A Structural Basis for a Phosphoramide Mustard-Induced DNA Interstrand Cross-Link at 5'-D(GAC) QING DONG*, DANIEL Barskyt, MICHAEL E
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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12170-12174, December 1995 Biochemistry A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC) QING DONG*, DANIEL BARSKYt, MICHAEL E. COLVINt, CARL F. MELIUSt, SUSAN M. LUDEMANt, JONATHAN F. MORAVEK§, 0. MICHAEL COLVINO, DARELL D. BIGNER*, PAUL MODRICHII, AND HENRY S. FRIEDMAN*§** Departments of *Pathology and §Pediatrics, IDuke Comprehensive Cancer Center, ItHoward Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, NC 27710; tM.S. 9214, Sandia National Laboratories, Livermore, CA 94550-0969; and tOncology Center, Johns Hopkins University, Baltimore, MD 21287 Contributed by Paul Modrich, September 14, 1995 ABSTRACT Phosphoramide mustard-induced DNA in- and quantum chemistry were applied to study the structural terstrand cross-links were studied both in vitro and by com- basis for the linkage induced by nitrogen mustards. puter simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross- MATERIALS AND METHODS links were defined by using different pairs of synthetic oligo- nucleotide duplexes, each of which contained a single poten- In Vitro Studies for PM-Induced DNA Cross-link Forma- tially cross-linkable site. Phosphoramide mustard was found tion. T4 polynucleotide kinase was purchased from New to cross-link dG to dG at a 5'-d(GAC)-3'. The structural basis England Biolabs. [,y-32P]ATP (6000 Ci/mmol; 1 Ci = 37 GBq) for the formation of this 1,3 cross-link was studied by molec- was purchased from Amersham. Oligonucleotides were pur- ular dynamics and quantum chemistry. Molecular dynamics chased from Oligo, etc. (Wilsonville, OR). PM (cyclohexylam- indicated that the geometrical proximity of the binding sites monium salt) was a gift from the Drug Synthesis and Chem- also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG istry Branch, National Cancer Institute. Dechlorophosphor- linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of amide mustard [HOP(O)(NH2)N(CH2CH2Cl)(CH2CH3)] was 1,2 and 1,3 mustard cross-linked DNA were found to be very made by a multistep organic synthesis procedure. (The syn- close, a 1,3 structure was more flexible and may therefore be thesis protocol will be made available by S.M.L.) PM or in a considerably higher entropic state. dechlorophosphoramide mustard was dissolved in 50 mM sodium phosphate, pH 7.0, as 500 mM stock immediately Cyclophosphamide is an important alkylating agent that has before drug treatment. been widely used in treating various malignant tumors (1). Oligonucleotides were purified by electrophoresis in a de- DNA interstrand cross-links appear to be the primary cyto- naturing 20% polyacrylamide gel (ref. 13, pp. 11.23-11.28) and toxic adduct and the base of antitumor activity of phosphor- eluted by soaking the crushed gel pieces containing the correct amide compounds (2, 3). Many studies have suggested that size in distilled water at 14°C. The oligonucleotide eluate was DNA interstrand cross-links induced by alkylating agents passed through a Sephadex G-25 column to remove urea and prevent the separation of the strands of the DNA helix and thus salts, and the oligonucleotides were concentrated in a Speed- inhibit DNA replication (4-6). Further clarification of the Vac (Savant), divided into aliquots, and stored at -70°C. One molecular interactions between DNA and cross-linking agents of the oligonucleotides in each pair was phosphorylated at the is necessary to understand the cellular responses to DNA 5' end by T4 polynucleotide kinase (ref. 13, pp. 5.68-5.71). The interstrand cross-links and the manner by which such lesions reaction was terminated by adding EDTA to a final concen- are recognized and repaired in mammalian cells. tration of 10 mM, and the solution was passed through a G-25 Phosphoramide mustard [PM, HOP(O)(NH2)N(CH2CH2Cl)2], column to remove ATP. The labeled oligonucleotide was the pharmacologically active metabolite of cyclophosphamide, purified again as described above. primarily alkylates the N7 atom of guanines in DNA (7, 8). It Two complementary oligonucleotides (1 ,uM each) were has been postulated that the DNA sequence 5'-CG-3' imparts mixed in 50 mM sodium phosphate, pH 7.0, heated at 68°C for a topologic and geometric condition favoring formation of 3 min, and allowed to cool to room temperature over several DNA interstrand cross-links by nitrogen mustard compounds hours just before the drug treatment. The annealed DNA (4). An early computer modeling study suggested that PM oligonucleotide duplexes or single-strand oligonucleotides (at favors a 5'-GC-3' DNA sequence (resulting in a 1,2 cross-link) 1 j,M) were treated with various concentrations of PM in 50 (9); however, several experimental studies with nitrogen mus- mM sodium phosphate for 4 hr at room temperature. The tard [CH3N(CH2CH2Cl)2] have indicated that 5'-GNC-3' (a reaction mixture was stored at -20°C or electrophoresed 1,3 cross-link) is the sequence for the nitrogen mustard- immediately. The gel was run at 1600 V in 90 mM Tris borate/2 induced DNA cross-link (10-12). Since DNA cross-links in- mM EDTA buffer, pH 8.0, for at least 2 hr before loading. An duced by nitrogen mustard and PM are structurally similar, it end-labeled oligonucleotide ladder was loaded on the same gel is conceivable that PM can form a 1,3 instead of a 1,2 linkage. (Pharmacia). After electrophoresis at 1800 V for 2 hr at 50°C, However, no experimental evidence has been published to DNA species were visualized by autoradiography. Cross-linked support either model. oligonucleotide duplexes visualized in this manner were ex- We have designed several pairs of synthetic oligonucleotide cised from the gel and purified as described above. Oligonu- duplexes to define the local determinants of PM-induced cleotides were sequenced by the Maxam-Gilbert method, cross-link formation in vitro. In addition, molecular dynamics using the G, G+A, C, and T+C reactions (14, 15). The publication costs of this article were defrayed in part by page charge Abbreviations: PM, phosphoramide mustard; NNM, nornitrogen mus- payment. This article must therefore be hereby marked "advertisement" in tard. accordance with 18 U.S.C. §1734 solely to indicate this fact. **To whom reprint requests should be addressed. 12170 Downloaded by guest on September 24, 2021 Biochemistry: Dong et al. Proc. Natl. Acad. Sci. USA 92 (1995) 12171 Structural Studies of Nornitrogen Mustard (NNM) DNA The internal energies of the bare NNM linkages were deter- Cross-link The structures of both 1,2 and 1,3 linkages were mined by ab initio quantum chemistry calculations. To this end, simulated by using molecular dynamics and were analyzed by the NNM cross-link was isolated from the DNA at regular quantum chemistry. Three 12-bp duplexes-i.e., pairs P1 and P3 intervals along the molecular dynamics trajectory, and the gua- (see Table 1) and a control pair, d(CATGTAG3G2CTAA)-d- nine N7s were replaced with hydrogens whose positions were (TTAG'CCTACATG)-were built in the canonical B-DNA adjusted to the appropriate C-H bond distance. Each 16-atom form by using the program QUANTA4.o/CHARMm (16). Because structure was then optimized with the GAUSSLAN 94 program (30) the primary interest was the effect of the physical linkage on at the Hartree-Fock level oftheory, using a 6-31G* basis set while the DNA and because molecular dynamics parameters for the constraining the terminal hydrogens and the ,3 carbons to their phosphoramide moiety have not been developed, all modeling molecular dynamics-derived positions (31). was performed on NNM [HN(CH2CH2Cl)2] instead of PM- i.e., HN in place of HOP(O)(NH2)N (1). This substitution could affect steric and electrostatic interactions. NNM, like RESULTS other nitrogen mustard diadducts, nitrogen mustard (17), and Design of Duplex Oligonucleotides Containing Only a Single melphalan, is expected to have a neutral net charge at pH 7. Cross-linkable Site. Different pairs of oligonucleotides were However, chlorambucil (18) and PM have been shown to be synthesized that contained a single potentially cross-linkable site. anions at pH 7 (19). Sterically, the much larger phosphoramide As shown in Table 1, pairs P1, P2, and P4 were expected to be moiety may reduce the range of available conformations of the link, but this is not expected to alter the computational results (see discussion below). The interstrand NNM cross-link was added to pairs P1 and P3 by replacing the terminal chloride atoms with the appropriate N7 atoms, whereas the control pair was left unmodified. For visualization purposes a PM cross- link was built into pair P1 as shown in Fig. 1. In our simulations the NNM link atoms were only weakly charged, and all three systems had a net charge of -22 owing to the 22 phosphate groups along the backbones. Counter-ions were not included because they may require a long equilibration time in the presence of explicit water (more than 50 ps) (20), and a comparative study found that inclusion of counter-ions in the absence of explicit water did not significantly improve the resulting DNA structural properties (21). The linked structures were minimized slightly to remove the unusually large strains introduced by inserting the link. Using the program X-PLOR (22), we added a sphere of radius 48 A ofTIP3P (23) water molecules within 2.6 A of the solute (DNA and NNM) molecules. Each total system consisted of approximately 6150 atoms. The original water density was maintained by applying a 49.2-A repulsive spherical shell having a harmonic force constant of 20 kcal/mol per A2 (1 kcal = 4.18 kJ).