The reaction of platinum(II) complexes with DNA. Kinetics of intrastrand crosslink formation in vitro.

The kinetics of the formation of bifunctional DNA platinum(II) adducts (DNA-crosslinks) have been investigated by endonuclease digestion and subsequent HPLC analysis of the soluble nucleotides and nucleotide platinum(II) adducts. The results indicate two waves of crosslinking [rate constants (0.2-0.3) min-1 and (0.015-0.025) min-1] that correlate with changes in ultra violet absorbance and ethidium bromide dependent fluorescence intensity, previously interpreted in terms of two consecutive, local conformational rearrangements of platinum-DNA (Schaller, W., Reisner, H., and Holler, E. (1987) Biochemistry 26, 943-950). The formation of crosslinks at sequences d(GpG) and d(GpNpG) follows identical kinetics. A minimal reaction mechanism is proposed for the binding of cis-diamminedichloroplatinum(II) to DNA under in vitro conditions. The approximately 3-fold higher rate for meso-[1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]diaquaplatinum(II) in comparison to the rate for cis-diamminediaquaplatinum(II) indicates that crosslink formation is affected by the nature of the non-leaving platinum ligand(s).     

DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin.     

DNA adducts of antitumor trans-[PtCl2 (E-imino ether)2].

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.     

The binding affinity of HMG1 protein to DNA modified by cis-platin and its analogs correlates with their antitumor activity.

The antitumor activity of cis-platin is believed to result from its interaction with cellular DNA and subsequent processing of DNA adducts by damage recognition proteins. Among them are the high mobility group (HMG) proteins 1 and 2, which have been hypothesized to mediate the effect of cis-platin. One possibility suggests that the tight binding of HMG1 to DNA adducts blocks the repair of damaged DNA. In order to further evaluate such a mechanism, several cis-platinum complexes with known antitumor activity have been used to treat DNA and the affinity of HMG1 to the DNA adduct induced by each drug was determined. The dissociation constants for the complexes of HMG1 with the platinated probe were obtained by gel mobility shift assays. The antitumor activity of the tested platinum compounds was found to correlate with the binding affinity of HMG1 to the respective drug-DNA adduct. These findings support the view that HMG1 contributes to cytotoxicity of cis-platin by shielding damaged DNA from repair. In addition, they offer a fast test for screening new platinum compounds for antitumor activity.     

Ring-substituted diaqua(1,2-diphenylethylenediamine)platinum(II) sulfate reacts with DNA through a dissociable complex.

Ring-substituted diaqua(1,2-diphenylethylenediamine)platinum(II) sulfate shows unusual kinetics in its reaction with salmon testis DNA. The mechanism for diaqua[meso-1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]platinum(II) sulfate, [Pt(H2O)2(meso-6)]2+SO4(2-), a representative of this series, has been investigated and compared with that for cis-[Pt(NH3)2(H2O)2]2+. Reactions were followed by atomic absorption, analytical HPLC of Pt-DNA digests, arrest of enzymatic DNA synthesis/degradation, ultraviolet and fluorescence spectrophotometry. Except for the formation of monofunctional DNA adducts, the kinetics of the platinum(II) complexes are comparable. The pseudo-first-order rate constant for the attack of DNA by [Pt(H2O)2(meso-6)]2+ follows the concentration of DNA in a hyperbolic fashion, which is in contrast to the linear dependence for cis-[Pt(NH3)2(H2O)2]2+. The hyperbolic dependence is typical for a dissociable DNA/drug complex preceding the coordination reaction. By studying the binding of free ligand to DNA, and by correlating ligand structures and electrostatic charges with effects on adduct formation, both the phenyl residues and the positive charge of the platinum(II) complex are shown to be crucial for the stability of the dissociable complex. A non-intercalative mode of binding to the DNA backbone is suggested. At the high concentrations of DNA found in cell nuclei, the reaction of the dissociable complex can, principally, become rate-limiting in the attack of DNA and thus reduce the cytotoxic efficiency of a drug.     

Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair.

Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide.     

Effects of monofunctional adducts of platinum(II) complexes on thermodynamic stability and energetics of DNA duplexes

Effects of adducts of [PtCl(NH3)3]Cl or chlorodiethylenetriamineplatinum(II) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes (15-bp) containing the single, site-specific monofunctional adduct at G-residues of the central sequences TGT/ACA or 5'-AGT/5'-ACT were prepared and analyzed by differential scanning calorimetry, temperature-dependent ultraviolet absorption and circular dichroism. The unfolding of the platinated duplexes was accompanied by relatively small unfavorable free energy terms. This destabilization was enthalpic in origin. On the other hand, a relatively large reduction of melting temperature (T(m)) was observed as a consequence of the monofunctional adduct in the TGT sequence, whereas T(m) due to the adduct in the AGT sequence was reduced only slightly. We also examined the efficiency of the mammalian nucleotide excision repair system to remove from DNA the monofunctional adducts and found that these lesions were not recognized by this repair system. Thus, rather thermodynamic than thermal characterization of DNA adducts of monofunctional platinum compounds is a property implicated in the modulation of downstream effects such as protein recognition and repair.     

Monofunctional DNA-platinum(II) adducts block frequently DNA polymerases.

The question of whether monofunctional DNA platinum(II) adducts block synthesis of DNA by purified DNA polymerases of different types and origin has been investigated by comparing the time dependence of synthesis arrest and of DNA adduct formation. Activated salmon testis DNA is used as a suitable substrate for DNA synthesis allowing to probe inhibition by platinum(II) monoadducts for the variety of inherent template-primers. Reaction amplitudes are related to defined mixtures of dichloro and chloroaqua platinum(II) complexes. It is found that (i) all investigated DNA polymerases seem arrested (100% efficiency) at bifunctional DNA adducts. (ii) human DNA polymerase beta bypasses most of the monofunctional lesions of the three platinum(II) complexes investigated. (iii) Klenow fragment is blocked by monoadducts with increasing efficiency in the order cis-diamminechloroaquaplatinum(II) (0%) less than meso-[1,2-bis(2,6- dichloro-4-hydroxyphenyl)ethylenediamine] chloroaquaplatinum(II) (50%) less than trans-diamminechloro-aquaplatinum(II) (75%). (iv) Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, Physarum polycephalum DNA polymerase alpha, and calf thymus DNA polymerase alpha appear to be arrested by monoadducts. According to these examples, blocking efficiencies depend on the cis/trans-stereogeometry of fixation of the carrier ligands at platinum(II) residues, on the size/chemical nature of the platin(II) carrier ligand and on the type/origin of DNA polymerase.     

Specificity of platinum-DNA adduct repair

Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216). HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt-DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion synthesis. Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt-GG adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma and HIV-1 RT had a similar specificity for translesion synthesis past Pt-DNA adducts (oxaliplatin > or = cisplatin > JM216). In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts. Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216). These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt-DNA adducts. Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.     

Protein interactions with platinum-DNA adducts: from structure to function

Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol beta and pol eta catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.     

The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study.

Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.     

A novel technique to assay adducts of DNA induced by anticancer agent cis-diamminedichloroplatinum(II).

The dideoxynucleotides d(pGpG) and d(pApG) and the tetradeoxynucleotide d(CpTpApG) were synthesized in solution phase by a modified phosphotriester technique and reacted with the anticancer agent cis-diamminedichloroplatinum(II) (cisplatin). The major products were isolated by HPLC and characterized by NMR and mass spectrometry as cross-link adducts of cisplatin with the neighboring purine bases. The cross-link adducts of d(pGpG) and d(pApG) were dansylated through a 5'-phosphoramidate linkage with ethylenediammine. The labeling efficiency of the adducts was quantitative as in the case of the normal dinucleotides. The modified tetramer was digested with nuclease P1. The excised adduct was enriched by HPLC and labeled with dansyl chloride. The analysis of the postlabeled adduct by HPCL, using a fluorescence detector, detected a peak with retention time corresponding to that of the dansylated cis-Pt(NH3)2d(pApG). Cochromatography with the authentic marker confirmed the identification. The same overall procedure was used to assay calf thymus DNA exposed to cisplatin. The major adducts were identified as cis-Pt(NH3)2d(pGpG) and cis-Pt(NH3)2d(pApG). The quantitative labeling efficiency of platinum adducts combined with highly sensitive fluorescence detection technique (subfemtomol) suggests that fluorescence postlabeling assay could be a novel approach for real-time analysis of DNA modification induced by platinated drugs in biological system.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Role of isomerization of initial complexes in the binding of inhibitors to dihydrofolate reductase

Stopped-flow measurements of protein fluorescence quenching when methotrexate (MTX) binds to dihydrofolate reductase (isoenzyme II) of Streptococcus faecium (SFDHFR II) analyze as the sum of two differentials: a rapid binding phase and a second phase for which the observed rate constant is independent of methotrexate concentration. Analysis of variation of the ratio of the amplitude of the fast and slow phases with methotrexate concentration indicates that the second phase is an isomerization of the initial binary complex. At pH 7.3, the equilibrium constant for this isomerization is 21.9, and the forward and reverse rate constants are 0.57 and 0.026 s-1, respectively. Similar results were obtained for binding of 3-deazamethotrexate to SFDHFR II, but the forward rate constant is greater (2.9 s-1 at pH 7.3). The equilibrium constants for these isomerizations are pH independent, but the rate constants decrease as the pH is raised, probably due to deprotonation of one or more groups on the enzyme. Analysis of progress curves obtained by the development of inhibition when SFDHFR II is added last to reaction mixtures containing dihydrofolate, NADPH, and MTX gives an association constant for initial reactions of 4.3 X 10(7) M-1. Since a preliminary estimate of the association constant for the binding reaction is 7.6 X 10(5) M-1, this suggests an isomerization of the ternary complex(es) with an equilibrium constant of about 56. In addition, analysis of the progress of development of inhibition indicates a further very slow isomerization with equilibrium constant 419 and forward rate constant 2.6 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence properties,thermal duplex stability,and kinetics of formation of cyanin platinum DNAs

Fluorescent and haptenized, monofunctionally binding platinum compounds are increasingly used for chemically labeling nucleic acids for hybridization detection purposes. Here we analyze in detail the effect of labeling density of the cyanin-3 and -5 platinum DNA adducts on fluorescence and thermal stability. We also analyzed the kinetics of the reaction of the cyanin platinum compounds with DNA. The data provided are important for the design of optimal platinum DNA labeling and hybridization conditions for fluorescence hybridization applications.     

The kinetic differences between sodium nitrite, amyl nitrite and nitroglycerin oxidation of hemoglobin

The effect of sodium nitrite, amyl nitrite and nitroglycerin (glyceryl trinitrate) on the hemoglobin of adult erythrocytes was examined in vitro. Both amyl nitrite and nitroglycerin reacted immediately with oxyhemoglobin to effect oxidation into methemoglobin while sodium nitrite required an inductionary period (lag phase) prior to the reaction. Kinetic studies of the biomolecular rate law for each of the preceding reaction's reactionary periods (log phases) allowed rate constant calculations to be made. The values are 1.14 x 10(4) M-1 min-1, 7.45 x 10(4) M-1 min-1, and 3.50 x 10(1) M-1 min-1 for sodium nitrite, amyl nitrite and nitroglycerin, respectively. A comparison of the amyl nitrite and nitroglycerin rate constants reveals that amyl nitrite is approximately 2000-fold more toxic to oxyhemoglobin than nitroglycerin. These oxidant's effect on in vitro hemoglobin solutions are comparable since both reactions approximate to rectangular hyperbolae. Sodium nitrite reacts about 300-fold faster with oxyhemoglobin than does nitroglycerin. However, the sodium nitrite reaction proceeds in a sigmoidal fashion which makes a strict comparison between these compounds relative toxicities less clear cut.     

Kinetic studies of the hydrolysis of platinum-DNA complexes by nuclease S1

The antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP) reacts covalently with DNA and disrupts its secondary structure. Damaged DNA, but not native DNA, is readily digested by S1 nuclease, an endonuclease specific for single stranded polynucleotides. We have measured S1 nuclease digestion of platinated DNA by the release of platinum-DNA adducts and compared it with digestion of unplatinated DNA. The rate of hydrolysis of damaged substrate from platinum-DNA complexes was less than the overall rate of digestion of nucleotides. Similar results were observed for platinum-DNA complexes in native, denatured or renatured conformations. The hydrolysis of denatured platinum-DNA complexes, rb = 0.075 platinum per nucleotide, obeyed Michaelis-Menten kinetics. Taking into account the level of DNA damage, Vm, for the release of platinated adducts was 0.6 times smaller than for digestion of unplatinated DNA. Km values and competition experiments indicated that the enzyme bound equally well to platinated and unplatinated substrates. Similar results were obtained for denatured DNA complexes with trans-DDP while [PtCl(diethylenetriamine)]Cl had no influence on nuclease digestion. These results suggest that bifunctional platinum-DNA lesions have contradictory effects on the hydrolysis of double stranded DNA by S1 nuclease. On one hand they create nuclease sensitive substrate by disrupting DNA secondary structure. On the other, they inhibit digestion of the damaged strand by increasing the activation energy for hydrolysis.     

Reaction of cis-diamminedichloroplatinum (II) and DNA in B or Z conformation

The nature of the adducts and the conformational changes produced in poly(dG-m5dC).poly(dG-m5dC) by cis-diamminedichloroplatinum(II) (cisPt) have been studied. In the reaction of cisPt and B-DNA, the main adduct is bidentate and arises from an intrastrand cross-link between two guanine residues separated by a cytosine. This was deduced from the study of the compounds by t.l.c. after acid hydrolysis of the polymer. The platinated polymer is not digested by S1 nuclease. The antibodies to Z-DNA bind to the platinated polymer with a smaller affinity than to poly (dG-br5dC).poly(dG-br5dC). The c.d. spectrum differs from that of poly(dG-br5dC).poly(dG-br5dC) or poly(dG-m5dC).poly-(dG-m5dC) in Z conformation. It is concluded that the bidentate adduct induces a conformational change from the B form towards a distorted Z form. In the reaction of cisPt and Z-DNA, a monodentate adduct is formed. This adduct stabilizes the Z conformation as shown by c.d. and binding to the anti-Z-DNA antibodies. At room temperature, the second function of the drug can still react with small ligands such as NH4HCO3. By heating, the second function reacts with a guanine residue. A bidentate adduct is formed as in the reaction of cisPt and B-DNA and it induces a transition from the Z form to the distorted Z form.