone torsion angles and deoxyribose sugar puckering upon cross-link formation. The most substantial change from natural DNA is the orientation of the nucleobases. First, although U remains in the native anti orientation, G adopts a syn orientation, which has been similarly predicted for other purinepyrimidine intrastrand cross-links with analogous connectivity.46,49,75,76 Second, the cross-link adopts a perpendicular orientation relative to the native stacked base arrangement. Molecular dynamics simulations were performed on dsDNA with the 50 -d(GCATGGCG[85]UGCTATGC)-30 sequence to establish the cross-link structure within double helices. The crosslinked dinucleotide adopts the same conformation throughout
Figure 6. Overlay of (a) the natural 50 GT30 sequence (gray) and 50 G[85]U30 (red) obtained using the DFT dinucleoside monophosphate model and (b) the natural 50 GT30 (gray) and 50 G[85]U30 (orange) dinucleotides obtained from the MD double stranded DNA model.
the simulation (355 ns), which is characterized by a highly distorted and unwound backbone (Figure 6b and Table S-3, Supporting Information). These changes to the backbone allow the cross-link to fall in the same plane as G in natural DNA (Figure 6). Therefore, 50 G[85]U30 does not have a large impact on the flanking base pairs, which is supported by similar hydrogen-bonding occupancies found in cross-linked (7899%) and natural (8399%) DNA. The conformation of the cross-link and flanking base pairs are unchanged throughout the course of the simulation, while the orientations of the nucleotides complementary to the cross-link change to give different interstrand base interactions. Specifically, three main conformations of cross-linked DNA (designated conformers 13; see Supporting Information, page S-7) were identified (Figure 7), each of which is stable for at least 8 consecutive nanoseconds over the course of the 55 ns simulation. Conformer 1 is characterized by hydrogenbonding interactions between O4 in the WatsonCrick face of the cross-linked U and the amino groups in the WatsonCrick faces of the opposing C and A bases, which occur with occupancies of 89% and 34%, respectively. This creates an opening 50 to the cross-link (Figure 7a) and local unwinding of the complementary strand. The helix width (interstrand C10 3 3 3 C10 distance between complementary bases) at the cross-link remains relatively unchanged at G (10.763 ( 0.635 Å) and decreases at U (9.765 ( 0.785 Å) compared to that of natural DNA (10.731 ( 0.189 Å and 10.614 ( 0.277 Å, respectively) and increases in flexibility. In conformer 2 (Figure 7b), both complementary bases form ππ interactions with the cross-link. Specifically, C adopts an anti orientation about the glycosidic bond (χ = 196.6 ( 8.8) but directs the C5 and C6 atoms toward the cross-link-containing strand. This is accompanied by large distortions in the backbone of the C nucleotide as C intercalates between the cross-link and the 50 -flanking C 3 3 3 G base pair. A remains in the anti orientation (χ = 258.9 ( 11.1) and intercalates between the cross-link and the 30 -flanking G 3 3 3 C base pair. This creates a zipper-like conformation, which is known to naturally occur for mismatched DNA.77 The helix narrows at both G (8.622 ( 0.301 Å) and U (6.776 ( 0.333 Å), and there are significant distortions to the complementary A and C backbone. Finally, in conformer 3 (Figure 7c), the complementary A is intercalated 30 of the cross-link in a ππ interaction, while the C base is flipped out of the helix and displays significant flexibility in the extrahelical 2193
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Figure 7. MD three conformers of 50 G[85]U30 -containing DNA obtained from molecular dynamics simulations, highlighting the cross-linked dinucleotide (red) and complementary cytosine (blue) and adenine (green) nucleotides.
groove disappears in all conformations since the G moiety of the cross-link protrudes into this region.
’ DISCUSSION Comparison of UV Bond Formation Pathways: Multiple Pathways Possible. Two unique UV formation pathways for the
Figure 8. Illustrations of the global change to the DNA helix when cross-linked DNA adopts conformers 13 (ac, respectively), where the helix axis (left, blue) and backbone (red), as well as major (purple) and minor (green) grooves (right), are identified. The axis bend is provided in brackets and is compared to a value of 9.5 for natural DNA (black).
position (for example, see Figures 7c and S-5 (Supporting Information) (45 ns)). C primarily adopts the anti orientation (χ = 229.5 ( 28.1) as the N4 amino group hydrogen bonds with the backbone phosphate oxygen in the A nucleotide (62.5% occupancy). However, more disordered states are also observed due to the flexibility in the backbone torsion angles. The interstrand distance remains compact at the cross-linked U (7.927 ( 0.544 Å), whereas the extrahelical orientation of C causes the helix to widen substantially at the cross-linked G (11.954 ( 0.841 Å). In addition to distortions localized to the region of the crosslink, a bend occurs toward the minor groove in all conformations (Figure 8 and Table S-4, Supporting Information). Furthermore, at the cross-linked region, the minor groove broadens (conformers 1 and 3) or even disappears (conformer 2), while the major
50 G[85]U30 intrastrand cross-link were characterized in the present work where bond formation can occur on the triplet surface or can be coupled with an ISC. Both pathways involve spontaneous associative and dissociative steps (Table 1), and the overall reaction decreases in energy (ΔERxn = 250.7 kJ mol1, Table 1), is exothermic (ΔHRxn = 241.0 kJ mol1), and is exergonic (ΔGRxn = 307.9 kJ mol1). The majority of this stabilization is gained upon transition from the lowest-lying triplet state to the singlet ground state (Figure 4a), where energy is released vibrationally (ISC) or as a photon (phosphorescence). Regardless of the mechanism, bond formation is the ratedetermining step with a 7076 kJ mol1 barrier (Figure 4a), which is comparable to other DNA reactions.41,43,7476 Both pathways are viable mechanisms for cross-link formation from exposure to UV radiation and may occur experimentally. It should be possible to determine whether bond formation occurs on the triplet surface due to the emission of phosphorescence (at approximately 668 nm). In theory, this signal should be distinguishable from the fluorescence (327 nm)78 or phosphorescence (400550 nm)79 of natural DNA. However, detection of phosphorescence will likely be complicated due to the low temperatures required for a measurable intensity79 and low cross-link yields.17 Cross-Link Formation by Ionizing Radiation: An Alternate Generation Pathway. As mentioned in the Introduction, 50 G[85]U30 has not yet been experimentally detected following ionizing irradiation of BrU-containing DNA even though indirect evidence in the literature suggests generation under these conditions is possible. Our calculations indicate that cross-link formation by ionizing radiation has a rate-limiting step of hydrogen atom removal, which involves a large barrier (ΔGq = 165.3 kJ mol1), and the overall reaction is slightly thermodynamically favored (ΔGRxn = 14.5 kJ mol1). While attempts to include a molecule to facilitate hydrogen atom removal were unsuccessful, it is anticipated that a mediating molecule from the surroundings or within DNA will decrease this calculated barrier, indicating 50 G[85]U30 formation from ionizing radiation will be more favorable under cellular conditions than our calculations predict. Regardless, the magnitude of the barrier for hydrogen atom removal compared to the small barriers for the U rotation and bond formation steps suggests that hydrogen removal will be the rate-limiting step for this reaction in DNA. 2194
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Chemical Research in Toxicology Conclusions regarding whether 50 G[85]U30 will form upon exposure of BrU-containing DNA to ionizing radiation, with particular emphasis on the demanding hydrogen atom removal step (ΔGq = 165.3 kJ mol1), can be drawn by comparing our calculated pathway to those previously reported for a related cross-link generated via ionizing radiation. Specifically, the formation of an experimentally observed45,49,80 intrastrand cross-link between C8 of a 50 G and the C5-methyl radical of a 30 T (50 G[85-Me]T30 ) was calculated to be highly unfavorable (ΔGRxn ≈ 167 kcal mol1), and hydrogen removal along the formation pathway of 50 G[85-Me]T3, modeled without a mediating molecule, was determined to be the rate-determining step (ΔGq ≈ 170 kJ mol1).76 Taken together, the facts that 50 G[85]U30 formed from ionizing radiation is more kinetically and thermodynamically favored than the experimentally observed 50 G[85-Me]T30 suggests that 5 0 G[85]U3 0 is a plausible product upon exposure of X U-containing DNA to ionizing radiation. Comparison of Cross-Link Formation from UV and Ionizing Radiation: Therapeutic Importance. There are substantial differences in the calculated reaction pathways for cross-link formation upon exposure of XU-containing DNA to UV or ionizing radiation (Figure 4). First, 50 G[85]U30 formation from UV radiation is highly exergonic (ΔGRxn = 307.9 kJ mol1), while formation from ionizing radiation is only slightly exergonic (ΔGRxn = 14.5 kJ mol1). Second, the rate-determining step in the UV pathways involves bond formation and a small barrier (ΔEq = 7076 kJ mol1), while that for ionizing radiation involves hydrogen atom removal and a comparatively large barrier (ΔEq = 171.8 kJ mol1). Therefore, both kinetic and thermodynamic factors indicate that 50 G[85]U30 formation is more likely upon exposure to UV radiation. Although the production of 50 G[85]U30 from ionizing radiation has never been detected, the discussion in the previous section suggests that 50 G[85]U30 is likely to form under therapeutically relevant conditions. This includes DNA containing Cl U, which is currently the most promising XU for clinical applications.8,9 Indeed, many of the same lesions (strand breaks and interstrand cross-links) can be formed by U• generated from either UV or ionizing radiation.12,13,16,18,19,2126 Future experimental work should both verify and quantify the formation of 50 G[85]U30 in XU-substituted cellular DNA upon exposure to ionizing radiation. Cross-Link Structure and DNA Distortion: Biological Implications. Given the evidence that 50 G[85]U30 is likely to form during the therapeutic use of XU, it is especially important to understand the structure of 50 G[85]U30 and its effect on the DNA helix, which will help reveal the biological consequences of this product including its potential to contribute to cell death during anticancer treatments. Although 50 G[85]U30 has been experimentally identified as a product of radiation in a dinucleoside monophosphate,15 synthetic oligonucleotides,15,20 and cellular human DNA,17 its exact structure and orientation in DNA, as well as potential distortions inflicted upon the DNA helix, have not been determined. The dinucleoside monophosphate and dsDNA models predict unique structures for the cross-link. The most notable differences occur in the backbone conformation and orientation of the cross-linked bases relative to the natural nucleobases. Although the small model predicts that the cross-link adopts a perpendicular base arrangement that would disrupt intrastrand basebase interactions and potentially neighboring base pairs, the large model suggests that
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the cross-linked nucleobases are well-aligned in the plane of G in natural DNA (Figure 6). The twist between the G and U bases (θ, Table S-3, Supporting Information) also decreases and occurs in the opposite direction when the cross-link is in DNA compared to that in a dinucleoside monophosphate. Furthermore, the large model predicts significant unwinding of the backbone of the cross-linked dinucleotide. Although backbone unwinding was not observed along the formation pathways studied using the dinucleoside monophosphate model, backbone flexibility is known to facilitate conformational changes within DNA through low transitional pathways involving many minima,81,82 and therefore, it is unclear how unwinding would affect the barriers for crosslink formation calculated in the present work. Regardless of these differences, both models predict that the WatsonCrick face of G is unavailable for hydrogen bonding with the opposing strand, which may cause a loss of genetic information at the cross-linked G upon replication. Calculations on dsDNA predict that 50 G[85]U30 causes significant distortions to the helix, which might have implications in DNA repair.83 For example, extrahelical bases, such as C in conformer 3, can aid in the recognition of DNA mismatches (Figure 7c).84 Although distortions may enhance the excision of 50 G[85]U30 in vivo, cell-cycle checkpoints and DNA repair are generally compromised in tumors, and lesions often persist.9 Therefore, the conformations of cross-linked DNA are likely to have a greater effect on DNA replication, a hyperactive process in rapidly dividing tumor cells.85 Indeed, related Gpyrimidine cross-links with [85] or [85-Me] connectivity were found to stall DNA polymerases,48,49,52,53 and 50 G[85]U30 may behave similarly. Some DNA polymerases stall before the cross-link,48,53 while others insert the correct nucleotide opposite the cross-linked pyrimidine and stall at the cross-linked G due to the lack of an appropriate hydrogen-bonding partner.48,49,52 A DSB may result if the stalled polymerase is not resolved.86 This supports the observation that the cytotoxicity of XU following irradiation cannot be attributed to the direct formation of strand breaks from U• but may instead involve cellular processing of the initial damage leading to a strand-break.22,25 When translesion bypass synthesis is successful, steric considerations cause a purine nucleotide to be inserted opposite the cross-linked G,46,49,53,54 which is promutagenic and may thereby hamper the functioning of tumor cells. Additionally, translesion synthesis is less efficient than high-fidelity polymerase replication, which may slow tumor growth.51 This indicates that DNA damage caused by U•, including intrastrand cross-links, may have broad biological implications through a variety of cellular pathways. The global changes to the DNA helix calculated in the present work have implications for the effects of the 50 G[85]U30 cross-link in tumor cells. First, the bending of the helix axis observed for all three conformers (Figure 8) is a significant finding since DNA bending plays a role in regulating replication and transcription, and alterations to this property are commonly observed in the use of many efficient antitumor therapies.87 For example, cisplatin forms an intrastrand cross-link between adjacent G nucleotides that causes an ∼35 bend toward the major groove.2,87 Similarly, anticancer drugs like nitrogen mustard mechlorethamine form interstrand cross-links that cause up to a 17 bend in DNA,88 while the intrastrand cross-links induced by mitomycin C cause a 15 bend.89 The bend in 50 G[85]U30 containing DNA has a substantial effect on the grooves of the helix (Figure 8 and Table S-4, Supporting Information). Second, the observed alterations to the grooves of the helix in the presence of 2195
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Chemical Research in Toxicology the cross-link (Figure 8 and Table S-4, Supporting Information) are also important findings. Disappearance of a groove is known to occur in highly deformed structures67 and may significantly affect the cell cycle since many proteins bind to DNA through interactions with the grooves.90 Overall, alterations to both the bending and the grooves of DNA when the cross-link is present parallel conformational changes induced by binding of cisplatin2,87 and are therefore indicative of the potential of 50 G[85]U30 to contribute to cell death through similar pathways.
’ CONCLUSIONS The formation pathways for 50 G[85]U30 following exposure of XU-containing DNA to UV or ionizing radiation has been examined. Following UV irradiation and the creation of the 50 G•+U•30 reactant, the reaction may proceed through bond formation on the triplet surface or bond formation via an intersystem crossing (ISC). Similar rate-limiting barriers (ΔEq = 7076 kJ mol1, bond formation) preclude computational identification of the preferred UV-formation pathway. Cross-link formation from ionizing radiation (or UV homolysis of 5-iodoracil) has a larger ratelimiting barrier and is less exoergonic than from that of UV radiation. However, since 50 G[85]U30 formation is kinetically and thermodynamically more favored than the formation of related experimentally observed purinepyrimidine cross-links under these same reaction conditions, it is likely that 50 G[85]U30 forms under therapeutically relevant conditions. The structure of 50 G[85]U30 has been presented here for the first time. Regardless of the model used, the cross-linked G adopts the syn orientation. This has important implications for DNA replication since similar cross-links stall polymerases or cause error-prone translesion synthesis. Distortions in crosslinked DNA are largely limited to the cross-linked dinucleotide and the opposing C and A nucleotides, with negligible effects on the flanking base pairs. Local distortions include unwinding of the backbone to accommodate the cross-link in the helix. Additionally, the complementary nucleotides adopt nonnative configurations in response to the damage, which results in three main conformations of cross-linked DNA. Global changes to the DNA helix upon cross-link formation include a bend in the helix axis and alterations to the major and minor grooves. These distortions parallel those induced by efficient antitumor therapies, such as cisplatin, which indicates that 50 G[85]U30 may contribute to the cytotoxicity of XU using similar mechanisms. The present study enhances our understanding of the formation of an important intrastrand cross-link, as well as sheds light on the biological implications of this cross-link in DNA, and therefore its potential contribution to the mechanism of action of X U in antitumor therapies. While the reaction pathways determined in this study have provided initial insight into cross-link formation and allowed for comparisons to related small-model work, future studies are required to characterize the effects of the surrounding DNA environment. It will also be important for experiments to confirm that these cross-links are products of ionizing radiation, as well as establish the role of these lesions in the therapeutic functions of the 5-halouracils. ’ ASSOCIATED CONTENT
bS
Supporting Information. Details of the ISC and MD simulations (including atom types and charges used), MD
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results, the coordinates of all DFT stationary points, and the coordinates of cross-linked DNA. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Funding Sources
This work was supported by the Natural Sciences and Engineering Research Council (NSERC), the Canadian Foundation for Innovation (CFI), the Canada Research Chairs program, and the National University of Ireland—Galway. C.D.M.C. thanks NSERC (Julie Payette), Alberta Innovates-Technology Futures, Alberta Scholarship Programs, and the University of Lethbridge for student scholarships. We thank the Upscale and Robust Abacus for Chemistry in Lethbridge (URACIL) for computing resources.
’ ABBREVIATIONS A, adenine; BrU, 5-bromouracil; C, cytosine; DFT, density functional theory; DSB, double strand break; dsDNA, doublestranded DNA; G, guanine; G•+, guanine radical cation; XU, 5-halouracil; ISC, intersystem crossing; MD, molecular dynamics; nm, nanometer; ns, nanosecond; ps, picosecond; SSB, single strand break; T, thymine; U•, uracil-5-yl radical ’ REFERENCES (1) Georgakilas, A. G. (2011) From Chemistry of DNA Damage to Repair and Biological Significance. Comprehending the Future. Mutat. Res., Fundam. Mol. Mech. Mutagen. 711, 1–2. (2) Rajski, S. R., and Williams, R. M. (1998) DNA Cross-Linking Agents As Antitumor Drugs. Chem. Rev. 98, 2723–2796. (3) Siddik, Z. H. (2003) Cisplatin: Mode of Cytotoxic Action and Molecular Basis of Resistance. Oncogene 22, 7265–7279. (4) Jung, Y., and Lippard, S. J. (2007) Direct Cellular Responses to Platinum-Induced DNA Damage. Chem. Rev. 107, 1387–1407. (5) Djordjevic, B., and Szybalski, W. (1960) Genetics of Human Cell Lines: III. Incorporation of 5-Bromo- and 5-Iododeoxyuridine into the Deoxyribononucleic Acid of Human Cells and Its Effect on Radiation Sensitivity. J. Exp. Med. 112, 509–531. (6) Szybalski, W. (1974) X-ray Sensitization by Halopyrimidines. Cancer Chemother. Rep. 1 (58), 539–557. (7) Kinsella, T. J., Mitchell, J. B., Russo, A., Morstyn, G., and Glatstein, E. (1984) The Use of Halogenated Thymidine Analogues As Clinical Radiosensitizers: Rationalle, Current Status, And Future Prospects: Non-Hypoxic Cell Sensitizers. Int. J. Radiat. Oncol., Biol., Phys. 10, 1399–1406. (8) Greer, S., Schwade, J., and Marion, H. S. (1995) Five-Chlorodeoxycytidine and Biomodulators of Its Metabolism Result in Fifty to Eighty Percent Cures of Advanced EMT-6 Tumors When Used with Fractionated Radiation. Int. J. Radiat. Oncol., Biol., Phys. 32, 1059–1069. (9) Greer, S., Alvarez, M., Mas, M., Wozniak, C., Arnold, D., Knapinska, A., Norris, C., Burk, R., Aller, A., and Dauphinee, M. (2001) Five-Chlorodeoxycytidine, a Tumor-Selective Enzyme-Driven Radiosensitizer, Effectively Controls Five Advanced Human Tumors in Nude Mice. Int. J. Radiat. Oncol., Biol., Phys. 51, 791–806. (10) Prados, M. D., Seiferheld, W., Sandler, H. M., Buckner, J. C., Phillips, T., Schultz, C., Urtasun, R., Davis, R., Gutin, P., Cascino, T. L., Greenberg, H. S., and Curran, W. J. (2004) Phase III Randomized Study of Radiotherapy Plus Procarbazine, Lomustine, and Vincristine with or without BUdR for Treatment of Anaplastic Astrocytoma: Final Report of RTOG 9404. Int. J. Radiat. Oncol. 58, 1147–1152. (11) von Sonntag, C. (2006) Free-Radical-Induced DNA Damage and Its Repair, Springer-Verlag, Berlin, Germany. 2196
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