Direct ratiometric detection of nitric oxide with Cu(ii)-based fluorescent probes.

We report the first Cu(ii)-based fluorescent sensors for ratiometric detection of nitric oxide. The two probes operate by energy transfer between hydroxycoumarin and fluorescein chromophores, contain polyproline or piperazine as rigid spacers, and elicit a rapid and selective ratiometric response upon direct reaction with nitric oxide.

Hydroxo-bridged Cubane-type tetrairon(II) clusters supported by sterically-hindered carboxylate ligands.

A series of hydroxo-bridged cubane-type tetrairon(II) clusters, [Fe(4)(mu-OH)(4)(mu-O(2)CAr(4)(-)t(BuPh))(2)(mu-OTf)(2)L(4)] (L = C(5)H(5)N (1), 4-(t)BuC(5)H(4)N (2), 3-FC(5)H(4)N (3)), were synthesized by using a sterically hindered carboxylate ligand, 2,6-di(4-tert-butylphenyl)benzoate (Ar(4)(-)t(BuPh)CO(2)(-)). Three different bridging units that mediate weak antiferromagnetic coupling interactions between the metal centers support the unprecedented cubane-type [Fe(4)(mu-OH)(4)](4+) cores in 1-3. The solution structures of 1 and 3 probed by FT-IR and (19)F NMR spectroscopy are consistent with the solid-state geometry determined by X-ray crystallography. Zero-field Mössbauer spectra of 1-3 at 4.2 K are characteristic of high-spin iron(II) centers in nearly identical coordination environments. Compound 1 undergoes two irreversible oxidation processes at ca -10 and +880 mV (vs Fc/Fc(+)), the former approaching quasi-reversible behavior with increased scan rates and a narrow potential sweep range. Comparisons are made with analogous known [Fe(4)X(4)](n)()(+) (X = O, S) units, and the structural integrity of tetrairon fragments upon a change in oxidation state is discussed together with some possible biological implications.

Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system.

2.4 A crystal structure of an oxaliplatin 1,2-d(GpG) intrastrand cross-link in a DNA dodecamer duplex.

(1R,2R-Diaminocyclohexane)oxalatoplatinum(II) (oxaliplatin) is a third-generation platinum anticancer compound that produces the same type of inter- and intrastrand DNA cross-links as cisplatin. In combination with 5-fluorouracil, oxaliplatin has been recently approved in Europe, Asia, and Latin America for the treatment of metastatic colorectal cancer. We present here the crystal structure of an oxaliplatin adduct of a DNA dodecanucleotide duplex having the same sequence as that previously reported for cisplatin (Takahara, P. M.; Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J. Nature 1995, 377, 649-652). Pt-MAD data were used to solve this first X-ray structure of a platinated DNA duplex derived from an active platinum anticancer drug other than cisplatin. The overall geometry and crystal packing of the complex, refined to 2.4 A resolution, are similar to those of the cisplatin structure, despite the fact that the two molecules crystallize in different space groups. The platinum atom of the [Pt(R,R-DACH)](2+) moiety forms a 1,2-intrastrand cross-link between two adjacent guanosine residues in the sequence 5'-d(CCTCTGGTCTCC), bending the double helix by approximately 30 degrees toward the major groove. Both end-to-end and end-to-groove packing interactions occur in the crystal lattice. The latter is positioned in the minor groove opposite the platinum cross-link. A novel feature of the present structure is the presence of a hydrogen bond between the pseudoequatorial NH hydrogen atom of the (R,R)-DACH ligand and the O6 atom of the 3'-G of the platinated d(GpG) lesion. This finding provides structural evidence for the importance of chirality in mediating the interaction between oxaliplatin and duplex DNA, calibrating previously published models used to explain the reactivity of enantiomerically pure vicinal diamine platinum complexes with DNA in solution. It also provides a new kind of chiral recognition between an enantiomerically pure metal complex and the DNA double helix.

Kinetic studies of the TATA-binding protein interaction with cisplatin-modified DNA.

The TATA-binding protein (TBP) recognizes the TATA box element of transcriptional promoters and recruits other initiation factors. This essential protein binds selectively to cisplatin-damaged DNA. Electrophoretic mobility shift assays were performed to study the kinetics of TBP binding both to the TATA box and to cisplatin-damaged DNA in different sequence contexts. TBP binds with high affinity (K(d) = 0.3 nm) to DNA containing site-specific cisplatin 1,2-intrastrand d(GpG) cross-links. The k(on) and k(off) values for the formation of these TBP complexes are 1-3 x 10(5) m(-1) s(-1) and approximately 1-5 x 10(-4) s(-1), respectively, similar to the corresponding values for the formation of a TBP-TATA box complex. In electrophoretic mobility shift assay competition assays, cisplatin-damaged DNA extensively sequesters TBP from its natural binding site, the TATA box. Nine DNA probes were prepared to determine the flanking sequence dependence of TBP binding to cisplatin-modified DNA. TBP clearly displays sequence context selectivity for platinated DNA, very similar to but not as dramatic as that of the high mobility group protein HMGB1. When TBP was added to an in vitro nucleotide excision repair assay, it specifically shielded cisplatin-modified 1,2-(GpG) intrastrand cross-links from repair. These results indicate that TBP is likely to be a key protein in mediating the cytotoxicity of cisplatin.

Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins.

The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.

Cisplatin: from DNA damage to cancer chemotherapy.

Cisplatin [cis-DDP, cis-diamminedichloroplatinum(II)] is a potent anticancer drug that has been used successfully to treat tumors of the head, neck, lungs, and genitourinary tract. The biological activity of cisplatin was discovered serendipitously more than 30 years ago, and since that time research efforts have focused on elucidating its mechanism of action. The present review provides a historical perspective of our attempts to understand this complex phenomenon and the results of recent work that guides our current activities in this field. Continued efforts to understand the mechanism of genotoxicity of cisplatin are expected to lead to the discovery of new drugs and combinations for the improvement of cancer chemotherapy.

Fluorescent sensors for Zn(2+) based on a fluorescein platform: synthesis, properties and intracellular distribution.

Two new fluorescent sensors for Zn(2+) that utilize fluorescein as a reporting group, Zinpyr-1 and Zinpyr-2, have been synthesized and characterized. Zinpyr-1 is prepared in one step via a Mannich reaction, and Zinpyr-2 is obtained in a multistep synthesis that utilizes 4',5'-fluorescein dicarboxaldehyde as a key intermediate. Both Zinpyr sensors have excitation and emission wavelengths in the visible range ( approximately 500 nm), dissociation constants (K(d1)) for Zn(2+) of <1 nM, quantum yields approaching unity (Phi = approximately 0.9), and cell permeability, making them well-suited for intracellular applications. A 3- to 5-fold fluorescent enhancement is observed under simulated physiological conditions corresponding to the binding of the Zn(2+) cation to the sensor, which inhibits a photoinduced electron transfer (PET) quenching pathway. The X-ray crystal structure of a 2:1 Zn(2+):Zinpyr-1 complex has also been solved, and is the first structurally characterized example of a complex of fluorescein substituted with metal binding ligands.

Synthesis and spectroscopic studies of non-heme diiron(III) species with a terminal hydroperoxide ligand: models for hemerythrin.

Two compounds, [Fe2(mu-OH)(mu-Ph4DBA)(TMEDA)2(OTf)] (4) and [Fe2(mu-OH)(mu-Ph4DBA)(DPE)2(OTf)] (7), where Ph4DBA(2-) is the dinucleating bis(carboxylate) ligand dibenzofuran-4,6-bis(diphenylacetate), have been prepared as synthetic models for the dioxygen-binding non-heme diiron protein hemerythrin (Hr). X-ray crystallography reveals that, in the solid state, these compounds contain the asymmetric coordination environment found at the diiron center in the reduced form of the protein, deoxyHr. Mössbauer spectra of the models (4, delta = 1.21(2), DeltaE(Q) = 2.87(2) mm s(-1); 7, delta(av) = 1.23(1), DeltaE(Qav) = 2.79(1) mm s(-1)) and deoxyHr (delta = 1.19, DeltaE(Q) = 2.81 mm s(-1)) are also in good agreement. Oxygenation of the diiron(II) complexes dissolved in CH2Cl2 containing 3 equiv of N-MeIm (4) or neat EtCN (7) at -78 degrees C affords a red-orange solution with optical bands at 336 nm (7300 M(-1) cm(-1)) and 470 nm (2600 M(-1) cm(-1)) for 4 and at 334 nm (6400 M(-1) cm(-1)) and 484 nm (2350 M(-1) cm(-1)) for 7. These spectra are remarkably similar to that of oxyHr, 330 nm (6800 M(-1) cm(-1)) and 500 nm (2200 M(-1) cm(-1)). The electron paramagnetic resonance (EPR) spectrum of the cryoreduced, mixed-valence dioxygen adduct of 7 displays properties consistent with a (mu-oxo)diiron(II,III) core. An investigation of 7 and its dioxygen-bound adduct by extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the oxidized species contains a (mu-oxo)diiron(III) core with iron-ligand distances in agreement with those expected for oxide, carboxylate, and amine/hydroperoxide donor atoms. The analogous cobalt complex [Co2(mu-OH)(mu-Ph4DBA)(TMEDA)2(OTf)] (6) was synthesized and structurally characterized, but it was unreactive toward dioxygen.

Interchain hydrogen-bonding interactions may facilitate translocation of K+ ions across the potassium channel selectivity filter, as suggested by synthetic modeling chemistry.

A 4-fold symmetric arrangement of TVGYG polypeptides forms the selectivity filter of the K+ channel from Streptomyces lividans (KcsA). We report the synthesis and properties of synthetic models for the filter, p-tert-butyl-calix[4]arene-(OCH(2)CO-XOBz)(4) (X = V, VG, VGY), 1-3. The first cation (Na+, K+) binds to the four -[OCH(2)CO]- units, a region devised to mimic the metal-binding site formed by the four T residues in KcsA. NMR studies reveal that cations and valine amide protons compete for the carbonyl oxygen atoms, converting NH(Val)...O=C hydrogen bonds to M+ ...O=C bonds (M+ = Na+ or K+). The strength of these interchain NH(Val)...O=C hydrogen bonds varies in the order 3 > 2 > 1. We propose that such interchain H-bonding may destabilize metal binding in the selectivity filter and thus help create the low energy barrier needed for rapid cation translocation.

Pentacoordinate cobalt(III) thiolate and nitrosyl tropocoronand compounds.

Reaction of [Co(TC-n,m)]+ with (Me4N)(SC6F5), where (TC-n,m) is a tropocoronand with n and m linker chain methylene groups, yielded the thiolate complexes [Co(SC6F5)(TC-3,3)] (1a), and [Co(SC6F5)(TC-4,4)] (2a), which were structurally characterized. Use of more electron-releasing thiolates afforded the [Co(TC-n,m)] reduction product and the corresponding disulfide. The bent nitrosyl complexes [Co(NO)(TC-3,3)] (1b) and [Co(NO)(TC-4,4)] (2b) were synthesized from [Co(TC-n,m)] and NO and their structures were also determined. Compounds 1a and 1b have square-pyramidal geometry like all other structurally characterized [MX(TC-3,3)] complexes. Compounds 2a and 2b have trigonal-bipyramidal stereochemistry, formerly rare for Co(III). Although 1a, 1b, and 2a are paramagnetic, 2b is diamagnetic due to the strong antibonding pi-interaction between the metal and NO pi* orbitals. In the presence of excess NO, [Co(TC-4,4)] exhibited novel reactivity in which a putative Co(N2) adduct formed.

Intermediate in beta-lactam hydrolysis catalyzed by a dinuclear zinc(II) complex: relevance to the mechanism of metallo-beta-lactamase.

Inactivation of beta-lactam antibiotics by metallo-beta-lactamase enzymes is a well-recognized pathway of antibiotic resistance in bacteria. As part of extensive mechanistic studies, the hydrolysis of a beta-lactam substrate nitrocefin (1) catalyzed by dinuclear zinc(II) model complexes was investigated in nonaqueous solutions. The initial step involves monodentate coordination of the nitrocefin carboxylate group to the dizinc center. The coordinated substrate is then attacked intramolecularly by the bridging hydroxide to give a novel intermediate (2') characterized by its prominent absorbance maximum at 640 nm, which affords a blue color. The NMR and IR spectroscopic data of 2' are consistent with it being zinc(II)-bound N-deprotonated hydrolyzed nitrocefin that forms from the tetrahedral intermediate upon C-N bond cleavage. Protonation of the leaving group is the rate-limiting step in DMSO solution and occurs after the C-N bond-breaking step. Addition of strong acids results in rapid conversion of 2' into hydrolyzed nitrocefin (3). The latter can be converted back to the blue species (2') upon addition of base. The low pK(a) value for the amino group in hydrolyzed nitrocefin is explained by its involvement in extended conjugation and by coordination to zinc(II). The blue intermediate (2') in the model system resembles well that in the enzymatic system, judging by its optical properties. The greater stability of the intermediate in the model, however, allowed its characterization by (13)C NMR and infrared, as well as electronic, spectroscopy.

Crystal structures of the soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath) demonstrating geometrical variability at the dinuclear iron active site.

The oxidation of methane to methanol is performed at carboxylate-bridged dinuclear iron centers in the soluble methane monooxygenase hydroxylase (MMOH). Previous structural studies of MMOH, and the related R2 subunit of ribonucleotide reductase, have demonstrated the occurrence of carboxylate shifts involving glutamate residues that ligate the catalytic iron atoms. These shifts are thought to have important mechanistic implications. Recent kinetic and theoretical studies have also emphasized the importance of hydrogen bonding and pH effects at the active site. We report here crystal structures of MMOH from Methylococcus capsulatus (Bath) in the diiron(II), diiron(III), and mixed-valent Fe(II)Fe(III) oxidation states, and at pH values of 6.2, 7.0, and 8.5. These structures were investigated in an effort to delineate the range of possible motions at the MMOH active site and to identify hydrogen-bonding interactions that may be important in understanding catalysis by the enzyme. Our results present the first view of the diiron center in the mixed-valent state, and they indicate an increased lability for ferrous ions in the enzyme. Alternate conformations of Asn214 near the active site according to redox state and a distortion in one of the alpha-helices adjacent to the metal center in the diiron(II) state have also been identified. These changes alter the surface of the protein in the vicinity of the catalytic core and may have implications for small-molecule accessibility to the active site and for protein component interactions in the methane monooxygenase system. Collectively, these results help to explain previous spectroscopic observations and provide new insight into catalysis by the enzyme.

Trinuclear Zn(II) and Cu(II) homo and heterotrimetallic complexes involving D-glucopyranosyl and biscarboxylate bridging ligands. A substrate binding model of xylose isomerases.

Reactions of MCl(2).nH(2)O with N,N'-bis(D-glucopyranosyl)-1,4,7-triazacyclononane ((D-Glc)(2)-tacn), which was formed from D-glucose and 1,4,7-triazacyclononane (tacn) in situ, afforded a series of mononuclear divalent metal complexes with two beta-D-glucopyranosyl moieties, [M((D-Glc)(2)-tacn)Cl]Cl (M = Zn (11), Cu (12), Ni (13), Co (14)). Complexes 11-14 were characterized by analytical and spectroscopic measurements and X-ray crystallography and were found to have a distorted octahedral M(II) center ligated by the pentacoordinate N-glycoside ligand, (beta-D-glucopyranosyl)(2)-tacn, and a chloride anion. Each D-glucose moiety is tethered to the metal center through the beta-N-glycosidic bond with tacn and additionally coordinated via the C-2 hydroxyl group, resulting in a lambda-gauche five-membered chelate ring. When L-rhamnose (6-deoxy-L-mannose) was used instead of D-glucose, the nickel(II) complex with two beta-L-rhamnopyranosyl moieties, [Ni((D-Man)(2)-tacn)(MeOH)]Cl(2) (15), was obtained and characterized by an X-ray analysis. Reactions of 11 (M = Zn) with [Zn(XDK)(H(2)O)] (21) or [Cu(XDK)(py)(2)] (22) (H(2)XDK = m-xylylenediamine bis(Kemp's triacid imide)) yielded homo and heterotrimetallic complexes formulated as [Zn(2)M'((D-Glc)(2)-tacn)(2)(XDK)]Cl(2) (M' = Zn (31), Cu (32)). The similar reactions of 12 (M = Cu) with complex 21 or 22 afforded [Cu(2)M'((D-Glc)(2)-tacn)(2)(XDK)]Cl(2) (M' = Cu (33), Zn (34)). An X-ray crystallographic study revealed that complexes 31 and 34 have either Zn(II)(3) or Cu(II)Zn(II)Cu(II) trimetallic centers bridged by two carboxylate groups of XDK and two D-glucopyranosyl residues. The M...M' separations are 3.418(3)-3.462(3) A (31) and 3.414(1)-3.460(1) A (34), and the M...M'...M angles are 155.18(8) degrees (31) and 161.56(6) degrees (34). The terminal metal ions are octahedrally coordinated by the (D-Glc)(2)-tacn ligand through three nitrogen atoms of tacn, two oxygen atoms of the C-2 hydroxyl groups of the carbohydrates, and a carboxylate oxygen atom of XDK ligand. The central metal ions sit in a distorted octahedral environment ligated by four oxygen atoms of the carbohydrate residues in the (D-Glc)(2)-tacn ligands and two carboxylate oxygen atoms of XDK. The deprotonated beta-D-glucopyranosyl unit at the C-2 hydroxyl group bridges the terminal and central ions with the C-2 mu-alkoxo group, with the C-1 N-glycosidic amino and the C-3 hydroxyl groups coordinating to each metal center. Complexes 31-34 are the first examples of metal complexes in which D-glucose units act as bridging ligands. These structures could be very useful substrate binding models of xylose or glucose isomerases, which promote D-glucose D-fructose isomerization by using divalent dimetallic centers bridged by a glutamate residue.

Laser-induced photo-cross-linking of cisplatin-modified DNA to HMG-domain proteins.

Laser-induced photo-cross-linking was investigated for DNA, modified with cisplatin at specific sites, bound to structure-specific recognition domains of proteins in the high-mobility group (HMG) class. The efficiency of photo-cross-linking depends on the wavelength and power of the laser, the nature of the protein domain, and the oligodeoxyribonucleotide sequences flanking the platinated site. Introduction of 5-iodouridine at thymine sites of the oligodeoxyribonucleotide as an additional photoreactive group did not increase the photo-cross-linking yield. Formation of platinum-mediated DNA-DNA interstrand cross-linking observed previously upon irradiation with 302 nm light [Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180-2188] was significantly reduced with laser irradiation. HMG1 domain B is superior to domain A for platinum-mediated photo-cross-linking, a result attributed to the different positioning of the proteins with respect to the platinum adduct and the greater ability of domain B to access photolabilized platinum in the major groove. Studies with proteins containing specifically mutated amino acids, and with DNA probes in which the sequences flanking the platinum cross-link site were varied, suggest that the most effective photo-cross-linking occurs for protein domains bound symmetrically and flexibly to cisplatin-modified DNA. The thermodynamic equilibrium between the protein-platinated DNA complex and its components, revealed in gel electrophoretic mobility shift assays (EMSAs), is significantly shifted to the right upon irreversible photo-cross-linking. Thus, only upon photo-cross-linking can the interaction of cisplatin-DNA 1,3-intrastrand d(GpTpG) or interstrand cross-links with HMG1 domain B protein be detected. Photo-cross-linking is thus an effective tool for investigating the interaction of cisplatin-modified DNA with damage-recognition proteins under heterogeneous conditions such those in cell extracts or living cells.

Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin.

The structure-specific recognition protein SSRP1, initially isolated from expression screening of a human B-cell cDNA library for proteins that bind to cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a single DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal block to transcription elongation by RNAPII in vitro. The affinity and specificity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were investigated by gel mobility shift assays. FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared with unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer. FACT binds the major 1,2-d(GpG) intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to form discrete, high affinity complexes with cisplatin-modified DNA under similar conditions. These results suggest that Spt16 primes SSRP1 for cisplatin-damaged DNA recognition by unveiling its HMG domain. As expected, the isolated HMG domain of SSRP1 is sufficient for specific binding to cisplatin-damaged DNA and binds the major cisplatin 1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT for cisplatin-modified DNA, as well as its importance for transcription of chromatin, suggests that the interaction of FACT and cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin.