Diepoxybutane and diepoxyoctane interstrand cross-linking of the 5S DNA nucleosomal core particle

Diepoxyalkanes form interstrand cross-links in DNA oligomers preferentially at 5'-GNC sites. We have examined cross-linking by 1,2,3,4-diepoxybutane (DEB) and 1,2,7,8-diepoxyoctane (DEO) within a fragment of the 5S RNA gene of Xenopus borealis in both the free and nucleosomal states. Sites and efficiencies of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. Both agents targeted 5'-GNC sites for cross-linking in the restriction fragment in its free state, and DEO also targeted 5'-GNNC sites. Monoalkylation occurred at all deoxyguanosines. The sites for both monoalkylation and interstrand cross-linking were similar in nucleosomal and free DNA, and cross-linked DNA was cleanly incorporated into the core particle structure. These findings suggest that the 5S core particle is able to tolerate any structural abnormalities induced by diepoxide cross-linking.     

cis-diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle

The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins.     

Superhelical torsion controls DNA interstrand cross-linking by antitumor cis- diamminedichloroplatinum(II).

Negatively supercoiled, relaxed and linearized forms of pSP73 DNA were modified in cell-free medium by cis-diamminedichloroplatinum(II) (cisplatin). The frequency of interstrand cross-links (ICLs) formed in these DNAs has been determined by: (i) immunochemical analysis; (ii) an assay employing NaCN as a probe of DNA ICLs of cisplatin; (iii) gel electrophoresis under denaturing conditions. At low levels of the modification of DNA (<1 Pt atom fixed per 500 bp) the number of ICLs formed by cisplatin was radically enhanced in supercoiled in comparison with linearized or relaxed DNA. At these low levels of modification, the frequency of ICLs in supercoiled DNA was enhanced with increasing level of negative supercoiling or with decreasing level of modification. In addition, the replication mapping of DNA ICLs of cisplatin was consistent with these lesions being preferentially formed in negatively supercoiled DNA between guanine residues in both the 5'-d(GC)-3' and the 5'-d(CG)-3' sites. Among the DNA adducts of cisplatin the ICL has the markedly greatest capability to unwind the double helix. We suggest that the formation of ICLs of cisplatin is thermodynamically more favored in negatively supercoiled DNA owing mainly to the relaxation of supercoils.     

Crystals of a nucleosome core particle containing defined sequence DNA

Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystallized, and the crystals examined by X-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a symmetrical arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange high-pressure liquid chromatography. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base-pairs from the molecular dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a approximately equal to 4 A, b approximately equal to 5 A and c approximately equal to 3 A, giving about a twofold increase in the number of measurable X-ray reflections over previous crystals containing mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.     

Thermal stability and energetics of 15-mer DNA duplex interstrand crosslinked by trans-diamminedichloroplatinum(II)

The effect of the location of the interstrand cross-link formed by trans-diamminedichloroplatinum(II) (transplatin) on the thermal stability and energetics of 15-mer DNA duplex has been investigated. The duplex containing single, site-specific cross-link, thermodynamically equivalent model structures (hairpins) and nonmodified duplexes were characterized by differential scanning calorimetry, temperature-dependent uv absorption, and circular dichroism. The results demonstrate that the formation of the interstrand cross-link of transplatin does not affect pronouncedly thermodynamic stability of DNA: the cross-link induces no marked changes not only in enthalpy, but also in "reduced" (concentration independent) monomolecular transition entropy. These results are consistent with the previous observations that interstrand cross-links of transplatin structurally perturb DNA only to a relatively small extent. On the other hand, constraining the duplex with the interstrand cross-link of transplatin results in a significant increase in thermal stability that is primarily due to entropic effects: the cross-link reduces the molecularity of the oligomer system from bimolecular to monomolecular. Importantly, the position of the interstrand cross-link within the duplex modulates cooperativity of the melting transition of the duplex and consequently its thermal stability.     

Effect of cis-, trans-diamminedichloroplatinum(II) and DBP on human serum albumin

Both isomers of diamminedichloroplatinum(II) bind to albumin and induce the formation of the albumin dimer (MW approximately 140 kDa). The trans isomer exhibits a much greater tendency to induce a protein dimerization than the cis isomer. Under similar experimental conditions, the phosphonic derivative of diammineplatinum(II) (DBP) does not induce any dimer formation. The amount of bound complex per mol of human serum albumin (HSA, for an incubation time of 7 days) was found to be 6, 10.5 and 1 mol for cis-, trans-DDP and DBP, respectively. The relative fluorescence intensity of platinum-bound HSA decreases to about 55% for cis-DDP, 45% for trans-DDP and to 85% for DBP when compared to the complex-free protein, suggesting that the binding occurs in the proximity of the Trp214 residue. The structural studies (CD) have shown that only DDP-isomers cause the distinct modification of HSA native structure (alpha-helical content). Pt(II) complexes binding to HSA affect the affinity of HSA towards heme and bilirubin. High excess of DDP prevents the heme and bilirubin binding, while DBP affects this binding much less effectively due to the low amount of the protein-bound complex. Reactions of platinum complexes with albumin are believed to play an important role in the metabolism of this anticancer drug. The minor effect of DBP on HSA may indicate that the toxicity of the phosphonate analog is much lower than toxicities of DDP isomers, most likely due to kinetic reasons.     

S1 nuclease sensitivity to cis- and trans-diamminedichloroplatinum(II) modified DNAS: influence of (G+C) content and nucleotide sequence

The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.     

DNA degradation after interaction of cis- and trans-diamminedichloroplatinum (II) with calf thymus nuclei

DNA samples isolated from control nuclei and nuclei treated by cis- and trans-diamminedichloroplatinum (DDP) were analyzed by gel electrophoresis. There were no changes in Mr when DNA isolated from nuclei treated with trans-DDP was analyzed. Scans of DNA isolated from nuclei treated with cis-DDP revealed significant changes in Mr. This DNA bears, however, no signs of regular fragmentation. The possible involvement of endonuclease activity in the degradation process is discussed.     

Interaction of poly C with cis- and trans-diamminedichloroplatinum (II): Secondary structure effects

The interactions of the ribonucleotide poly C with cis- and trans-diamminedichloroplatinum (II) were probed with Raman spectroscopy. Both platinum compounds attack the N-1 position of the cytosine residue, while the trans form appears to attack the PO₂^? as well. Raman difference spectra obtained from samples of poly C containing similar amounts of the respective Pt compounds indicate that the trans form is able to disrupt the secondary structure of poly C to a greater degree than the cis form. This latter observation may be a contributing factor in the low therapeutic index exhibited by the trans form.     

Interstrand DNA-platinum-DNA/cross-links induced by cis- and trans-diamminedichloroplatinum(II) at elevated temperature

Trans-diamminedichloroplatinum(II) (trans-DDP) forms with DNA at 37 degrees C, more numerous interstrand cross-links than cis-DDP in the isolated DNA and DNA in the chromatin complex. An increase in the temperature to 42.5 degrees C had no effect on the interstrand cross-links of DND-Pt-DNA formed by the two isomers, both in DNA and in chromatin.     

In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein cross-linking, and inhibition of replication

The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the approximately 5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II)

DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA.     

Covalent binding and interstrand cross-linking of duplex DNA by dirhodium(II,II) carboxylate compounds

The study of the interactions of double-stranded (ds) DNA with the dirhodium carboxylate compounds Rh(2)(O(2)CCH(3))(4)(H(2)O)(2) (Rh1), [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) (Rh2), and Rh(2)(O(2)CCF(3))(4) (Rh3) supports the presence of covalently linked DNA adducts, including stable DNA interstrand cross-links. The present biochemical study refutes earlier claims that no reaction between dirhodium compounds and dsDNA occurs. The reversal behavior of these interstrand cross-links in 5 M urea at 95 degrees C (for different heating times) implies the presence of various coordination modes involving ax/ax, ax/eq, and eq/eq DNA interactions with the dirhodium core. The reaction rates of the dirhodium compounds with dsDNA were determined spectroscopically and are in the order Rh1 < Rh2 < Rh3. This difference in behavior of the three dirhodium compounds correlates with the lability of the leaving groups and corresponds to the extent of interstrand cross-link formation by these compounds on a 123 bp DNA fragment, as observed by denaturing polyacrylamide gel electrophoresis (dPAGE). Since all three dirhodium compounds form covalent Rh-DNA adducts, including interstrand cross-links, it is important that DNA be considered a potential target for biological activity of these dirhodium carboxylate compounds.     

Repair of plasmid DNA damaged in vitro with cis- or trans-diamminedichloroplatinum(II) in Escherichia coli

Plasmid pBR322 was modified in vitro with the antitumor compound cis-diamminedichloroplatimum(II) (cis-DDP) or the isomeric trans-DDP. The numbers of platinum adducts were determined by atomic absorption spectrophotetry. DNA-repair-proficient and various DNA-repair-deficient (uvrB, uvrD, recB and recA) strains of Escherichia coli were transformed by the damaged plasmids and the ratios of the transformation frequencies of cells by damaged plasmids relative to those by untreated plasmids were determined. Results of transformation assays indicated that the uvrB gene function was essential for repair of plasmid DNA damaged with cis-DDP. A functional recA gene product seemed to be of minor importance for repair of plasmids damaged with cis-DDP. trans-DDP had a different effect on plasmid DNA. trans-DDP-modified DNA was better able to transform cells than cis-DDP-modified DNA, and the DNAs appeared to be repaired differently. Prior induction of SOS functions increased the survival of plasmids treated with cis-DDP in wild-type and uvrD mutants, but did not increase the survival of plasmids damaged with trans-DDP in these strains. In in vitro repair experiments, plasmid DNA modified with cis-DDP was more readily incised by the UVRABC excinuclease than that modified with trans-DDP.     

Binding of cis- and trans-diamminedichloroplatinum(II) to deoxyribonucleic acid exposes nucleosides as measured immunochemically with anti-nucleoside antibodies

We report the use of anti-nucleoside antibodies to probe for local denaturation of calf thymus DNA upon binding of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, and the biologically inactive analogues trans-diamminedichloroplatinum(II), trans-DDP, and chloro(diethylenetriamine)platinum(II) chloride, [Pt(dien)Cl]Cl. These antibodies specifically recognize each of the four DNA nucleosides. They bind well to denatured DNA, but not to native DNA in which the bases are less accessible owing to Watson-Crick duplex structure. At relatively high levels of modification (D/N approximately 0.1), cis-DDP causes significant disruption of DNA base pairing as reflected by the increased binding of anti-cytidine, anti-adenosine, and anti-thymidine antibodies. At lower levels of platinum adduct formation, however, all four anti-nucleoside antibodies bind more to DNA modified with trans-DDP. This result indicates that adducts formed by trans-DDP disrupt the DNA structure to a greater extent than those formed by cis-DDP at low D/N ratios. Modification of DNA by the monofunctional complex [Pt(dien)Cl]Cl does not affect its recognition by anti-nucleoside antibodies, demonstrating that base pair disruption is a consequence of bifunctional binding. The relative anti-nucleoside antibody recognition of cis-DDP-modified DNA is anti-cytosine greater than anti-adenosine approximately anti-thymidine much greater than anti-guanosine, consistent with the major adduct being an intrastrand d(GpG) cross-link. These results reveal that base pair disruption in a naturally occurring DNA modified by either cis-DDP or trans-DDP is sufficient to be detected by protein (antibody) binding. The relevance of these findings to current ideas about the molecular mechanism of action of cis-DDP is discussed.     

Unwinding of supercoiled DNA by cis- and trans-diamminedichloroplatinum(II): influence of the torsional strain on DNA unwinding.

The effective unwinding angle, phi, for cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP was determined by utilizing high resolution gel electrophoresis and supercoiled phi X174 RF DNA as a substrate. The effective unwinding angle was calculated by equating the reduction in mobility of the DDP-modified DNA to the removal of a number of superhelical turns. The value of the effective unwinding angle for both DDP isomers was greatest at the low levels of DDP bound and decreased with increasing amounts of unwinding agent. The cis-isomer is a better unwinding agent than is the trans-isomer, being nearly twice as effective in unwinding the supercoiled DNA at the DDP levels investigated. A comparison of the magnitude of phi below rb values of 0.005 and those at high levels of binding reveals that the extent of torsional strain in the supercoiled DNA influences the magnitude of the unwinding of the DNA by these complexes. When this method is used in the analysis of the unwinding angle for a covalently bound species on supercoiled DNA, it may provide a more reliable estimate of the magnitude of phi at high degrees of supercoiling and at low levels of modification.     

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.     

Estimation of the extent of platination and the sites of Pt binding after interaction of cis- and trans-diamminedichloroplatinum(II) with DNA and chromatin

Differences in the mode of binding of cis- and trans-diamminedichloroplatinum(II) complexes (cis and trans-DDP) with DNA and chromatin were studied with the use of [14C]methylbromphenvinphos as an alkylating agent which attacks the sites in purines bases involved also in the reaction with cis-DDP (Oliński et al., J. Biochem. Biophys. Meth., 7, 171-173, 1983). Methylation of pre-formed DDP-DNA and DDP-chromatin complexes, followed by qualitative and quantitative analysis of the methylation products in DNA hydrolysates, permitted evaluation of the distribution and extent of platination of the bases. No major differences were found between the action of the two DDP isomers on DNA. However, a significant decrease in binding of trans-DDP to adenine moieties was observed when the interaction of cis- and trans-DDP on chromatin was compared.     

Modification of DNA dynamics by platinum drug binding: a time-dependent fluorescence depolarization study of the interaction of cis- and trans-diamminedichloroplatinum(II) with DNA

The interaction of calf thymus DNA with the antitumor drug cis-diamminedichloroplatinum(II), and with the clinically ineffective trans isomer, is studied by time-dependent fluorescence depolarization spectroscopy of intercalated ethidium. The effect of the platinum compounds on the rapid torsional motions of DNA in solution is observed via depolarization of the ethidium fluorescence. The depolarization data are successfully analyzed with an elastic model of DNA dynamics and yield a value for the product of the torsional rigidity of the DNA and the friction factor for DNA twisting. The dependence of this quantity on the degree of platination of the DNA is determined for each isomer. At low levels of platination, the cis isomer increases the solute-solvent friction acting on the DNA torsional motions, which we attribute to local kinking of the helix axis at the sites of platination. At high levels of platination, the cis isomer decreases the torsional rigidity of the DNA, indicating that disruption of DNA duplex structure occurs under these conditions. The binding of the trans isomer to DNA has no effect on the torsional rigidity or the friction. The present results are compared with other findings on the interaction of these platinum compounds with DNA.     

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn²⁺ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.