Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.

In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases.     

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.     

Recognition of cisplatin-DNA interstrand cross-links by replication protein A

Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.     

Binding discrimination of MutS to a set of lesions and compound lesions (base damage and mismatch) reveals its potential role as a cisplatin-damaged DNA sensing protein

The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate. The rate of association, kon, for binding to the 1,2-d(GpG) adduct was 3.1 x 104 m-1 s-1 and the specificity of binding was essentially dependent on koff. DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized. Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra. Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutY-dependent mismatch repair pathway. The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.     

Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2] [3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.     

DNA bending and unwinding due to the major 1,2-GG intrastrand cross-link formed by antitumor cis-diamminedichloroplatinum(II) are flanking-base independent

Antitumor cisplatin [cis-diamminedichloroplatinum(II)] forms on DNA predominantly intrastrand cross-links between neighboring purine residues. Several discoveries suggested that the toxicity of cisplatin originated from these lesions. The formation of 1,2-GG intrastrand cross-link of cisplatin leads to marked conformational alterations in DNA including a directional, rigid bend toward the major groove and local unwinding. These altered structures attract various cellular proteins. This phenomenon has been postulated to mediate antitumor properties of cisplatin. Importantly, the binding affinity of several proteins that specifically recognize 1,2-GG intrastrand cross-link to platinated DNA is modulated by the nature of the base pairs that immediately flank the platinated d(GpG) site. However, the influence of sequence context on DNA bending and unwinding due to the formation of the 1,2-GG intrastrand cross-link has not been extensively investigated. In the present study we have employed electrophoretic retardation (phasing) assay to analyze bending and unwinding induced by the single, site-specific 1,2-GG intrastrand cross-link immediately flanked by various bases formed by cisplatin in nine oligodeoxyribonucleotide duplexes. The results indicate that bending and unwinding of DNA as a consequence of the formation of the major adduct of cisplatin is, in the first approximation, independent of the base pairs flanking the platinated d(GpG) site.     

Recognition of cisplatin adducts by cellular proteins

Cisplatin is a widely used chemotherapeutic agent. It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts. The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin. The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins. Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival.     

Cisplatin adducts inhibit 1,N(6)-ethenoadenine repair by interacting with the human 3-methyladenine DNA glycosylase

The human 3-methyladenine DNA glycosylase (AAG) is a repair enzyme that removes a number of damaged bases from DNA, including adducts formed by some chemotherapeutic agents. Cisplatin is one of the most widely used anticancer drugs. Its success in killing tumor cells results from its ability to form DNA adducts and the cellular processes triggered by the presence of those adducts in DNA. Variations in tumor response to cisplatin may result from altered expression of cellular proteins that recognize cisplatin adducts. The present study focuses on the interaction between the cisplatin intrastrand cross-links and human AAG. Using site-specifically modified oligonucleotides containing each of the cisplatin intrastrand cross-links, we found that AAG readily recognized cisplatin adducts. The apparent dissociation constants for the 1, 2-d(GpG), the 1,2-d(ApG), and the 1,3-d(GpTpG) oligonucleotides were 115 nM, 71 nM, and 144 nM, respectively. For comparison, the apparent dissociation constant for an oligonucleotide containing a single 1,N(6)-ethenoadenine (epsilonA), which is repaired efficiently by AAG, was 26 nM. Despite the affinity of AAG for cisplatin adducts, AAG was not able to release any of these adducts from DNA. Furthermore, it was demonstrated that the presence of cisplatin adducts in the reactions inhibited the excision of epsilonA by AAG. These data suggest a previously unexplored dimension to the toxicological response of cells to cisplatin. We suggest that cisplatin adducts could titrate AAG away from its natural substrates, resulting in higher mutagenesis and/or cell death because of the persistence of AAG substrates in DNA.     

Nucleotide excision repair from site-specifically platinum-modified nucleosomes

Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is approximately 2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level.     

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.     

End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion

The study of DNA repair has been facilitated by the development of extract-based in vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3'-ends of the duplex repair substrate with nuclease-resistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3'-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3'-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5'-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.     

Recognition of DNA interstrand cross-link of antitumor cisplatin by HMGB1 protein

Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands.     

Structural, energetic and dynamic properties of guanine(C8)-thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742-755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5'-d(…G*pT*…) and 5'-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson-Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.     

Solution structure of a DNA duplex containing a cis-diammineplatinum(II) 1,3-d(GTG) intrastrand cross-link, a major adduct in cells treated with the anticancer drug carboplatin.

The platinum 1,3-d(GXG) intrastrand cross-link is one of the adducts formed in the reaction of the antitumor drug cisplatin with DNA, and in fact the major adduct found in cells treated with the cisplatin analogue carboplatin. To determine the 3D structure of this adduct, the duplex d(CTCTGTGTCTC).d(GAGACACAGAG)], where GTG denotes a platinum 1,3-intrastrand cross-link, was prepared and studied with high-resolution (1)H NMR. The solution structure was determined using the SPEDREF protocol, which includes an iterative NOE-restrained refinement procedure. Calculated and recorded NOE spectra were found to be in good agreement (NMR R factor 22%). The studied duplex is more distorted from B-DNA than previously determined structures of the 1,2-d(GG) intrastrand adducts. The base pairing is lost for the 5'G-C and the central T-A base pair in the GTG lesion, and the central thymine is extruded from the minor groove. To accommodate this lesion, the minor groove is widened, and the 5'-guanine ribose adopts an N-type conformation. The helix is unwound locally and is significantly bent toward the major groove. Significant difference between the structural distortion of the 1, 3-d(GTG) cross-link and other Pt-DNA cross-links sheds new light on the observed differences in protein recognition of these lesions, and thus on the possible differences in mechanisms of action of the various Pt-DNA adducts formed in treatment with platinum anticancer complexes.     

Redox state-dependent interaction of HMGB1 and cisplatin-modified DNA

HMGB1, one of the most abundant nuclear proteins, has a strong binding affinity for cisplatin-modified DNA. It has been proposed that HMGB1 enhances the anticancer efficacy of cisplatin by shielding platinated DNA lesions from repair. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. The reduced domain A protein binds to a 25-bp DNA probe containing a central 1,2-d(GpG) intrastrand cross-link, the major platinum-DNA adduct, with a 10-fold greater binding affinity than the oxidized domain A. The binding affinities of singly and doubly mutated HMGB1 domain A, respectively deficient in one or both cysteine residues that form the disulfide bond, are unaffected by changes in external redox conditions. The redox-dependent nature of the binding of HMGB1 domain A to cisplatin-modified DNA suggests that formation of the intradomain disulfide bond induces a conformational change that disfavors binding to cisplatin-modified DNA. Hydroxyl radical footprinting analyses of wild-type domain A bound to platinated DNA under different redox conditions revealed identical cleavage patterns, implying that the asymmetric binding mode of the protein across from the platinated lesion is conserved irrespective of the redox state. The results of this study reveal that the cellular redox environment can influence the interaction of HMGB1 with the platinated DNA and suggest that the redox state of the A domain is a potential factor in regulating the role of the protein in modulating the activity of cisplatin as an anticancer drug.     

Modulation of MutS ATP-dependent functional activities by DNA containing a cisplatin compound lesion (base damage and mismatch)

DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versus a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3' platinated guanine. MutS exhibited substantial affinity for this compound lesion in hydrolytic and in non-hydrolytic conditions of ATP, contrasting with the normal nucleotide inhibition effect of mispair binding. The cisplatin compound lesion was also shown to stimulate poorly MutS ATPase activity to approach the hydrolysis rate induced by nonspecific DNA. Moreover, MutS undergoes distinct conformation changes in the presence of the compound lesion and ATP under hydrolytic conditions as shown by limited proteolysis. In the absence of MutS, the cisplatin compound lesion was shown to induce a 39 degrees rigid bending of the DNA double helix contrasting with an unbent state for DNA containing a GT mispair. Furthermore, an unbent DNA substrate containing a monofunctional adduct mimicking a cisplatin residue failed to form a persistent nucleoprotein complex with MutS in the presence of adenine nucleotide. We propose that DNA bending could play a role in MutS biochemical modulations induced by a compound lesion and that cisplatin DNA damage signaling by the MMR system could be modulated in a direct mode.