Biomedical subjects

Stephen J Lippard

Publications and source records attributed to Stephen J Lippard.

At least 37 records · Page 2Linked to original sources

PubMed2006

Selective Hg(II) detection in aqueous solution with thiol derivatized fluoresceins.

The syntheses and photophysical properties of mercury sensors 2 and 3 (MS2 and MS3), two asymmetrically derivatized fluorescein-based dyes designed for Hg(II) detection, are described. These sensors each contain a single pyridyl-amine-thiol metal-binding moiety, form 1:1 complexes with Hg(II), and exhibit selectivity for Hg(II) over other Group 12 metals, alkali and alkaline earth metals, and most divalent first-row transition metals. Both dyes display superior brightness (Phi x epsilon) and fluorescence enhancement following Hg(II) coordination in aqueous solution. At neutral pH, the fluorescence turn-on derives from greater brightness due to increased molar absorptivity. At higher pH, photoinduced electron transfer quenching of the free dye is enhanced, and the Hg(II)-induced turn-on also benefits from alleviation of this pathway. MS2 can detect ppb levels of Hg(II) in aqueous solution, demonstrating its ability to identify environmentally relevant concentrations of Hg(II).

Cations, Divalent↗

PubMed2006

Synthesis and oxidation of carboxylate-bridged diiron(II) complexes with substrates tethered to primary alkyl amine ligands.

The synthesis and crystallographic characterization of a series of diiron(II) complexes with sterically hindered terphenyl carboxylate ligands and alkyl amine donors are presented. The compounds [Fe(2)(mu-O(2)CAr(Tol))(4)(L)(2)] (L=NH(2)(CH(2))(2)SBn (1); NH(2)(CH(2))(3)SMe (2); NH(2)(CH(2))(3)CCH (3)), where (-)O(2)CAr(Tol) is 2,6-di(p-tolyl)benzoate, and [Fe(2)(mu-O(2)CAr(Xyl))(2)(O(2)CAr(Xyl))(2)(L)(2)] (L=NH(2)(CH(2))(3)SMe (4); NH(2)(CH(2))(3)CCH (5)), where (-)O(2)CAr(Xyl) is 2,6-di(3,5-dimethylphenyl)benzoate, were prepared as small molecule mimics of the catalytic sites of carboxylate-bridged non-heme diiron enzymes. The compounds with the (-)O(2)CAr(Tol) carboxylate form tetrabridged structures, but those containing the more sterically demanding (-)O(2)CAr(Xyl) ligand have only two bridging ligands. The ancillary nitrogen ligands in these carboxylate-rich complexes incorporate potential substrates for the reactive metal centers. Their oxygenation chemistry was studied by product analysis of the organic fragments following decomposition. Compound 1 reacts with dioxygen to afford PhCHO in approximately 30% yield, attributed to oxidative dealkylation of the pendant benzyl group. Compound 3 decomposes to form Fe(II)Fe(III) and Fe(III)Fe(IV) mixed-valence species by established bimolecular pathways upon exposure to dioxygen at low temperatures. Upon decomposition, the alkyne-substituted amine ligand was recovered quantitatively. When the (-)O(2)CAr(Tol) carboxylate was replaced by the (-)O(2)CAr(Xyl) ligand in 5, different behavior was observed. The six-coordinate iron(III) complex with one bidentate and two monodentate carboxylate ligands, [Fe(O(2)CAr(Xyl))(3)(NH(2)(CH(2))(3)CCH)(2)] (6), was isolated from the reaction mixture following oxidation.

Amines↗

PubMed2006

6-methylpyridyl for pyridyl substitution tunes the properties of fluorescent zinc sensors of the Zinpyr family.

To prepare fluorescent zinc sensors with binding affinities lower than that of the parent 9-(o-carboxyphenyl)-2,7-dichloro-4,5-bis(bis(2-pyridylmethyl)methylaminomethyl)-6-hydroxy-3-xanthenone (ZP1), dimethylated and tetramethylated derivatives were synthesized having either two or four of the pyridyl subunits methylated at the 6-position. Like the parent ZP1, both Me(2)ZP1 and Me(4)ZP1 exhibit increased fluorescence in the presence of Zn(2+). The integrated emission of Me(2)ZP1 increases 4-fold in the presence of excess zinc, whereas Me(4)ZP1 displays 2.5-fold enhanced fluorescence for Zn(2+). Methylating the 6-positions of the pyridyl rings raises the dissociation constant of the sensors and lowers the pK(a) values associated with the tertiary amine ligands in a systematic manner. The properties of the dimethylated Me(2)ZP1 dye resemble those of ZP1, but the tetramethylated Me(4)ZP1 differs greatly from ZP1 in terms of its brightness, affinity toward Zn(2+), exchange kinetics, and metal sensitivity. Both Me(2)ZP1 and Me(4)ZP1 can enter HeLa cells and signal the presence of Zn(2+). Staining caused by both dyes is punctate, with localization patterns resembling that observed for ZP1.

Cell Membrane Permeability↗

PubMed2006

Synthesis, characterization, and preliminary oxygenation studies of benzyl- and ethyl-substituted pyridine ligands of carboxylate-rich diiron(II) complexes.

In this study benzyl and ethyl groups were appended to pyridine and aniline ancillary ligands in diiron(II) complexes of the type [Fe(2)(mu-O(2)CAr(R))(2)(O(2)CAr(R))(2)(L)(2)], where (-)O(2)CAr(R) is a sterically hindered terphenyl carboxylate, 2,6-di(p-tolyl)- or 2,6-di(p-fluorophenyl)benzoate (R = Tol or 4-FPh, respectively). These crystallographically characterized compounds were prepared as analogues of the diiron(II) center in the hydroxylase component of soluble methane monooxygenase (MMOH). The use of 2-benzylpyridine (2-Bnpy) yielded doubly bridged [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(2-Bnpy)(2)] (1) and [Fe(2)(mu-O(2)CAr(4)(-)(FPh))(2)(O(2)CAr(4)(-)(FPh))(2)(2-Bnpy)(2)] (4), whereas tetra-bridged [Fe(2)(mu-O(2)CAr(Tol))(4)(4-Bnpy)(2)] (3) resulted when 4-benzylpyridine (4-Bnpy) was employed. Similarly, 2-(4-chlorobenzyl)pyridine (2-(4-ClBn)py) and 2-benzylaniline (2-Bnan) were employed as N-donor ligands to prepare [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(2-(4-ClBn)py)(2)] (2) and [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(2-Bnan)(2)] (5). The placement of the substituent on the pyridine ring had no effect on the geometry of the diiron(II) compounds isolated when 2-, 3-, or 4-ethylpyridine (2-, 3-, or 4-Etpy) was introduced as the ancillary nitrogen ligand. The isolated [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(2-Etpy)] (6), [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(3-Etpy)] (7), [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(4-Etpy)] (8), and [Fe(2)(mu-O(2)CAr(4)(-FPh))(2)(O(2)CAr(4)(-)(FPh))(2)(2-Etpy)(2)] (9) complexes all contain doubly bridged metal centers. The oxygenation of compounds 1-9 was studied by GC-MS and NMR analysis of the organic fragments following decomposition of the product complexes. Hydrocarbon fragment oxidation occurred for compounds in which the substrate moiety was in close proximity to the diiron center. The extent of oxidation depended upon the exact makeup of the ligand set.

Carboxylic Acids↗

PubMed2006

Dioxygen-initiated oxidation of heteroatomic substrates incorporated into ancillary pyridine ligands of carboxylate-rich diiron(II) complexes.

Progress toward the development of functional models of the carboxylate-bridged diiron active site in soluble methane monooxygenase is described in which potential substrates are introduced as substituents on bound pyridine ligands. Pyridine ligands incorporating a thiol, sulfide, sulfoxide, or phosphine moiety were allowed to react with the preassembled diiron(II) complex [Fe(2)(mu-O(2)CAr(R))(2)(O(2)CAr(R))(2)(THF)(2)], where (-)O(2)CAr(R) is a sterically hindered 2,6-di(p-tolyl)- or 2,6-di(p-fluorophenyl)benzoate (R = Tol or 4-FPh). The resulting diiron(II) complexes were characterized crystallographically. Triply and doubly bridged compounds [Fe(2)(mu-O(2)CAr(Tol))(3)(O(2)CAr(Tol))(2-MeSpy)] (4) and [Fe(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)(2-MeS(O)py)(2)] (5) resulted when 2-methylthiopyridine (2-MeSpy) and 2-pyridylmethylsulfoxide (2-MeS(O)py), respectively, were employed. Another triply bridged diiron(II) complex, [Fe(2)(mu-O(2)CAr(4)(-)(FPh))(3)-(O(2)CAr(4)(-)(FPh))(2-Ph(2)Ppy)] (3), was obtained containing 2-diphenylphosphinopyridine (2-Ph(2)Ppy). The use of 2-mercaptopyridine (2-HSpy) produced the mononuclear complex [Fe(O(2)CAr(Tol))(2)(2-HSpy)(2)] (6a). Together with that of previously reported [Fe(2)(mu-O(2)CAr(Tol))(3)(O(2)CAr(Tol))(2-PhSpy)] (2) and [Fe(2)(mu-O(2)CAr(Tol))(3)(O(2)CAr(Tol))(2-Ph(2)Ppy)] (1), the dioxygen reactivity of these iron(II) complexes was investigated. A dioxygen-dependent intermediate (6b) formed upon exposure of 6a to O(2), the electronic structure of which was probed by various spectroscopic methods. Exposure of 4 and 5 to dioxygen revealed both sulfide and sulfoxide oxidation. Oxidation of 3 in CH(2)Cl(2) yields [Fe(2)(mu-OH)(2)(mu-O(2)CAr(4)(-)(FPh))(O(2)CAr(4)(-FPh))(3)(OH(2))(2-Ph(2)P(O)py)] (8), which contains the biologically relevant {Fe(2)(mu-OH)(2)(mu-O(2)CR)}(3+) core. This reaction is sensitive to the choice of carboxylate ligands, however, since the p-tolyl analogue 1 yielded a hexanuclear species, 7, upon oxidation.

Carboxylic Acids↗

PubMed2006

Selective labeling of extracellular proteins containing polyhistidine sequences by a fluorescein-nitrilotriacetic acid conjugate.

The compound NTA-DCF consists of two components, a dichlorofluorescein (DCF) reporter and a nitrilotriacetic acid (NTA) functionality. The latter binds polyhistidine sequences selectively through a bridging metal ion. The NTA-DCF conjugate has photophysical properties similar to those of the parent DCF fluorophore both by itself and as its nickel(II) complex. The insensitivity of the emission to paramagnetic ions allows the probe to label His6-tagged proteins fluorescently on the extracellular surfaces of HEK 293-T and HeLa cells.

Fluoresceins↗

PubMed2006

Nitric oxide-induced fluorescence enhancement by displacement of dansylated ligands from cobalt.

The cobalt complexes [Co(Ds-AMP)(2)] (1) and [Co(Ds-AQ)(2)] (2), where Ds-AMP and Ds-AQ are the conjugate bases of dansyl aminomethylpyridine (Ds-HAMP) and dansyl aminoquinoline (Ds-HAQ), respectively, were synthesized in two steps as fluorescence-based nitric oxide (NO) sensors and characterized by X-ray crystallography. The fluorescence of the two complexes was significantly quenched in CH(3)CN or CH(3)OH compared to that of the free Ds-HAMP or Ds-HAQ ligands. Addition of NO to a CH(3)CN solution of 1 or 2 enhanced the integrated fluorescence emission by factors of 2.1(+/-0.3) or 3.6(+/-0.4) within 35 or 20 min, respectively. Introduction of NO to methanolic solutions of the complexes similarly increased the fluorescence by 1.4(+/-0.1) for 1 or 6.5(+/-1.4) for 2 within 1 h. These studies demonstrate that 1 and 2 can monitor the presence of NO with turn-on emission and that their fluorescence responses are more rapid than those of previously reported cobalt systems in coordinating solvents such as CH(3)CN and CH(3)OH. (1)H NMR and IR spectroscopic data revealed the formation of a [Co(NO)(2)](10) cobalt-dinitrosyl adduct, with concomitant dissociation of one ligand from the cobalt center, as the metal-containing product of the NO reactions, a result indicating NO-induced ligand release to be the cause of the fluorescence increase.

Cobalt↗

PubMed2006

Correlating structure with function in bacterial multicomponent monooxygenases and related diiron proteins.

Bacterial multicomponent monooxygenases (BMMs) catalyze the O2-dependent hydroxylation of hydrocarbons at a carboxylate-bridged diiron center similar to those that occur in a variety of dimetallic oxygen-utilizing enzymes. BMMs have found numerous biodegradation and biocatalytic applications. Recent investigations have begun to reveal how BMMs perform their C-H bond activation chemistry and why these enzymes may be mechanistically different from other related diiron proteins. The structures of the BMM component proteins and of complexes between them provide insights into the tuning of the dinuclear iron center and the enzyme mechanism. Selected findings are compared and contrasted with the properties of other carboxylate-bridged diiron proteins, revealing common structural and functional themes.

Bacteria↗

PubMed2006

The inorganic side of chemical biology.

Bioinorganic chemistry remains a vibrant discipline at the interface of chemistry and the biological sciences. Metal ions function in numerous metalloenzymes, are incorporated into pharmaceuticals and imaging agents, and inspire the synthesis of catalysts used to achieve many chemical transformations.

Biochemical Phenomena↗

PubMed2005

Intermolecular electron-transfer reactions in soluble methane monooxygenase: a role for hysteresis in protein function.

Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) to the hydroxylase component (MMOH) of soluble methane monooxygenase (sMMO) primes its non-heme diiron centers for reaction with dioxygen to generate high-valent iron intermediates that convert methane to methanol. This intermolecular electron-transfer step is facilitated by a reductase (MMOR), which contains [2Fe-2S] and flavin adenine dinucleotide (FAD) prosthetic groups. To investigate interprotein electron transfer, chemically reduced MMOR was mixed rapidly with oxidized MMOH in a stopped-flow apparatus, and optical changes associated with reductase oxidation were recorded. The reaction proceeds via four discrete kinetic phases corresponding to the transfer of four electrons into the two dinuclear iron sites of MMOH. Pre-equilibrating the hydroxylase with sMMO auxiliary proteins MMOB or MMOD severely diminishes electron-transfer throughput from MMOR, primarily by shifting the bulk of electron transfer to the slowest pathway. The biphasic reactions for electron transfer to MMOH from several MMOR ferredoxin analogues are also inhibited by MMOB and MMOD. These results, in conjunction with the previous finding that MMOB enhances electron-transfer rates from MMOR to MMOH when preformed MMOR-MMOH-MMOB complexes are allowed to react with NADH [Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768-12785], suggest that isomerization of the initial ternary complex is required for maximal electron-transfer rates. To account for the slow electron transfer observed for the ternary precomplex in this work, a model is proposed in which conformational changes imparted to the hydroxylase by MMOR are retained throughout the catalytic cycle. Several electron-transfer schemes are discussed with emphasis on those that invoke multiple interconverting MMOH populations.

Electron Transport↗

PubMed2005

QZ1 and QZ2: rapid, reversible quinoline-derivatized fluoresceins for sensing biological Zn(II).

QZ1, 2-[2-chloro-6-hydroxy-3-oxo-5-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, and QZ2, 2-[6-hydroxy-3-oxo-4,5-bis-(quinolin-8-ylaminomethyl)-3H-xanthen-9-yl]benzoic acid, two fluorescein-based dyes derivatized with 8-aminoquinoline, have been prepared and their photophysical, thermodynamic, and zinc-binding kinetic properties determined. Because of their low background fluorescence and highly emissive Zn(II) complexes, QZ1 and QZ2 have a large dynamic range, with approximately 42- and approximately 150-fold fluorescence enhancements upon Zn(II) coordination, respectively. These dyes have micromolar K(d) values for Zn(II) and are selective for Zn(II) over biologically relevant concentrations of the alkali and alkaline earth metals. The Zn(II) complexes also fluoresce brightly in the presence of excess Mn(II), Fe(II), Co(II), Cd(II), and Hg(II), offering improved specificity for Zn(II) over di(2-picolyl)amine-based Zn(II) sensors. Stopped-flow kinetic investigations indicate that QZ1 and QZ2 bind Zn(II) with k(on) values of (3-4) x 10(6) M(-1) s(-1), compared to (6-8) x 10(5) M(-1) s(-1) for select ZP (Zinpyr) dyes, at 4.3 degrees C. Dissociation of Zn(II) from QZ1 and QZ2 occurs with k(off) values of 150 and 160 s(-1), over 5 orders of magnitude larger than those for ZP probes, achieving reversibility on the biological (millisecond) time scale. Laser scanning confocal and two-photon microscopy studies reveal that QZ2 is cell-permeable and Zn(II)-responsive in vivo. Because of its weaker affinity for Zn(II), QZ2 responds to higher concentrations of intracellular Zn(II) than members of the ZP family, illustrating that binding affinity is an important parameter for Zn(II) detection in vivo.

Aminoquinolines↗

PubMed2005

Iron substitution for sodium in a carboxylate-bridged, heterodinuclear sodium-iron complex.

The synthesis and structural characterization of the carboxylate-bridged, heterodinuclear iron-sodium complex [NaFe(PIC2DET)(mu-O2CTrp)3] (2), where PIC2DET (1) is a 2,3-diethynyltriptycene-linked dipicolinic methyl ester ligand and Trp is 9-triptycenyl, are described. The metal ions in 2 are bridged by three triptycene carboxylates with an Fe...Na distance of 3.181(2) A, and each is coordinated to a pyridine nitrogen and carbonyl oxygen atom of 1, forming two five-membered chelate rings. A linkage isomer in which Fe1 is bound by the other ester oxygen atom of 1 was identified by X-ray crystallographic analysis. Treatment of 2 with Fe(OTf)2.2MeCN resulted in substitution of sodium by iron(II) to give the cationic diiron(II) complex [Fe2(PIC2DET)(mu-O2CTrp)3][OTf] (3). This reaction was investigated by UV-vis, IR, MS, and stopped-flow spectroscopy. The substitution is first order with respect to 2 and zero order with respect to Fe(OTf)2.2MeCN (kobs = 21 +/- 2 s-1), consistent with a dissociative mechanism. A positive enthalpy of activation (DeltaH = 59 +/- 6 kJ mol-1) and negative entropy of activation (DeltaS = -20 +/- 6 J mol-1 K-1) were calculated from the temperature dependence of the rate-determining dissociation step.

Carboxylic Acids↗

PubMed2005

RNA polymerase II blockage by cisplatin-damaged DNA. Stability and polyubiquitylation of stalled polymerase.

The consequences of human RNA polymerase II (pol II) arrest at the site of DNA damaged by cisplatin were studied in whole cells and cell extracts, with a particular focus on the stability of stalled pol II and its subsequent ubiquitylation. Site-specifically platinated DNA templates immobilized on a solid support were used to perform in vitro transcription in HeLa nuclear extracts. RNA elongation was completely blocked by a cisplatin intrastrand cross-link. The stalled polymerase was quite stable, remaining on the DNA template in nuclear extracts. The stability of pol II stalled at the site of cisplatin damage was also observed in live cells. A cell fractionation experiment using cisplatin-treated HeLa cells revealed an increased level of chromatin-associated pol II proteins following DNA damage. The stalled polymerase was transcriptionally active and capable of elongating the transcript following chemical removal of platinum from the template. Transcription inhibition by alpha-amanitin in vitro enhanced pol II ubiquitylation at ubiquitin residues Lys-6, Lys-48, and Lys-63. In live cells expressing epitope-tagged ubiquitin mutants, several ubiquitin lysines also participated in pol II ubiquitylation following DNA damage. Cisplatin treatment triggered ubiquitylation-mediated pol II degradation in HeLa cells, which could be prevented by the proteasomal inhibitor MG132. Fractionation of pol II from cells co-treated with MG132 and cisplatin indicated that the undegraded ubiquitylated polymerase was mostly unbound or only loosely associated with chromatin. These data are consistent with a model in which only a fraction of pol II, ubiquitylated in response to cisplatin damage of DNA, dissociates from the sites of platination. This altered polymerase is rapidly destroyed by proteasomes.

Cell Fractionation↗

PubMed2005

Zinc-secreting Paneth cells studied by ZP fluorescence.

We have used a new family of zinc-specific-responsive fluorescent dyes (ZPs) to study the sequestration and secretion of zinc from Paneth cells, which are located in the bases of the crypts of Lieberkühn within the rat small intestine. Vivid ZP fluorescence zinc staining of Paneth cell secretory granules is seen in both cryostat sections and isolated crypts, providing firm evidence for a pool of labile (rapidly exchangeable) zinc within these cells. We further demonstrate that this ionic zinc pool is secreted under physiological conditions. In vivo stimulation of the small intestine by IP injection of the secretagogue pilocarpine results in discrete zinc staining within the lumens of subsequently isolated crypts, concomitant with a decrease in the zinc staining of Paneth cell granules located within the same crypts. In contrast, the secretion of zinc into the lumens of isolated crypts stimulated in vitro with either carbachol or LPS (lipopolysaccharide) is not observed. However, a distinct change in Paneth cell morphology, suggesting attempted secretion, is seen in response to the direct application of cholinergics but not LPS. These findings suggest that zinc is coreleased with other Paneth cell anti-microbials, and that the intact intestine is necessary for secretion into the crypt lumen.

Animals↗

PubMed2005

Synthesis of diethynyltriptycene-linked dipyridyl ligands.

[reaction: see text] An efficient route to a new family of dinucleating ligands has been developed. A convergent strategy to these ligands involved dual Sonogashira cross-coupling of 2,3-diethynyltriptycene with a variety of functionally diverse 5-bromopyridines. The resultant ligands were accessed in four steps and 40-50% overall yield from 1,2,4,5-tetrabromobenzene. Synthesis of an imidazole and a quinoline derivative by this method is also described.

Bridged-Ring Compounds↗

PubMed2005

Reactions of the diiron(IV) intermediate Q in soluble methane monooxygenase with fluoromethanes.

Soluble methane monooxygenases utilize a carboxylate-bridged diiron center and dioxygen to convert methane to methanol. A diiron(IV) oxo intermediate Q is the active species for this process. Alternative substrates and theoretical studies can help elucidate the mechanism. Experimental results for reactions with derivatized methanes were previously modeled by a combination of quantum mechanical/molecular mechanical techniques and the model was extended to predict the relative reactivity of fluoromethane. We therefore studied reactions of Q with CF(n)H(4-n) (n=1-3) to test the prediction. The kinetics of single-turnover reactions of Q with these substrates were monitored by double-mixing stopped-flow optical spectroscopy. For fluoro- and difluoromethane, conversion to the alcohols occurred with second-order rate constants less than that of methane, the values being 28,700 (CH4)>25,000 (CFH3)>9300 (CF2H2) M(-1) s(-1). KIE values for C-H versus C-D activation above the classical limit were observed, requiring modification of the theoretical predictions.

Catalysis↗

PubMed2005

Copper complexes for fluorescence-based NO detection in aqueous solution.

Two copper(II) complexes containing dansylated ligands were investigated as turn-on fluorescence-based nitric oxide (NO) sensors. Upon addition of NO (g), the quenched fluorescence of both complexes was restored in both organic and buffered aqueous solutions, which is caused by the formation of a diamagnetic Cu(I) species and protonation of the sulfonamide functionality of the ligands. The NO detection limit of these Cu(II) complexes is 10 nM.

Copper↗

PubMed2005

Platinum anticancer drug damage enforces a particular rotational setting of DNA in nucleosomes.

We constructed two site-specifically modified nucleosomes containing an intrastrand cis-{Pt(NH3)2}2+ 1,3-d(GpTpG) cross-link, similar to one formed by the anticancer drugs carboplatin and cisplatin on DNA, and investigated their structures by hydroxyl radical footprinting and exonuclease III digestion. Hydroxyl radical footprinting demonstrated that the presence of the platinum cross-link selects out a specific rotational setting of DNA on the histone octamer core in each of two reconstituted nucleosomes in which the platinum positions differ by half a DNA helical turn. The {Pt(NH3)2}2+ cross-link is situated in a structurally similar location, with the undamaged strand projecting outward, forcing the DNA to adopt opposite rotational settings in its wrapping around the histone octamer in the two nucleosomes. Enzymatic digestion by exonuclease III of the nucleosome substrates revealed that the platinum cross-link affects the translational positioning of the DNA, forcing it into an asymmetric arrangement with respect to the core histone proteins. We suggest that these phasing phenomena may be central to the recognition and processing of platinum-DNA adducts in cancer cells treated with these drugs and possibly may be common to other DNA damaging events.

Animals↗