DNA Distortion Caused by Uracil-Containing Intrastrand Cross-Links

Feb 1, 2016 - Four uracil-containing intrastrand cross-links have been detected in human cells upon UV irradiation of 5-bromouracil-containing DNA, na...
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DNA Distortion Caused by Uracil-Containing Intrastrand Cross-Links Cassandra D. M. Churchill,† Leif A. Eriksson,‡ and Stacey D. Wetmore*,† †

Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, Alberta T1K 3M4, Canada ‡ Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, Göteborg 405 30, Sweden S Supporting Information *

ABSTRACT: Four uracil-containing intrastrand cross-links have been detected in human cells upon UV irradiation of 5bromouracil-containing DNA, namely 5′-G[8-5]U-3′, 5′-U[58]G-3′, 5′-A[8-5]U-3′, and 5′-A[2-5]U-3′. These lesions feature unique composition and connectivity compared with other intrastrand cross-links reported in the literature. For the first time, structural information obtained using molecular dynamics (MD) simulations reveal that all four lesions distort the DNA helix, which can involve an extrahelical location of the cross-link, changes in the helical interactions of the complementary nucleotides, or disruption of hydrogen bonding in the flanking base pairs up to two positions from the cross-linked site; however, the degree of distortion varies between the cross-links, being affected by the sequence, nucleobase−nucleobase connectivity, and the purine involved. Most importantly, the relative distortion of the damaged DNA provides the first structural explanation for the observed abundances of the four uracil-containing cross-links. Furthermore, the highly distorted conformations suggest that these lesions will likely have severe implications for DNA replication and repair processes in cells.

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INTRODUCTION The induction of DNA damage is one strategy clinically employed to kill cancer cells.1−3 For example, platinumcontaining anticancer drugs, such as cisplatin, carboplatin, and oxaliplatin, bind to DNA in vivo and thereby trigger cell death. Research has shown that complex lesions are the most desirable product of DNA-targeting anticancer therapies because these are difficult to repair,3−6 especially when coupled to the compromised repair pathways in tumors.7 As a result, radiation has also been commonly used to fight cancer by inducing single -strand breaks, double-strand breaks, tandem lesions, and clustered lesions.4,5,8,9 Radiosensitizing agents can be coadministered with radiation to amplify DNA damage to enhance the effectiveness of existing cancer therapies. One important class of radiosensitizing agents is the 5-halopyrimidines, which include chloro-, bromo-, and iodo-substituents.10−14 Of particular interest are the thymidine analogues, the 5-halouracils (XU), which produce the uracil-5-yl radical (U·, Figure 1) upon radiation-induced addition of an electron and halide dissociation.6,12 Clinical trials in the 1990s focused on the halouracils due to their relatively low toxicity in nonradiated tissue,15 the ability for their preferential incorporation into rapidly dividing tumor cells,12,13 and the reactivity of U· under the hypoxic conditions in tumor cells.16 Although XU agents were widely examined in clinical trials,12−14,17−21 little survival advantage was observed in these previous studies.13 This outcome was largely attributed to poor incorporation of XU into tumors relative to normal tissues;13,22 however, recent advances have improved the © XXXX American Chemical Society

Figure 1. Structure of the uracil-5-yl radical produced upon irradiation of 5-halouracil (XU).

incorporation of XUs into tumor DNA.11,23,24 Specifically, 5chloro-2′-deoxycytidine can be administered, where kinases, like deoxycytidine kinase, and cytosine deaminase convert this prodrug to its active form, 5-chloro-2′-deoxyuridine triphosphate, which can be incorporated into DNA. Elevated levels of deoxycytidine kinase and cytosine deaminase in tumor cells can be used to preferentially incorporate 5-chloro-2′-deoxyuridines into tumor tissue when coadministered with several enzyme Received: October 22, 2015 Revised: January 29, 2016

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The Journal of Physical Chemistry B inhibitors.11,23 Mutations to human deoxycytidine kinase have also been identified that can increase the specificity of this enzyme for 5-halo-2′-deoxycytidines.24 In addition to unique approaches that use the existing cellular machinery to selectively incorporate XUs into tumor DNA, interest in the use of 5-halouracils in anticancer strategies has rematerialized due to recent detailed investigations of the cellular reactions of U·. Specifically, although it is confirmed in the literature that single- and double-strand breaks result from this radical species and lead to cell death,15,25−35 strand breaks alone cannot account for the survival curves observed in cells where the uracil-5-yl radical is produced.25,32,33 This indicates that other types of damage are also formed and persist in cells25,33 or this other damage progresses into strand breaks.25,32 Cross-links have more recently been identified as pr od uc t s f o l lo w in g ir r a d ia tio n of X U-containing DNA.15,26−29,36−39 Specifically, interstrand cross-links have been detected by Hunting and coworkers,15,26−29 while Wang et al. have identified intrastrand cross-links.37−39 Because intrastrand cross-links have been shown to pose a particular challenge for cellular processing of DNA, such as replication and damage excision,40−47 these advances make it particularly important to revisit the use of halouracils as antitumor therapies. Furthermore, because the simultaneous formation of several types of DNA damage may help evade resistance currently associated with many anticancer drugs by forming clustered lesions that are particularly difficult to repair,48−50 the mechanism of action of the DNA lesions associated with XU needs to be uncovered. Our work focuses on the intrastrand cross-links that have been identified following UV irradiation of 5-bromouracilcontaining cellular DNA (Figure 2).38 Similar to other intrastrand DNA cross-links discussed in the literature,40,43,44,51,52 the lesions from XU form exclusively with purine nucleobases and exhibit sequence-selective formation preferences. The most abundant lesion contains a covalent bond between C8 of a 5′-guanine and C5 of a 3′-uracil (denoted 5′-G[8-5]U-3′, Figure 2a), while the abundance for the remaining cross-links decreases as 5′-U[5-8]G-3′ > 5′-A[85]U-3′ > 5′-A[2-5]U-3′ (Figure 2b−d).38 Unfortunately, there are no crystal structures or NMR data available to provide structural information about the uracil−purine intrastrand cross-links in DNA. Nevertheless, computer simulations can provide critical information about the structure of cross-links and their effects on DNA helices. Indeed, Dumont and coworkers have used computational methods to gain critical information about the 5′-G[8-5m]T-3′, 5′-G[8-5m]mC-3′, and 5′-G[8-5]C-3′ lesions formed in natural DNA helices.53,54 In this light, we previously used density functional theory (DFT) and molecular dynamics (MD) simulations to determine the structure, as well as the formation pathway, of the most abundant 5′-G[8-5]U-3′ lesion.55 Our calculations uncovered three conformations of 5′-G[8-5]U-3′ cross-linked DNA, which most predominantly differ in the conformations of the complementary bases. Nevertheless, all conformations of 5′G[8-5]U-3′ cross-linked DNA contain major helical distortions (such as bending and groove alterations at the lesion site), which emphasize that this cross-link may contribute to the cytotoxicity of halouracils. The present study extends upon our previous work by examining the influence of the experimentally detected, but less abundant, cross-links arising from irradiation of 5-bromouracil-containing DNA to further explore the structural impact of cross-link formation. Specifically, MD

Figure 2. Structure and numbering of the uracil-containing intrastrand cross-links known to form upon irradiation of XU in cellular DNA. Definitions are provided for the θ dihedral angle (red), which determines the relative orientation of the cross-linked nucleobases, and the χ dihedral angle (blue; ∠(O4′C1′N9C4) for purines and (∠(O4′C1′N1C2) for pyrimidines), which dictates the glycosidic bond orientation to be anti (χ = 180 ± 90°) or syn (χ = 0 ± 90°).

simulations are used to assess the structure of double-stranded DNA (dsDNA) containing 5′-U[5-8]G-3′, 5′-A[8-5]U-3′, or 5′-A[2-5]U-3′. For the first time, our work provides a structural explanation for the observed formation preferences of these uracil-containing DNA cross-links. Furthermore, knowledge of the structures of the cross-links allows the potential impact of each DNA damage product on biological pathways to be discussed, with an emphasis placed on the effects of cross-link formation on DNA replication and repair.

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COMPUTATIONAL METHODS Our previous work on the 5′-G[8-5]U-3′ cross-link successfully used MD simulations on a DNA model to gain accurate structural information about the cross-link and clues regarding the biological consequences of cross-link formation.55 Therefore, MD simulations were performed on dsDNA containing the 5′-U[5-8]G-3′, 5′-A[8-5]U-3′, or 5′-A[2-5]U-3′ intrastrand cross-links using protocols coded in the macro md_run.mcr module of the YASARA program.56 The 5′-d(GCATGGCGTGCTATGC)-3′ and 5′-d(GCATGGCATGCTATGC)-3′ sequences were initially generated using the nucgen module of AmberTools.57 Cross-linked DNA for simulations was obtained by overlaying (all-electron M06-2X/6-31G(d,p) optimized) cross-linked dinucleotide monophosphates onto the central dinucleotides (in bold) in the corresponding canonical dsDNA. The AMBER03 force field58 was used to describe the natural DNA components, while the cross-linked dinucleotide was described with GAFF.59 (GAFF parameters and atom types are provided in Table S1, Supporting Information.) Each crosslinked dsDNA was explicitly solvated in a 21 Å periodic box extending from the solute in each direction. Na+ and Cl− ions B

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Figure 3. Structures of (a) 5′-U[5-8]G-3′ Conformer 1, (b) 5′-U[5-8]G-3′ Conformer 2, (c) 5′-A[8-5]U-3′, (d) 5′-A[2-5]U-3′, (e) 5′-G[8-5]U-3′ Conformer 1, (f) 5′-G[8-5]U-3′ Conformer 2, and (g) 5′-G[8-5]U-3′ Conformer 3 obtained from molecular dynamics simulations. The cross-linked dinucleotide is indicated in red, and the complementary bases are indicated in blue and green. Other distorted base pairs are indicated with orange and cyan as appropriate.

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RESULTS 5′-U[5-8]G-3′. In both conformers of 5′-U[5-8]G-3′containing dsDNA that occur over the 55 ns simulation (Figure 3a,b), the 3′-G nucleoside adopts the anti orientation about the glycosidic bond of canonical DNA (the χ dihedral angle (Figure 2) equals 180 ± 90°), while the 5′-U nucleoside adopts the syn orientation (χ = 0 ± 90°; Table S2). Despite the syn orientation of U, the ∠(O4′C1′N1) angle is wider than natural DNA and thereby exposes O2, N3, and O4 of the U Watson−Crick edge to the opposing strand, albeit in an inverted arrangement. In both conformers, the anti orientation of G coupled with widening of ∠(O4′C1′N9) directs N7 and O6 of the G Hoogsteen edge toward the opposing strand. Nevertheless, the cross-linked nucleotides in the two DNA conformers are in part distinguished by significant differences in the average χ dihedral angle, which deviates by up to 30° (Table S2). The θ dihedral angle (Figure 2) that defines the relative orientation of the cross-linked nucleobases deviates from planarity (180°) in both conformers (Table S2); however, the average θ for each conformation deviates by up to 25° over the course of the MD simulations, with greater nonplanarity exhibited in conformer 1 (θ = 160.3 ± 7.8°) than conformer 2 (θ = 185.4 ± 10.0°). In terms of the sugar−phosphate backbone, both conformers have C2′-endo puckering of the 5′sugar and C1′-exo puckering of the 3′-sugar. Therefore, the backbone of the cross-linked dinucleotide in conformers 1 and 2 differs most substantially at the αU, αG, and ζG dihedral angles (Table S2), where the subscript indicates the nucleotide involved. Furthermore, the backbone of the cross-linked dinucleotide is unwound with respect to natural DNA in both conformers (Figure 3a,b). Despite some similarities, the two conformations of 5′-U[58]G-3′-containing dsDNA significantly differ in the orientation of the cross-linked dinucleotides, complementary nucleotides, and flanking base pairs. In conformer 1, the cross-link adopts an extrahelical position, and, as a result, the complementary A and

were added to neutralize the system and achieve a physiologically relevant NaCl concentration of 0.9% (mass percent). The solvated complexes were first minimized, followed by a simulation at 298 K and 1 atm in explicit water. The particle-mesh Ewald (PME) method was used to describe long-range electrostatics, with a 10.54 Å cutoff for van der Waals forces. Dual time steps of 1.25 and 2.5 fs were employed for intra- and intermolecular forces, respectively. Snapshots were saved every 25 ps. Each simulation was conducted for 55 ns, with structural equilibration reached after 4 ns (determined by rmsd; Figure S1), which resulted in 51 ns of production-phase data. Stable conformations were identified in each of the three simulations based on structural similarities and the requirement that a conformation be present for a minimum of 8 consecutive nanoseconds. Two unique conformations were identified for dsDNA containing 5′-U[5-8]G-3′, with conformer 1 occurring from 4.000 to 39.375 ns and conformer 2 from 39.875 to 55.000 ns. Extending the simulation to 70 ns showed that conformer 2 also prevails over this extended time frame. Regardless of which structure dominates the conformational space, both conformers are discussed in the main text because they may have differing cytotoxic effects. For 5′-A[8-5]U-3′containing dsDNA, a stable conformation was identified between 19.600 and 43.000 ns. For a brief period following 43.000 ns, a small perturbation was detected in the backbone of the cross-link, after which time the same stable conformer was present for the remainder of the simulation. 5′-A[2-5]U-3′containing DNA adopted a stable conformation over the entire (4.000 to 55.000 ns) simulation. Simulations were also performed for the corresponding natural dsDNA sequences, resulting in 25 ns of production-phase data. For each crosslinked DNA conformer and the natural dsDNA strands, average structures were generated for analysis, including evaluation of backbone distortions (Table S2) and the calculation of the helical axis and groove parameters using the Curves+ program60 (Table S3). C

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The Journal of Physical Chemistry B C nucleotides do not interact with the cross-link (Figure 3a). Instead, the complementary A adopts the syn orientation, moves further into the helix, and forms inter- and intrastrand stacking interactions with its 3′ base pair, while the complementary C adopts the anti orientation and interacts with the sugar−phosphate backbone of the opposing strand. These significant distortions widen the helix at the cross-linked U nucleotide (d(C1′−C1′) = 13.272 ± 0.695 Å). Nevertheless, the helix width is similar to, but more flexible than, natural DNA (10.672 ± 0.244 Å) at the cross-linked G nucleotide (10.785 ± 0.507 Å). Although the G···C base pair flanking the 5′ side of the cross-link is essentially undisturbed (hydrogenbonding occupancy of 88−98%), significant changes occur to the 3′-flanking base pairs (Figure 3a). Most notably, G of the flanking C···G base pair (Figure 3a, orange) adopts the high anti orientation (χ = 295.5 ± 14.4°) and interacts with the cross-link via π−π stacking interactions rather than hydrogen bonding to its complementary C, which adopts a syn orientation (χ = 306.7 ± 14.0°). These disruptions push the A in the next T···A pair (Figure 3a, cyan) out of the helix and thereby eliminate Watson−Crick hydrogen bonding in this base pair. Thus, significant distortions occur in the DNA backbone of both the cross-linked and complementary strands within a four base-pair region that includes the cross-link and the −1 and −2 base pairs with respect to the cross-link. In conformer 2, the cross-link is extrahelical and does not interact with the complementary bases, which remain in similar orientations as described for conformer 1 (Figure 3a); however, the helix is wider at both cross-linked nucleotides (13.658 ± 0.524 Å and 12.161 ± 0.584 Å at the U and G nucleotides, respectively) compared with conformer 1. As with conformer 1, the 5′-flanking G···C base pair of the cross-link is undisturbed and maintains Watson−Crick hydrogen bonding (89−99%); however, the C···G base pair flanking the 3′ side of the crosslink (Figure 3b, orange) also engages in Watson−Crick-like hydrogen bonding in conformer 2 (84−96%) despite the syn conformation of C (χ = 307.3 ± 11.7°) and the high anti orientation of G (χ = 294.0 ± 27.2°). Furthermore, the entire C···G base pair interacts with the cross-link through π−π stacking. Consequently, only small structural changes occur in the next T···A base pair (Figure 3b, cyan), which engages in Watson−Crick hydrogen bonding (61−96%). Nevertheless, significant distortion occurs to the backbone of both strands at the cross-link and the two base pairs on its 3′ side (Table S2). The local distortions in the 5′-U[5-8]G-3′-containing DNA previously described cause global changes to the helix. Specifically, the significant alterations to the backbone bend the helix axis toward the minor groove in both conformers (Figure 4a,b), which alters both the major and minor grooves. The helical bend of conformer 1 is 51.2°, which substantially narrows the major groove at the cross-link and significantly widens the minor groove. In fact, the minor groove is wider than the major groove (Table S2), and the major groove disappears in the region 3′ with respect to the cross-link in conformer 1. Although the helical bend of conformer 2 (15.3°) is much less than conformer 1, the major groove also disappears at the cross-linked dinucleotide in conformer 2, while the minor groove narrows. 5′-A[8-5]U-3′. One conformation of 5′-A[8-5]U-3′-containing DNA occurs over the 55 ns simulation. In this cross-link (Figure 3c), A adopts the syn orientation about the glycosidic bond (χA = 42.0 ± 7.5°), while U adopts the anti orientation (χU = 212.8 ± 8.7°). In addition to deviations about χ, the

Figure 4. Global distortions induced to the DNA helix by (a) 5′-U[58]G-3′ Conformer 1, (b) 5′-U[5-8]G-3′ Conformer 2, (c) 5′-A[85]U-3′, and (d) 5′-A[2-5]U-3′.

backbone conformation of the 5′-A[8-5]U-3′ dinucleotide is unwound compared with natural DNA (Table S2), and both deoxyribose moieties of the cross-linked dinucleotide adopt C1′-exo puckering. The cross-linked bases are twisted with respect to each other (θ = 165 ± 7.5°), which is similar to the relative orientation in conformer 1 of 5′-U[5-8]G-3′ crosslinked DNA. The 5′-A[8-5]U-3′ cross-link adopts an extrahelical position, which prevents direct hydrogen bonding between the cross-link and the complementary bases. As a result, the backbones of the complementary T and A nucleotides are distorted. Furthermore, the helix widens at the cross-linked A (11.420 ± 0.878 Å) and U (12.499 ± 0.781 Å) nucleotides compared with natural DNA (10.716 ± 0.277 Å and 10.609 ± 0.288 Å for the A and T nucleotides, respectively). Unlike as discussed for 5′-U[5-8]G3′, all flanking base pairs are unperturbed in the presence of 5′A[8-5]U-3′. Indeed, both the 3′ and 5′ flanking pairs with respect to the cross-link maintain a hydrogen-bonding occupancy (79−98%) similar to that observed in the corresponding natural DNA sequence (80−98%). Nevertheless, global distortions occur to the helix due to the presence of 5′A[8-5]U-3′. Most significantly, the cross-link generates a 26.4° bend in the helix axis (Figure 4c), which removes the major groove in several locations along the DNA strand (Table S3). 5′-A[2-5]U-3′. Only one conformation of 5′-A[2-5]U-3′containing DNA occurs over the course of the 55 ns MD D

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U[5-8]G-3′ is unable to interact with the opposing strand, which inevitably decreases the overall strand stability. Furthermore, this distortion is coupled to significant unwinding of the backbone in the complementary strand opposing the cross-link. As a result, the 5′-U[5-8]G-3′ lesion distorts the helix outside of the lesion site, specifically disrupting hydrogen bonding in the 3′-flanking base pair with respect to the crosslink (Figure 3a, orange) and even interrupting the pairing in the next 3′ base pair (Figure 3a, cyan). Therefore, although the 5′G[8-5]U-3′ and 5′-U[5-8]G-3′ cross-links are isomeric, each lesion induces very different local structural changes to DNA. This suggests that the structural effects of the lesion depend on the direction of the sequence (i.e., 5′-purine versus 3′-purine). Unlike the G-containing cross-links, which can adopt two or three conformations, the A-containing lesions adopt a single stable conformation over the course of the MD simulations. Although both nucleosides in the 5′-A[2-5]U-3′ lesion assume the canonical anti orientation about the glycosidic bond, A in the 5′-A[8-5]U-3′ cross-link adopts the syn orientation, while U remains in the anti conformation. Furthermore, larger deviations in important backbone dihedral angles occur for 5′A[2-5]U-3′ compared with 5′-A[8-5]U-3′, indicating that some unwinding of the strand occurs upon incorporation of 5′-A[25]U-3′ (Figure 3c,d). These deviations suggest that the structure of damaged DNA depends on the nucleobase− nucleobase connectivity. Comparison of the guanine and adenine-containing lesions reveals that the purine involved in the uracil cross-link can significantly affect the structure at the lesion site in the damaged helix. For example, although the 5′-G[8-5]U-3′ crosslink remains in the helix, the 5′-A[8-5]U-3′ adenine-containing lesion adopts an extrahelical position, which eliminates discrete interactions with the opposing strand. Therefore, nucleobase composition also has a significant effect on structure. U Cross-Links Bend the DNA Helix Similar to Other DNA-Targeting Anticancer Agents. Regardless of differences in the local conformation of the cross-links and surrounding bases, the incorporation of each U-containing cross-link into DNA leads to global helical distortions. Specifically, DNA containing any of the uracil cross-links exhibits a bend in the helical axis; however, there is a significant range in the bend (15−51°), that indicates that intrastrand cross-links can have unique effects on the helix. The most significant bend occurs upon the formation of 5′-U[5-8]G-3′ (51.2°), with the bend induced by the remaining cross-links decreasing as 5′-G[8-5]U-3′ (23−41°)55 > 5′-A[8-5]U-3′ (26.4°) > 5′-A[2-5]U-3′ (15.8°). Regardless of the cross-link formed, the global distortions induced by the uracil-containing cross-links parallel those previously reported for DNA crosslinks containing a thymine (5′-G[8-5m]T-3′; 21°),53,54 5methylcytosine (5′-G[8-5m]mC-3′; 10°),53 or cytosine (5′G[8-5]C-3′; 24°).53,54 Most importantly, the distortions to the helical axis induced by the uracil-containing intrastrand crosslinks are similar to or even greater than those induced by successful anticancer agents, like cisplatin (35°),61 mechlorethamine (17°),62 and mitomycin C (15°).63 Furthermore, the damage induced by the uracil-5-yl radical has the potential to form of a wide variety of lesions beyond intrastrand cross-links (including strand breaks, interstrand cross-links, alkali labile lesions, and clustered lesions),15,25−33,35,36 which may help evade the resistances currently associated with many drugs.4,5 Deviations in Local and Structural Distortions Induced by U Cross-Links Provide Clues about the

simulation (Figure 3d). In 5′-A[2-5]U-3′, both A and U adopt the anti orientation (χA = 203.4 ± 16.8° and χU = 201.7 ± 11.3°). Despite this canonical DNA conformation, the backbone of the 5′-A[2-5]U-3′ dinucleotide is unwound with respect to natural DNA. Furthermore, deviations are observed in the canonical sugar puckering, where the 5′ sugar adopts a C1′-exo pucker and the 3′ sugar adopts the C2′-endo conformation. The twist between the cross-linked bases (θ = 166.1 ± 15.0°) is similar to that in 5′-A[8-5]U-3′. As observed for 5′-A[8-5]U-3′, the 5′-A[2-5]U-3′ cross-link adopts an extrahelical position and is unable to hydrogen bond with its complementary bases. Distortions occur in the backbone of the complementary nucleotides, which are generally larger and exhibit more significant standard deviations than for 5′-A[8-5]U-3′. Indeed, some unwinding occurs to 5′A[2-5]U-3′-containing DNA (Figure 3d). There is also significant widening of the helix at the cross-linked A (11.396 ± 1.277 Å) and U (12.732 ± 1.323 Å) nucleotides compared with natural DNA; however, the flanking base pairs are unperturbed in the presence of 5′-A[2-5]U-3′, with hydrogenbonding occupancies of 81−99% (compared with 80−98% in natural DNA). Finally, the helix bends by 15.5° due to this cross-link (Figure 4d), which alters several of the major and minor grooves in the helix (Table S3).

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DISCUSSION Local Distortions Induced by U Cross-Links in DNA Depend on the Nucleobase−Nucleobase Direction, Connectivity, and Composition. In addition to the 5′U[5-8]G-3′, 5′-A[2-5]U-3′, and 5′-A[8-5]U-3′ cross-links considered in the present work, the structure of the most abundant 5′-G[8-5]U-3′ lesion in dsDNA has been previously studied using analogous computational approaches.55 In the three conformers of 5′-G[8-5]U-3′ previously characterized (Figure 3e−g), both nucleosides in the cross-link adopt the anti orientation, and the lesion remains in the helix, protruding into the major groove. As a result, the cross-link interacts with at least one of the original complementary bases in all three conformers. In conformer 1 (Figure 3e), the Watson−Crick hydrogen-bonding face of uracil is exposed to the complementary strand, and therefore U forms hydrogen-bonding interactions with the opposing A. In addition, U hydrogen bonds with the opposing C, which causes an opening 5′ with respect to the lesion and local unwinding in the complementary strand. In the other two conformers, the cross-link forms π−π interactions with the complementary bases. In conformer 2 (Figure 3f), 5′-G[8-5]U-3′ engages in π−π interactions with both the opposing C and A, with the latter stacking between the cross-link and the 3′-base pair. This causes significant distortion in the backbone of the complementary C nucleotide and a local zipper-like DNA conformation. In conformer 3 (Figure 3g), the lesion stacks in a similar conformation with A, as discussed for conformer 2, while the opposing C adopts an extrahelical position. Regardless of the conformer considered, 5′-G[8-5]U-3′ does not have a large impact on the flanking base pairs, and therefore lesion-induced distortions are limited to the cross-link formation site. The direction of the DNA sequence (i.e., the 5′-purine versus 3′-purine) affects intrastrand cross-link structure. In contrast with the 5′-G[8-5]U-3′ lesion, U in the 5′-U[5-8]G-3′ crosslink is in a non-native syn orientation, and the entire cross-link adopts an extrahelical position in dsDNA regardless of the strand conformation considered (Figure 3a,b). Therefore, 5′E

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The Journal of Physical Chemistry B Experimentally Observed Relative Yields. The observed yields of U cross-links formed following UV-radiation of BrUcontaining DNA decrease as 5′-G[8-5]U-3′ > 5′-U[5-8]G-3′ > 5′-A[8-5]U-3′ > 5′-A[2-5]U-3′.38 This indicates a dependence on the direction of the DNA sequence, with a preference for a 5′-purine over a 3′-purine, and on the nucleobase composition, with a preference for G over A. Although the factors that dictate this preference have yet to be identified, several ideas have been put forward to explain the experimental formation preference of the 5′-G[8-5-Me]T-3′ and 5′-T[5-Me−8]G-3′ cross-links produced following exposure of natural DNA to ionizing radiation.44,51,64 These proposals include alternate reaction mechanisms and ease of hydrogen atom removal along the formation pathway, which is related to the accessibility of the linkage (C8) site for hydrogen abstraction;65,66 however, intrastrand cross-links between purines and pyrimidines in dsDNA exhibit a 5′-purine preference regardless of the method of generation (UV or ionizing radiation)67 or type of DNA (natural or modified, cellular or synthetic, dsDNA or dinucleoside monophosphate), 37,38,40,41,43−45,51,52,64,67−69 which suggests that more general selection rules must dictate observed cross-link formation preferences. Experimental studies have noted a correlation between the sequence preference of purine−pyrimidine intrastrand crosslinks and the proximity of the nucleobase sites forming the bond,51,52,67−69 with higher yields resulting from a reduced distance between the reactive centers. For example, the distance between C8 of the purine and the C5-methyl group of thymine is shorter in the 5′-purine-T-3′ (3.8 Å) sequence than the 5′-Tpurine-3′ (6.3 Å) sequence,51 which correlates with the 5′purine formation preference of thymine-containing cross-links. Proximity may also be a governing factor in the formation of 5′G[N2-5]U-3′ and 5′-U[5−N2]G-3′ in a (flexible) dinucleoside monophosphate but not cellular DNA,37,38 where fiber diffraction data indicates d(N2−C5) = 5.2 Å for the 5′-GU-3′ sequence and d(C5−N2) = 7.8 Å for the 5′-UG-3′ sequence in B-DNA.57 Most importantly, the preference for 5′-purine-U-3′ cross-links is supported by proximity of sites in (undamaged) dsDNA. Specifically, fiber diffraction data57 indicate that the C8 site of the purine and the C5 site of the pyrimidine are closer in a 5′-purine (3.9 Å) than a 3′-purine (5.1 Å) sequence due to the natural twist of DNA. Our simulations on natural DNA helices similarly indicate the C8−C5 distance is shorter for a 5′G or 5′-A sequence (4.02 ± 0.31 or 4.00 ± 0.32 Å, respectively) than a 3′-G or 3′-A sequence (4.50 ± 0.74 Å or 4.64 ± 0.40 Å, respectively). In addition to proximity between the sites forming the crosslink, the present work highlights the importance of the structural features of the cross-linked DNA in determining cross-link formation preferences. For example, prior to 5′-G[85]U-3′, both nucleosides adopt the native anti orientation, while 5′-U[5-8]G-3′ formation requires U to first adopt the syn orientation, which suggests an additional rotational barrier must be overcome before the 5′-U[5-8]G-3′ lesion can form in DNA. Furthermore, 5′-G[8-5]U-3′ remains intrahelical (protruding into the major groove) in all DNA conformations, which changes the DNA grooves and helix axis but causes little disruption to the flanking base pairs. As a result, interactions between the cross-link and the opposing bases are highly probable (with the exception of the conformer with an extrahelical opposing C). In contrast, the 5′-U[5-8]G-3′ lesion is extrahelical in both DNA conformers and significantly disrupts (one or two) 3′-flanking base pairs. As a result, the

helical bend induced by 5′-U[5-8]G-3′ (51.2°) is greater than that for any conformation of 5′-G[8-5]U-3′-containing DNA (23−41°). Combined, these structural features indicate that the 5′-G[8-5]U-3′ lesion is better accommodated in the DNA helix. Most importantly, the greater distortion observed for 5′-U[58]G-3′ may result in less favorable kinetic and thermodynamic parameters for the formation reaction, which may explain why there is a greater preference for 5′-G[8-5]U-3′ formation. Similarly, although both adenine-containing cross-links adopt an extrahelical position, there are more significant structural changes in the 5′-A[2-5]U-3′ than 5′-A[8-5]U-3′ cross-link, including backbone distortion, which correlates with the greater preference for 5′-A[8-5]U-3′ formation. Thus, greater distortions in the structure of the cross-link itself or cross-linked DNA helix decrease the formation preference. This proposal agrees with previous literature, concluding that helical distortions contribute to the energetic barrier for cross-link formation more than intrinsic base reactivity.53 Unfortunately, neither proximity based on B-DNA structure nor differences in structural features explain the preference for G- over A-containing uracil lesions; however, this is not surprising because the generation mechanism of the uracil-5-yl radical is dependent on the identity of the neighboring purine.34,70,71 Specifically, during UV-mediated cross-link formation, G-containing lesions originate from long-lived triplet states (G·+U·), which allow large conformational changes to occur. In contrast, A-containing cross-links originate from reactive doublet states (AU·), which prefer strand break formation (via hydrogen abstraction from the nearby 5′-sugar by the U radical) to cross-link formation. This difference in the mechanism for reaction initiation may account for the preference of G over A intrastrand cross-link formation following UV radiation of BrU-containing DNA. In summary, despite the qualitative agreement between the measured proximity of the two sites forming the cross-link in BDNA and the experimentally observed formation preferences of various cross-links, we propose that proximity is only one aspect that influences the final outcome. For example, the distance between coupling atoms may be significant due to the high reactivity of nucleobase radicals. Alternatively, the mechanism for reaction initiation may play a critical role. The present work underscores the importance of the structural features of the cross-link and the associated damaged DNA. There is a direct correlation between the calculated distortion of the cross-linked DNA and the experimentally observed formation preference for U-containing cross-links. Proximity and structural distortions are likely closely linked. For example, a greater separation between two sites in dsDNA may require large and energetically expensive conformational changes prior to or during cross-link formation. Large distances in canonical (anti) pyrimidine−purine sequences can require major conformational changes prior to cross-link formation, which can include anti-to-syn nucleoside transitions or global distortions to the DNA duplex. Biological Implications. Because cross-link yield cannot be directly correlated with cytotoxicity, all four U-containing intrastrand cross-links may contribute to tumor cell death upon irradiation of XU. Our calculations reveal that each uracilcontaining cross-link adopts a unique structure, which implies that the cross-link composition may have different cellular consequences. In addition to the implications in cancer therapies previously discussed, alterations to the intrinsic bending of DNA may affect the regulation of fundamental F

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more readily repaired in vivo.38 The 5′-G[8-5]C-3′ lesion, similar to 5′-G[8-5]U-3′, was found to be easily recognized and incised in E. coli.42 The structures of 5′-G[8-5]C-3′, 5′-G[85m]mC-3′ and 5′-G[8-5m]T-3′,53,54 compared with data on in vivo repair of these lesions,42 suggest a correlation between the magnitude of DNA duplex distortion and damage recognition, where the greater distortion occurring in the absence of a methylene linker leads to better recognition. Therefore, we expect greater recognition of U-containing lesions compared with T- and mC-containing lesions and greater recognition of extra-helical cross-links that induce larger dsDNA bending. Purine−pyrimidine intrastrand cross-link repair has been studied only in E. coli cells,42,45 and examination of cross-link repair, uracil-containing or otherwise, in mammalian cell lines would provide a better understanding of the biological implications of this class of lesions.

biological processes, such as repair, replication, or transcription. Unfortunately, however, neither the replication nor repair of uracil-containing intrastrand cross-links has been studied to date. Nevertheless, in vitro and in vivo studies of other purine− pyrimidine cross-links (i.e., 5′-G[8-5m]T-3′, 5′-G[8-5m]mC3′, or 5′-G[8-5]C-3′) and pyrimidine−pyrimidine dimers have revealed a plethora of effects ranging from stalling polymerases 41,43,44,46 or incorrect nucleotide incorporation47,51,52,67,69 upon replication, successful translesion synthesis (TLS),47 and removal by nucleotide excision repair (NER).42,45 The structures of purine−pyrimidine that do not contain a methylene linker, such a those in the present work and elsewhere,53−55 are structurally unique from those containing a methylene linkage53,54 or cyclobutane pyrimidine dimers.72,73 Therefore, different biological implications should be expected among these lesions. It is difficult to predict the biological implications of major distortions like extrahelical bases or bends in the helix axis due to the intricate pathways involved in cellular responses to DNA damage, which are further complicated in tumor cells; however, the structures of U-containing cross-linked DNA predicted for the first time in the present work provide important clues about possible biological implications. In terms of replication, previous work has shown that standard replicative polymerases have difficulty bypassing lesions with locked orientations about the glycosidic bond74 due to the inability to fit in the enzyme active site. Thus, the rigidity of the cross-linked structures revealed in the present work, which is similar to the rigidity found for 5′-G[8-5]C-3′ and less flexible than 5′-G[8-5m]T-3′ containing a methylene spacer,54 suggests that U-containing cross-links may stall replicative polymerases. If a stalled polymerase is not resolved, the complex can collapse into a double-strand break (DSB).75 This supports previous observations that direct formation of DSBs from U• cannot fully account for the observed cell death and that cellular processing of another lesion to a DSB may be involved. Alternatively, error-prone TLS may facilitate replication past DNA crosslinks, as observed experimentally for 5′-G[8-5m]T-3′ and 5′G[8-5]C-3′.40,44,46,47 The structures elucidated for the first time in the present work suggest that the Watson−Crick purine face in the 5′-G[8-5]U-3′, 5′-U[5-8]G-3′, and 5′-A[8-5]U-3′ crosslinks is unavailable for interactions with the opposing base, which explains why polymerases tend to stall at the cross-linked purine41,43,44,46 or why TLS introduces a mutation at this site.40,44,46 For example, purines tend to be misincorporated across cross-linked guanines40,44,46 and therefore genetic information is potentially lost upon TLS of cross-linked DNA; however, the replication of DNA containing an intrastrand cross-link is highly dependent on the cell line and polymerases considered. For example, hpol η correctly bypasses 5′-G[8-5m]T-3′ and 5′-T[5m-8]G-3′, even though mutations are still observed in human cells. 47 Therefore, future experimental studies must focus attention on the replication of uracil-containing cross-links to understand their mutagenic potential. This will be particularly important for low-fidelity polymerases, which are overexpressed in tumor cells.75 Although the repair of U-containing intrastrand cross-links has not been studied to date, their formation in cellular DNA shows increased yields of 5′-U[5-8]G-3′ and 5′-A[8-5]U-3′ with increased irradiation time but decreased yields of 5′-G[85]U-3′ and 5′-A[2-5]U-3′. Although this may be attributed to the preferential decomposition of 5′-G[8-5]U-3′ and 5′-A[25]U-3′, Zeng and Wang proposed that these lesions may be

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CONCLUSIONS The structures of three uracil-containing intrastrand cross-links (5′-U[5-8]G-3′, 5′-A[8-5]U-3′, and 5′-A[2-5]U-3′) are elucidated for the first time using molecular dynamics simulations and compared with the structure of the most abundant lesion previously investigated by our group (5′-G[8-5]U-3′). For all cross-links, lesion formation is accompanied by changes in the available hydrogen-bonding faces of the cross-linked nucleobases. With the exception of the 5′-G[8-5]U-3′ cross-link, the uracil-containing cross-linked dinucleotides adopt extrahelical positions. This is accompanied by large backbone distortions and likely a high energetic cost associated with the formation of these lesions. The calculated structural differences provide the first potential explanation for the previously observed formation preferences of these cross-links. The incorporation of these cross-links into DNA induces a bend in the helical axis ranging from 15−51°. These lesions also alter the DNA major or minor grooves as well as substantially distort the backbone in the region local to the cross-linked nucleotides. These structural effects may have implications in DNA damage replication, recognition, and repair because bending is known to play an important role regulating DNA−protein interactions. Ucontaining cross-links induce bends to DNA helices comparable to known anticancer agents and also induce strand breaks and interstrand cross-links, suggesting that incorporation of BrU in DNA followed by irradiation may be an effective tool in cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b10381. Parameters used in molecular dynamics simulations (Table S1), rmsd for each MD simulation (Figure S1), backbone torsion angles in the cross-link (Table S2), and groove analysis (Table S3). (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 1 403 329 2323. Notes

The authors declare no competing financial interest. G

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ACKNOWLEDGMENTS Financial support was provided by the Natural Sciences and Engineering Research Council (NSERC), the Canada Foundation for Innovation (CFI), the Canada Research Chairs program, and the Faculty of Natural Sciences at University of Gothenburg. 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.

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