Mutagenic and genotoxic effects of DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II).

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.     

Bending studies of DNA site-specifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-[Pt(NH3)2(N3-cytosine)Cl]+

Duplex oligonucleotides containing a single intrastrand [Pt(NH3)2]2+ cross-link or monofunctional adduct and either 15 or 22 bp in length were synthesized and chemically characterized. The platinum-modified and unmodified control DNAs were polymerized in the presence of DNA ligase and the products studied on 8% native polyacrylamide gels. The extent of DNA bending caused by the various platinum-DNA adducts was revealed by their gel mobility shifts relative to unplatinated controls. The bifunctional adducts cis-[Pt(NH3)2[d(GpG)]]+, cis-[Pt(NH3)2[d(ApG)]]+, and cis-[Pt(NH3)2[d(G*pTpG*)]], where the asterisks denote the sites of platinum binding, all bend the double helix, whereas the adduct trans-[Pt(NH3)2[d(G*pTpG*)]] imparts a degree of flexibility to the duplex. When modified by the monofunctional adduct cis-[Pt(NH3)2(N3-cytosine)(dG)]Cl the helix remains rod-like. These results reveal important structural differences in DNAs modified by the antitumor drug cisplatin and its analogs that could be important in the biological processing of the various adducts in vivo.     

Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2(d(GpG]], built into a specific site in a viral genome

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH3)2(d(GpG]] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA).d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. The dodecamer d(TCTAGGCCTTCT) was synthesized by the solid-phase phosphotriester method and platinated by reaction with cis-[Pt(NH3)2(H2O)2]2+ (yield 39%). Characterization by pH-dependent 1H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [gamma-32P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. Approximately 80% of the gapped duplexes incorporated a dodecanucleotide in the ligation reaction. Of these, approximately half did so with the dodecanucleotide covalently joined to the genome at both 5' and 3' termini. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. The cis-[Pt(NH3)2(d(GpG]] cross-link completely inhibited StuI cleavage, which was fully restored following incubation of the platinated genome with cyanide to remove platinum as [Pt(CN)4]2-. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH3)2(d(GpG]] cross-link induces localized weakening of the DNA double helix. In addition, double- and single-stranded genomes, in which the cis-[Pt(NH3)2(d(GpG]] cross-link resides specifically in the plus strand, were constructed. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH3)2(d(GpG]] cross-link is lethal to the single-stranded bacteriophage.     

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts.

A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA unwinding produced by site-specific intrastrand cross-links of the antitumor drug cis-diamminedichloroplatinum(II)

The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedichloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-[Pt(NH3)2[d(GpG)]], cis-[Pt(NH3)2(d(ApG)]], and cis-[Pt(NH3)2[d(GpTpG)]] adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG. Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis. The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymers containing the adducts. The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained. We find that the cis-GG and cis-AG adducts both unwind DNA by 13 degrees, while the cis-GTG adduct unwinds DNA by 23 degrees. In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts. On the basis of an analysis of the present and other published studies of site-specifically modified DNA, we propose that local duplex unwinding is a major determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease. In addition, local duplex unwinding of 13 degrees and bending by 35 degrees are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.     

Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

As a widely used anticancer drug, cis-diamminedichloroplatinum(II) (cisplatin) reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH(3))(2){d(GpG)-N7(1),-N7(2)}] intrastrand cross-links. Drug resistance, one of the major limitations of cisplatin therapy, is partially due to the inherent ability of human Y-family DNA polymerases to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin-DNA adducts. To better understand the mechanistic basis of translesion synthesis contributing to cisplatin resistance, this study investigated the bypass of a single, site-specifically placed cisplatin-d(GpG) adduct by a model Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4). Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was decreased by 72- and 860-fold, respectively. Moreover, the fidelity at the lesion decreased up to two orders of magnitude. The cisplatin-d(GpG) adduct affected six downstream nucleotide incorporations, but interestingly the fidelity was essentially unaltered. Biphasic kinetic analysis supported a universal kinetic mechanism for the bypass of DNA lesions catalyzed by various translesion DNA polymerases. In conclusion, if human Y-family DNA polymerases adhere to this bypass mechanism, then translesion synthesis by these error-prone enzymes is likely accountable for cisplatin resistance observed in cancer patients.     

The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG)

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.     

H8 chemical shifts in oligonucleotide cross-linked at a GpG sequence by cis-Pt(NH3)2(2+): a clue to the adduct structure.

The origin of the anomalous H8 chemical shifts observed in 1H-NMR spectra of oligonucleotides cross-linked at a GpG sequence with cis-[Pt(NH3)2]2+ has been investigated and clarified. The main contributions that distinguish the H8 resonances of the two platinum-ligating guanines from other GH8 signals and from each other are: (a) the inductive effect of platinum binding which we have recently quantified as a downfield shift of 0.48 +/- 0.07 ppm (M. H. Fouchet, D. Lemaire, J. Kozelka and J.-C. Chottard, unpublished results); (b) the ring-current effect of one GpG guanine on the H8 resonance of the other guanine, which is negative (shielding) for the 5'-H8 and positive (deshielding) for the 3'-H8 in single-stranded adducts, but has the opposite sign in double-stranded adducts; (c) a deshielding polarization effect of the phosphate 5' to the GpG unit. The different signs of the ring-current effects in single-stranded and double-stranded oligonucleotides originate from the orientation of the guanines in the cis-[Pt(NH3)2(Gua)2]2+ moiety (Gua, guanine), which is left-handed helicoidal in single strands and right-handed helicoidal in double strands. In the platinated dinucleotides (cis-[Pt(NH3)2(GpG)]+, cis-[Pt(NH3)2(d(GpG))]+ and cis-[Pt(NH3)2(d(GpG))]), the guanines assume either the left-handed or the right-handed arrangement, depending on the sugar moiety (ribose or deoxyribose), protonation state at N1 and, in the solid state, on crystal forces. This work shows that chemical shifts contain valuable structural information which is complementary to that extracted from correlated spectroscopy and nuclear Overhauser spectroscopy data.     

Conformational analysis of the adduct cis-[Pt(NH3)2 d(GpG)]+ in aqueous solution. A high field (500-300 MHz) nuclear magnetic resonance investigation.

A 500, 400 and 300 MHz proton NMR study of the reaction product of cis-Pt(NH3)2Cl2 or cis-[Pt(NH3)2 (H2O)2] (NO3)2 with the deoxydinucleotide d(GpG): cis-[Pt(NH3)2 d(GpG)] was carried out. Complete assignment of the proton resonances by decoupling experiments and computer simulation of the high field part of the spectrum yield proton-proton and proton-phosphorus coupling constants of high precision. Analysis of these coupling constants reveal a 100% N (C3'-endo) conformation for the deoxyribose ring at the 5'-terminal part of the chelated d(GpG) moiety. In contrast, the 3'-terminal -pG part of the molecule displays the normal behaviour for deoxyriboses: the sugar ring prefers to adopt an S (C2'-endo) conformation (about 70%). Extrapolating from this model compound, it is suggested that Pt chelation by a -dGpdG- sequence of DNA would require a S to N conformational change of one deoxyribose moiety as the main conformational alteration and lead to a kink in one strand of the double-helical structure of DNA.     

Reaction of DNA oligonucleotides with [Pt(dien)GSMe]2+ (GSMe =S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+: evidence of oligonucleotide platination via sulfur-coordinated platinum intermediates

To study the possibility of DNA platination via platinum-sulfur coordinated intermediates, the reactions of the complexes [Pt(dien)GSMe]2+ (GSMe=S-methylated glutathione) and cis-[Pt(NH3)2(GSMe)2]2+ with the synthetic oligonucleotides d(ATATGCATAT), d(ATTACCGGTAAT), and d(ATCCTATTTTTTTTAGGAT) have been investigated. The reactions were studied using FPLC, NMR, and mass spectrometry. It was found that the sulfur atom of the platinum-thioether adduct is substituted by these oligonucleotides. For the reactions with [Pt(dien)GSMe]2+ at 310 K, half-lives were determined to be t 1/2 =147+/-7 h for d(ATATGCATAT), t 1/2 =84+/-4 h) for d(ATTACCGGTAAT), and t 1/2 = 21+/-1 h for d(ATCCTATTTTTTTTAGGAT. This study clearly shows that it is indeed possible for oligonucleotides to be platinated via Pt-thioether coordinated intermediates. The rates at which such substitutions occur, however, makes it improbable that such a mechanism contributes significantly to the antitumor activity of cisplatin.     

Fixing the conformations of diamineplatinum(II)-GpG chelates: NMR and CD signatures of individual rotamers

The bulky, asymmetric analog of the antitumor drug cisplatin, [PtCl(2)(tmen)] (tmen = N,N,N'-trimethylethylenediamine), was used to produce crosslinks with the dinucleotide d(GpG), modeling the most frequent lesions that cisplatin and its analogs cause to DNA. The ligand tmen was chosen because it is expected to constrain the guanine cis to the NMe(2) group in the adduct [Pt(tmen){d(GpG)}](+) to an orientation perpendicular to the coordination plane and to stabilize the other guanine in an oblique orientation, thus maintaining a head-to-head geometry typical of cisplatin-d(GpG) crosslinks within single- and double-stranded DNA. Of the four possible combinations of tmen chirality (R or S symmetry of the coordinated NHMe group) and crosslink direction (5'-G bound cis to the secondary or the tertiary amino group of tmen), two isomers were preponderantly formed, [Pt(R-tmen){d(GpG)}](+) with 5'-G bound cis to NMe(2) and [Pt(S-tmen){d(GpG)}](+) with 5'-G bound cis to NHMe. The former was shown to have a right-handed R2 orientation of guanines similar to that found in duplex DNA, whereas the latter had a left-handed L1 orientation that modeled cisplatin-d(GpG) adducts within single-stranded DNA. The R2 rotamer was found to be in an equilibrium (as observed using EXSY spectroscopy) with a minor fraction (< or =4%) of a Delta-HT rotamer related to R2 by rotation of the 3'-G about the Pt-N7 bond. The major rotamers R2 and L1 were isolated using reverse-phase HPLC, and their NMR and CD signatures were compared to those of the corresponding rotamers of the less hindered adduct [Pt(dmen)(GpG)](+) (dmen = N,N-dimethylethylenediamine). From this and other comparisons with previously reported platinum dinucleotide complexes, and from molecular modeling, it could be concluded that both steric repulsion between guanine and substituents of the cis amino group and N-H...O6 hydrogen bonding are significant effects favoring the oblique orientation of one guanine base typical of the HH rotamers of [Pt(diamine){d(GpG)}](+) and [Pt(diamine)(GpG)](+) complexes.     

Guanine-06 methylation reduces the reactivity of d(GpG) towards platinum complexes.

6-methylated guanine dinucleotides were used to study the influence of hydrogen bonding on the specific binding of the antitumor drug cDDP, cis-PtCl2(NH3)2, to DNA. In this interaction, the guanine-06 site appears to be important in explaining the preference for a pGpG-N7(1),N7(2) chelate, which results from H-bridge formation with the ammine ligand of cDDP. Guanine-06 methylated dinucleotides and the nonmodified dinucleotides were reacted with [Pt(dien)Cl]+, cis-PtCl2(NH3)2, and cis-[Pt(NH3)2(H2O)2]2+ and the reaction products were characterized by 1H NMR using pH titrations. Methylation at guanine-06 clearly reduces the preference for the guanine. In competition experiments monitored by NMR and experiments using UV spectrophotometry a decreasing reactivity towards [Pt(dien)(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+ was found, in the order of d(GpG) greater than d(GomepG) greater than d(GpGome) greater than d(GomepGome). The difference in reactivity between 5' guanine methylation and 3' guanine methylation is ascribed to differences in the H-bond formation with the backbone phosphate. The resulting reduced stacking of the bases in both modified dinucleotides, compared to the bases in d(GpG), results in a preference for the 3' guanine over 5'.     

DNA oligonucleotide duplexes containing intramolecular platinated cross-links: energetics,hydration, sequence,and ionic effects

The anticancer activity of cisplatin arises from its ability to bind covalently to DNA, forming primarily intrastrand cross-links to adjacent purine residues; the most common adducts involve d(GpG) (65%) and d(ApG) (25%) intrastrand cross-links. The incorporation of these platinum adducts in a B-DNA helix induces local distortions, causing bending and unwinding of the DNA. In this work, we used temperature-dependent UV spectroscopy to investigate the unfolding thermodynamics, and associated ionic effects, of two sets of DNA decamer duplexes containing either cis-[Pt(NH(3))(2)[d(GpG]] or cis-[Pt(NH(3))(2) [d(ApG]] cross-links, and their corresponding unmodified duplexes. The platinated duplexes are less stable and unfold with lower T(M)s (and Delta G degrees s) in enthalpy-driven reactions, which indicates a loss of favorable base-pair stacking interactions. The folding thermodynamics and hydration effects for the first set of decamers containing the d(GpG) cross-link was investigated by a combination of titration calorimetry, density, and ultrasound techniques. The hydration parameters showed an uptake of structural water by the platinated duplex and a release of electrostricted water by the control duplex. Relative to the unmodified duplex, the folding of the platinated duplex at 20 degrees C yielded a positive Delta Delta G degrees term [and positive Delta Delta H-Delta(T Delta S) compensation] and a negative differential volume change. The opposite signs of the Delta Delta G degrees and Delta Delta V terms confirmed its uptake of structural water. Further, solvent-accessible surface areas calculations for a similar pair of dodecamer duplexes indicated that the modified duplex has a 503 oeA(2) higher polar and nonpolar surface area that is exposed to the solvent. Therefore, the incorporation of a platinum adduct in duplex DNA disrupts favorable base-pair stacking interactions, yielding a greater exposure of aromatic bases to the solvent, which in turn immobilizes structural water. The overall results correlate nicely with the results reported in the available structural data of nuclear magnetic resonance solution studies.     

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.     

Effect of geometric isomerism in dinuclear platinum antitumour complexes on the rate of formation and structure of intrastrand adducts with oligonucleotides.

The dinuclear platinum complexes [[trans -PtCl (NH3)2]2[mu]-[NH2(CH2) n NH2]](NO3)2[1,1/t,t ( n = 4,6)] and [[cis-PtCl(NH3)2]2[mu];-[NH2(CH2) n NH2](NO3) 2[1,1/c,c ( n = 4,6)] exhibit antitumour activity comparable with cisplatin. 1,1/c,c complexes do not form 1,2 GG intrastrand adducts, the major adduct of cisplatin, with double-stranded DNA. This 1H NMR spectroscopy study shows that, in the absence of a complementary strand, 1,1/c,c ( n = 4,6) form a 1,2 GG (N7, N7) intrastrand adduct with r(GpG), d(GpG) and d(TGGT). Initial binding to r(GpG) (and also reaction with GMP) at 37 degrees C was slower for 1,1/c,c compared with 1,1/t,t, whereas the second binding step (adduct closure) was faster for 1,1/c,c. However, the 1H NMR spectra of the 1,1/c,c adducts at 37 degrees C show two H8 signals, one of which is broad and becomes sharper on increasing the temperature, indicating restricted rotation around the Pt-N7 bond. For the d(GpG)-1,1/c,c ( n = 4) adduct, 2D NMR spectroscopy assigned the broad H8 signal to the 3' G, which has syn base orientation and 60% S-type/40% N-type sugar conformation. The 5' G has anti base orientation and S-type sugar conformation. Apart from the restricted rotation around the 3' G, the structure is similar to that of 1,2 GG intrastrand adducts of 1,1/t,t. This steric hindrance may explain the inability of 1,1/c,c complexes to form 1,2 GG intrastrand adducts with sterically more demanding double-stranded DNA.     

Interaction of cis-[Pt(NH3)2(H2O)2](NO3)2 with ribose deoxyribose diguanosine phosphates

The three diguanosine phosphates GpG (4 X 10(-4) M), d(GpG) (10(-5) M), and d(pGpG) (10(-5) M) have been reacted with cis-[Pt(NH3)2(H2O)2](NO3)2 (1 Pt/dinucleotide) in water at pH 5.5 and 37 degrees C. In each case a single product is formed. The three complexes have been characterized by proton nuclear magnetic resonance (1H NMR) and circular dichroism (CD) analyses. They are N(7)-N(7) chelates of the metal with an anti-anti configuration of the bases. They present a conformational change upon deprotonation of guanine N(1)H whose pKa is ca. 8.7 (D2O). Their CD spectra, compared to those of the free dinucleotides, exhibit an increase of ellipticity in the 275-nm region, which can be qualitatively related to the characteristic increase reported for platinated DNA and poly(dG) . poly(dC). These results are in favor of the hypothesis of intrastrand cross-linking of adjacent guanines, by the cis-PtII(NH3)2 moiety, after a local denaturation of DNA.     

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-?Pt(NH(3))(2)?(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins.