Intrastrand cross-links are not formed in the reaction between transplatin and native DNA: relation with the clinical inefficiency of transplatin.

The reaction between trans-diamminedichloroplatinum(II) and single-stranded oligonucleotides containing the sequence d(GXG) (X being an adenine, cytosine or thymine residue) yields trans-[Pt(NH3)2[(GXG)-GN7,GN7]] intrastrand cross-links. These cross-links do not prevent the pairing of the platinated oligonucleotides with their complementary strands but they decrease the thermal stability of the duplexes. The thermal stability is not much affected by the chemical nature of the X residue and its complementary base. By gel electrophoresis, it is shown that the trans- [Pt(NH3)2[d(GTG)-GN7,GN7]] cross-link bends the DNA double helix (26 degrees) and unwinds it (45 degrees). The pairing of the platinated oligonucleotides with their complementary strands promotes the rearrangement of the 1,3-intrastrand cross-links into interstrand cross-links. At a given temperature, the nature of the X residue, its complementary base and of the base pairs adjacent to the adducts do not dramatically affect the rate of the reaction. To know whether trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links do not rearrange in some sequences, the location of these adducts was searched in double-stranded DNA after reaction with trans-diamminedichloroplatinum(II) by means of the 3'-5' exonuclease activity of T4 DNA polymerase. At low level of platination, trans-[Pt(NH3)2[d(GXG)-GN7,GN7]] cross-links were not detected. Monofunctional adducts and interstrand cross-links were mainly formed. These results are discussed in relation with the clinical inefficiency of trans-diamminedichloroplatinum(II).     

Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.

In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924].     

The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands

Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH₃NH₂)₂Cl₂], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand cross-links. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH₃NH₂)₂Cl₂] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced frequency of intrastrand cross-links yielded by trans-[Pt(CH₃NH₂)₂Cl₂] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH₃NH₂)₂Cl₂]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

Stereochemistry modulates the stability of reduced interstrand cross-links arising from R- and S-alpha-CH3-gamma-OH-1,N2-propano-2'-deoxyguanosine in the 5'-CpG-3' DNA sequence

The solution structures of 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' interstrand DNA cross-links in the 5'-CpG-3' sequence were determined by NMR spectroscopy. These were utilized as chemically stable surrogates for the corresponding carbinolamine interstrand cross-links arising from the crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts. The results provide an explanation for the observation that interstrand cross-link formation in the 5'-CpG-3' sequence by the R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts is dependent upon stereochemistry, favoring the R-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adduct [Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61]. Molecular dynamics calculations, restrained by NOE-based distances and empirical restraints, revealed that both the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-links were located in the minor groove and retained Watson-Crick hydrogen bonds at the tandem cross-linked C.G base pairs. However, for the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the center of the minor groove, whereas for the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the 3' direction, showing steric interference with the DNA helix. The 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link exhibited a lower thermal stability as evidenced by NMR spectroscopy as a function of temperature. The two cross-links also exhibited apparent differences in the conformation of the interstrand three-carbon cross-link, which may also contribute to the lower apparent thermodynamic stability of the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link.     

Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines.

We have studied several aspects of DNA damage formation and repair in human ovarian cancer cell lines which have become resistant to cisplatin through continued exposure to the anticancer drug. The resistant cell lines A2780/cp70 and 2008/c13*5.25 were compared with their respective parental cell lines, A2780 and 2008. Cells in culture were treated with cisplatin, and the two main DNA lesions formed, intrastrand adducts and interstrand cross-links, were quantitated before and after repair incubation. This quantitation was done for total genomic lesions and at the level of individual genes. In the overall genome, the initial frequency of both cisplatin lesions assayed was higher in the parental than in the derivative resistant cell lines. Nonetheless, the total genomic repair of each of these lesions was not increased in the resistant cells. These differences in initial lesion frequency between parental and resistant cell lines were not observed at the gene level. Resistant and parental cells had similar initial frequencies of intrastrand adducts and interstrand cross-links in the dihydrofolate reductase (DHFR) gene and in several other genes after cisplatin treatment of the cells. There was no increase in the repair efficiency of intrastrand adducts in the DHFR gene in resistant cell lines compared with the parental partners. However, a marked and consistent repair difference between parental and resistant cells was observed for the gene-specific repair of cisplatin interstrand cross-links. DNA interstrand cross-links were removed from three genes, the DHFR, multidrug resistance (MDR1), and delta-globin genes, much more efficiently in the resistant cell lines than in the parental cell lines. Our findings suggest that acquired cellular resistance to cisplatin may be associated with increased gene-specific DNA repair efficiency of a specific lesion, the interstrand cross-link.     

Interstrand cross-links of cisplatin induce striking distortions in DNA

In the reaction between cellular DNA and cisplatin, different bifunctional adducts are formed including intrastrand and interstrand cross-links. The respective role of these lesions in the cytotoxicity of the drug is not yet elucidated. This paper deals with the current knowledge on cisplatin interstrand cross-links and presents results on the formation, stability and structure of these adducts. A key step in the studies of these lesions is the recent determination of solution and crystallographic structures of double-stranded oligonucleotides containing a unique interstrand cross-link. The DNA distortions induced by this adduct exhibit unprecedented features such as the location of the platinum residue in the minor groove, the extrusion of the cytosines of the cross-linked d(GpC).d(GpC) site, the bending of the helix axis towards the minor groove and a large DNA unwinding. In addition to a detailed determination of the distortions, the high resolution of the crystal structure allowed us to locate the water molecules surrounding the adduct. The possible implications of this structure for the chemical properties and the cellular processing of cisplatin interstrand cross-links are discussed.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.     

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.     

The nature of inactivating lesions produced by platinum(II) complexes in phage lambda DNA

Lambda DNA loses transfectivity and acquires interstrand cross-links after treatment with either trans-Pt(II) or cis-Pt(II). With trans-Pt(II) there is close to an equivalence between the fraction of lambda DNA cross-linked and the fraction inactivated. In contrast, with cis-Pt(II) there are approx. 5 inactivating lesions for each lambda DNA interstrand cross-link. These results suggested that trans-PT(II) does not introduce intrastrand inactivating lesions into lambda DNA while cis-Pt(II) does so. To verify this conclusion, the cross-linked and uncross-linked fractions of lambda DNA treated with trans-PT(II) or cis-Pt(II) were separated on alkaline sucrose gradients. After trans-Pt(II) treatment, the uncross-linked fraction of lambda DNA was transfective when renaturated. However after cis-Pt(II) treatment the uncross-linked fraction of lambda DNA was not transfective when renatured. Thiourea treatment restored transfectivity to all inactivated fractions, showing that these lesions are reversible. We conclude that trans-Pt(II) inactivates lambda DNA primarily by introducing interstrand cross-links but that cis-Pt(II), although it also introduces interstrand cross-links, inactivates lambda DNA primarily by introducing intrastrand lesions.     

DNA interstrand cross-links of the novel antitumor trinuclear platinum complex BBR3464. Conformation,recognition by high mobility group domain proteins,and nucleotide excision repair

The novel phase II antitumor polynuclear platinum drug BBR3464 ([(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](NO(3))(4)) forms intra- and interstrand cross-links (CLs) on DNA (which is the pharmacological target of platinum drugs). We examined first in our recent work how various intrastrand CLs of BBR3464 affect the conformation of DNA and its recognition by cellular components (Zehnulova, J., Kasparkova, J., Farrell, N., and Brabec, V. (2001) J. Biol. Chem. 276, 22191-22199). In the present work, we have extended the studies on the DNA interstrand CLs of this drug. The results have revealed that the interstrand CLs are preferentially formed between guanine residues separated by 2 base pairs in both the 3' --> 3' and 5' --> 5' directions. The major 1,4-interstrand CLs distort DNA, inducing a directional bending of the helix axis and local unwinding of the duplex. Although such distortions represent a potential structural motif for recognition by high mobility group proteins, these proteins do not recognize 1,4-interstrand CLs of BBR3464. On the other hand, in contrast to intrastrand adducts of BBR3464, 1,4-interstrand CLs are not removed from DNA by nucleotide excision repair. It has been suggested that interstrand CLs of BBR3464 could persist considerably longer in cells compared with intrastrand adducts, which would potentiate the toxicity of the interstrand lesions to tumors sensitive to this polynuclear drug.     

Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines

A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon > 3-carbon > 4-carbon.     

Synthesis and 1H NMR spectroscopic characterization of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]]

The reaction of trans-[Pt(NH3)2Cl2] with the sodium salt of [d(ApGpGpCpCpT)]2 in aqueous solution at 37 degrees C was monitored by reversed-phase high-performance liquid chromatography and UV spectroscopy. Two intermediates, most likely monofunctional adducts, were observed, which subsequently formed one predominant single-stranded product, as well as several polymeric species proposed to be interstrand cross-linked products. The single-stranded adduct was structurally characterized by 1H NMR spectroscopy. From the pH dependence of the chemical shifts, two-dimensional homonuclear chemical shift correlation (COSY) spectroscopy, and one- and two-dimensional nuclear Overhauser effect (NOESY) experiments, the platinum(II) moiety was found to be coordinated to the N7 positions of adenine(1) and guanine(3), with the intervening guanine(2) base destacked from its neighboring residues. This intrastrand 1,3 adduct induces changes in the backbone torsion angles and causes the deoxyribose ring of adenine(1) to switch from a C2'-endo to a predominantly C3'-endo conformation. The other deoxyribose rings retain B DNA type conformations. The structure of trans-[Pt(NH3)2[d(ApGpGpCpCpT)-N7-A(1),N7-G(3)]] differs from those previously reported for cis-DDP 1,2- and 1,3-intrastrand oligonucleotide adducts but is consistent with the structures of trans-DDP 1,3-intrastrand adducts of two previously reported trinucleotides.     

Gene-specific formation and repair of cisplatin intrastrand adducts and interstrand cross-links in Chinese hamster ovary cells

We have used three methods to study the formation and repair of intrastrand adducts and interstrand cross-links in the DNA of Chinese hamster ovary cells induced by the anticancer drug cis-diamminedichloroplatinum II (cisplatin). Using atomic absorption spectroscopy, we found that 21% of the total genomic cisplatin adducts were removed at 8 h and 42% at 24 h. We used ABC excinuclease digestion, coupled with out previously reported methodology to quantify DNA in specific genomic regions. These adducts were removed faster in the transcribed dihydrofolate reductase and c-myc genes compared to a noncoding fragment, a region containing the little or nontranscribed c-fos oncogene, and to the overall genome. Interstrand cross-links in specific sequences were quantified by Southern hybridization of denatured-renatured DNA separated on a neutral gel. We found that cross-links were removed more efficiently from the gene regions than intrastrand adducts and, at high levels of cross-linking, removal was similar from transcribed and from nontranscribed regions.     

UVB-Induced formation of intrastrand cross-link products of DNA in MCF-7 cells treated with 5-bromo-2'-deoxyuridine

Nucleoside 5-bromo-2'-deoxyuridine (BrdU), after being incorporated into cellular DNA, is well-known to sensitize cells to ionizing radiation and UV irradiation. We reported here, for the first time, the sequence-dependent formation of intrastrand cross-link products from the UVB irradiation of BrdU-treated MCF-7 human breast cancer cells. Our results showed that BrdU replaced more than 30% dT in genomic DNA after the cells were treated with 10 microM BrdU for 48 h. LC-MS/MS data revealed that more than 50% of the incorporated BrdU was consumed during UVB irradiation, of which more than half was dehalogenated to yield dU. Low-dose (5.0 kJ/m2) UVB irradiation of BrdU-treated cells yielded four intrastrand cross-link products, where the C5 of uracil is covalently bonded to the C8 of its neighboring 5' or 3' guanine to give G[8-5]U and U[5-8]G, respectively, and the C5 of uracil could couple with the C2 or C8 of its vicinal 5' adenine to give A[2-5]U and A[8-5]U, respectively. All the above cross-link products except A[2-5]U could also be induced in BrdU-treated cells upon UVB irradiation at a dose of 39 kJ/m2. We further demonstrated, by using LC-MS/MS, that the yield of G[8-5]U was much greater than the total yields of A[2-5]U and A[8-5]U. In addition, our results revealed that BrdU treatment stimulated considerably the UVB-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in vivo. The formation of these intrastrand cross-link products and 8-oxo-dG in vivo underscores the importance of these products in the photosensitizing effect of BrdU.     

Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts.

During reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, a number of adducts are formed which may be discriminated by the excision-repair system. An in vitro excision-repair assay with human cell-free extracts has been used to assess the relative repair extent of monofunctional adducts, intrastrand and interstrand cross-links of cis-DDP on plasmid DNA. Preferential removal of cis-DDP 1,2-intrastrand diadducts occurred in the presence of cyanide ions. In conditions where cyanide treatment removed 85% of total platinum adducts while approximately 70% of interstrand cross-links remained in plasmid DNA, no significant variation in repair synthesis by human cell extracts was observed. Then, we constructed three types of plasmid DNA substrates containing mainly either monoadducts, 1,2-intrastrand cross-links or interstrand cross-links lesions. The three plasmid species were modified in order to obtain the same extent of total platinum DNA adducts per plasmid. No DNA repair synthesis was detected with monofunctional adducts during incubation with human whole cell extracts. However, a two-fold increase in repair synthesis was found when the proportion of interstrand cross-links in plasmid DNA was increased by 2-3 fold. These findings suggest that (i) cis-DDP 1,2-intrastrand diadducts are poorly repaired by human cell extracts in vitro, (ii) among other minor lesions potentially cyanide-resistant, cis-DDP interstrand cross-links represent a major lesion contributing to the repair synthesis signal in the in vitro assay. These results could account for the drug efficiency in vivo.     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

Measurement of interstrand cross-link frequency and distance between interruptions in DNA exposed to 4,5',8-trimethylpsoralen and near-ultraviolet light

Bifunctional psoralens react photochemically with DNA to form single-strand adducts and interstrand, chemical cross-links. Cross-link formation is first order with [P], the concentration of added psoralen, when [P] much less than Kd, the psoralen-DNA dissociation constant. DNA molecules containing interstrand cross-links are reversibly bihelical and so are readily detected. It was not heretofore possible to determine cross-link frequency in polydisperse DNA from the mass F of DNA spared cross-linkage. We have derived a statistical relation to calculate cross-link frequency at fixed light exposure and variable [P]. We show here that S, the initial slope of the curve described by -ln F as a function of [P], is proportional to Mw, the weight-average molecular weight of nick-free DNA. The cross-link frequency at any [P] can be determined from k, a constant measured for DNA of known Mw at low cross-linkage. This relation is valid for DNA of any molecular weight distribution. In experiments with uniform length DNA, -ln F (cross-link frequency) increased in simple proportion to [P]. Intact and restriction endonuclease HindIII digested phage lambda DNA molecules have discrete lengths. S for each was proportional to Mw of the twin helix even though the molecular weight distribution of the restriction fragments was skewed. S was proportional to Mw and to the median molecular weight of sheared cellular DNA over a wide range. Also, we found that 1/S was linear with exposure of cellular DNA to gamma radiation. S can therefore be used to calculate L, the average distance between interruptions in the double helix.     

Enhanced DNA repair as a mechanism of resistance to cis-diamminedichloroplatinum(II)

Murine leukemia L1210 cells, either sensitive or resistant to the toxic action of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II), have been studied for potential differences in the formation and repair of drug-induced DNA damage. The sensitivity for these experiments was obtained by using the radiolabeled analogue [3H]-cis-dichloro(ethylenediamine)platinum(II). The resistant cells demonstrated a 40% reduction in drug accumulation but a qualitatively similar profile of DNA-bound adducts. These adducts resembled those previously characterized in pure DNA and represented intrastrand cross-links at GG, AG, and GNG (N is any nucleotide) sequences in DNA. Repair of these cross-links occurred in a biphasic manner: rapid for the first 6 h and then much slower. The resistant cells removed up to 4 times as many adducts during the rapid phase of repair. The extent of this repair did not directly correlate with the degree of resistance in that cells with 100-fold resistance were only slightly more effective at repair than cells with 20-fold resistance. Therefore, although enhanced DNA repair is thought to contribute markedly to drug resistance, other mechanisms for tolerance of DNA damage may also occur in these cells.