Recognition of the DNA helix stabilizing anthramycin-N2 guanine adduct by UVRABC nuclease

The binding of the anti-tumor antibiotic anthramycin to a defined linear DNA fragment was investigated using both exonuclease III and lambda exonuclease. We show that most of the guanine residues are reactive toward anthramycin; however, several guanine residues showed preferential reactivity for the drug. Using purified UVRA, UVRB and UVRC proteins we present evidence that these three proteins in concert are able to recognize and produce specific strand cleavage flanking anthramycin-DNA adducts. The cleavage of anthramycin adducts by UVRABC nuclease is specific and results in strand breaks at five or six bases 5' and three or four bases 3'-flanking an adduct. At some guanine residues single incisions were observed only on one side of the adduct. The 5' strand breaks observed often occurred as doublet bands on sequencing gels, indicating plasticity in the site of 5' cleavage whereas the 3' cleavage did not show this effect. When DNA fragments modified with elevated levels of anthramycin were used as substrates the activity of the UVRABC nuclease toward the anthramycin adducts decreased. Possible mechanisms for the recognition and specific cleavage of the helix-stabilizing anthramycin DNA adduct and other helix destabilizing lesions by the UVRABC nuclease are discussed.     

Role of structure and dynamics of DNA with cisplatin and oxaliplatin adducts in various sequence contexts on binding of HMGB1a

Anti-cancer drugs, such as cisplatin and oxaliplatin, covalently bind to adjacent guanine bases in DNA to form intra-strand adducts. Differential recognition of drug–DNA adducts by the protein HMGB1a has been related to the differences in efficacy of these drugs in tumours. Additionally, the bases flanking the adduct (the sequence context) also have a marked effect on HMGB1a binding affinity. We perform atomistic molecular dynamics simulations of DNA with cisplatin and oxaliplatin adducts in four sequence contexts (AGGC, CGGA, TGGA and TGGT) in the absence and presence of HMGB1a. The structure of HMGB1a-bound drug–DNA molecules is independent of sequence and drug identity, confirming that differential recognition cannot be explained by the protein-bound structure. The differences in the static and conformational dynamics of the drug–DNA structures in the absence of the protein explain some but not all trends in differential binding affinity of HMGB1a. Since the minor groove width and helical bend of all drug–DNA molecules in the unbound state are lower than the protein-bound state, HMGB1a must actively deform the DNA during binding. The thermodynamic pathway between the unbound and protein-bound states could be an additional factor in the binding affinity of HMGB1a for drug–DNA adducts in various sequence contexts.     

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.     

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF–, AAF– and AP–DNA adducts, determined by gel mobility shift assay, are 33 ± 9, 8 ± 2 and 23 ± 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF–DNA was cleaved ~2-fold more efficiently than AF– or AP–DNA (AAF > AF ≈ AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF–DNA (but not for AP–DNA), making it equivalent to that for AAF–DNA. These results are consistent with a model in which DNA damage recognition by the E.coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF– and AAF–DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.     

Excision Repair of 2,5-Diaziridinyl-1,4-Benzoquinone (DZQ)-DNA Adduct by Bacterial and Mammalian 3-Methyladenine-DNA Glycosylases

The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formami-dopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.     

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 effect of chromatin structure on cisplatin damage in intact human cells

The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.     

DNA adducts of the enantiomers of the Pt(II) complexes of the ahaz ligand (ahaz=3-aminohexahydroazepine) and recognition of these adducts by HMG domain proteins

The bending, unwinding, and structural changes in DNA caused by the binding of each of the enantiomers of the platinum(II) complexes of the ahaz ligand (R- and S-[PtCl(2)(ahaz)], ahaz=3-aminohexahydroazepine) have been studied using 20-23 bp oligonucleotides containing TGGT and CGGA-binding sites as has the recognition of the adducts by HMG domain proteins. The domain A of HMGB1 (HMGB1a protein) binds to the adduct formed by the R enantiomer at the CGGA sequence with a similar high affinity as it does to the adduct of antitumor cisplatin, and to the adduct formed by the S enantiomer with a slightly lower affinity. In contrast, HMGB1a binds much more weakly to the ahaz adducts than to the cisplatin adducts formed at the TGGT sequence, with the binding to the adduct formed by the R enantiomer being weakest. Each enantiomer and cisplatin cause unwinding of both sequences that is in the narrow range, 19-22 degrees. There are modest but significant differences in the degree of bending induced, with the S enantiomer causing the least bending, cisplatin intermediate, and the R enantiomer the most. Molecular modeling of the {Pt(ahaz)}/GG adducts in 8-bp models reveals significant differences in the local distortion at the GG-binding sites depending on the flanking bases and shows that interactions between the thymine methyl groups and the ahaz ligand are likely to inhibit bending of the TGGT sequence.     

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.     

Molecular dynamic simulations of cisplatin- and oxaliplatin-d(GG) intrastand cross-links reveal differences in their conformational dynamics

Mismatch repair proteins, DNA damage-recognition proteins and translesion DNA polymerases discriminate between Pt-GG adducts containing cis-diammine ligands (formed by cisplatin (CP) and carboplatin) and trans-RR-diaminocyclohexane ligands (formed by oxaliplatin (OX)) and this discrimination is thought to be important in determining differences in the efficacy, toxicity and mutagenicity of these platinum anticancer agents. We have postulated that these proteins recognize differences in conformation and/or conformational dynamics of the DNA containing the adducts. We have previously determined the NMR solution structure of OX-DNA, CP-DNA and undamaged duplex DNA in the 5'-d(CCTCAGGCCTCC)-3' sequence context and have shown the existence of several conformational differences in the vicinity of the Pt-GG adduct. Here we have used molecular dynamics simulations to explore differences in the conformational dynamics between OX-DNA, CP-DNA and undamaged DNA in the same sequence context. Twenty-five 10 ns unrestrained fully solvated molecular dynamics simulations were performed starting from two different DNA conformations using AMBER v8.0. All 25 simulations reached equilibrium within 4 ns, were independent of the starting structure and were in close agreement with previous crystal and NMR structures. Our data show that the cis-diammine (CP) ligand preferentially forms hydrogen bonds on the 5' side of the Pt-GG adduct, while the trans-RR-diaminocyclohexane (OX) ligand preferentially forms hydrogen bonds on the 3' side of the adduct. In addition, our data show that these differences in hydrogen bond formation are strongly correlated with differences in conformational dynamics, specifically the fraction of time spent in different DNA conformations in the vicinity of the adduct, for CP- and OX-DNA adducts. We postulate that differential recognition of CP- and OX-GG adducts by mismatch repair proteins, DNA damage-recognition proteins and DNA polymerases may be due, in part, to differences in the fraction of time that the adducts spend in a conformation favorable for protein binding.     

Specific DNA structural attributes modulate platinum anticancer drug site selection and cross-link generation

Heavy metal compounds have toxic and medicinal potential through capacity to form strong specific bonds with macromolecules, and the interaction of platinum drugs at the major groove nitrogen atom of guanine bases primarily underlies their therapeutic activity. By crystallographic analysis of transition metal-and in particular platinum compound-DNA site selectivity in the nucleosome core, we establish that steric accessibility, which is controlled by specific structural parameters of the double helix, modulates initial guanine-metal bond formation. Moreover, DNA conformational features can be linked to both similarities and distinctions in platinum drug adduct formation between the naked and nucleosomal DNA states. Notably, structures that facilitate initial platinum-guanine bond formation can oppose cross-link generation, rationalizing the occurrence of long-lived therapeutically ineffective monofunctional adducts. These findings illuminate DNA structure-dependent reactivity and provide a novel framework for understanding metal-double helix interactions, which should facilitate the development of improved chromatin-targeting medicinal agents.     

Strong sequence-dependent polymorphism in adduct-induced DNA structure: analysis of single N-2-acetylaminofluorene residues bound within the NarI mutation hot spot

We have used a set of chemical probes to characterize and to compare the structural deformation of double-stranded oligomers bearing a single N-2-acetylaminofluorene (AAF) adduct covalently bound to each of the three guanine residues located within the frameshift mutation hot spot sequence -G1G2CG3CC-(NarI site). Two classes of chemical probes have been used, probes that sense the geometry of the helix, giving rise to cuts at every nucleotide (for example, 1,10-phenanthroline-copper), and probes that react with specific bases depending on their conformation (e.g., diethyl pyrocarbonate). For all probes that were tested, a distinct pattern of reactivity was observed according to the position of the adduct within the DNA sequence, revealing an important polymorphism in the adduct-induced DNA structure. With 1,10-phenanthroline-copper at least three base pairs 3' of the AAF-modified guanine were reactive on each strand, showing that the deformation of the DNA helix extends over a region of 4-6 bases pairs centered around the adduct and sensed by the probe in both strands. With the base-specific probes, reactivities were limited to the base complementary to the modified guanine and to adjacent bases. Within this sequence context, the three possible AAF adducts have previously been shown to exhibit strong differences in biological responses such as excision repair [Seeberg, E., & Fuchs, R. P. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 191-194] and mutagenesis [Burnouf, D., Koehl, P., & Fuchs, R. P. P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151].(ABSTRACT TRUNCATED AT 250 WORDS)     

The interactions of square platinum(II) complexes with guanine and adenine: a quantum-chemical ab initio study of metalated tautomeric forms

The influence of binding of square planar platinum complexes on tautomeric equilibria of the DNA bases guanine and adenine was investigated using the density functional B3LYP method. Neutral trans-dichloro(amine)-, +1 charged chloro(diamine)-, and +2 charged triamine-platinum(II) species were chosen for coordination to bases. Only the N₇ interaction site of the bases was considered. The calculations demonstrate that the neutral platinum adduct does not change the tautomeric equilibria of the bases. Furthermore, N₇ binding of the neutral Pt adduct moderately reduces the probability of protonation of the N₁ position of adenine. Larger effects can be observed for +1 and mainly +2 adducts, but these can be rationalized by electrostatic effects. Since the electrostatic effects are expected to be efficiently compensated for by a charged backbone of DNA and counterions in a polar solvent, no dramatic increase in mispair formation is predicted for Pt(II) adducts, which is in agreement with experiment. The interaction energies between Pt adducts and the nucleobases were also evaluated. These interaction energies range from ca. 210 kJ/mol for neutral adducts, interacting with both bases and their tautomers, up to 500 kJ/mol for the +2 charged adducts, interacting with the keto-guanine tautomer and the anti-imino-adenine tautomer. The surprisingly large interaction energy for the latter structure is due to the strong H-bond between the NH₃ ligand group of the metal adduct and the N₆ nitrogen atom of the base. Received: 6 July 1999 / Accepted: 7 December 1999     

Excision Repair of 2,5-Diaziridinyl-1,4-Benzoquinone (DZQ)-DNA Adduct by Bacterial and Mammalian 3-Methyladenine-DNA Glycosylases

The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level.     

Energetics,conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear Pt complex

Background

The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear Pt^II complexes, such as [{cis-Pt(NH₃)₂}₂(μ‐OH)(μ-pyrazolate)]²⁺ (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin.

Methods

Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins.

Results

Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin.

General significance

The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear Pt^II complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells.     

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.     

Effects of DNA adduct structure and sequence context on strand opening of repair intermediates and incision by UvrABC nuclease

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.     

Sequence specificity, conformation, and recognition by HMG1 protein of major DNA interstrand cross-links of antitumor dinuclear platinum complexes

Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [?trans-PtCl(NH(3))(2)?H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site-specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues.     

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

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

DNA damage by anti-cancer agents resolved at the nucleotide level of a single copy gene: evidence for a novel binding site for cisplatin in cells.

A new PCR based technique has been developed to investigate the sequence selectivity of adduct formation by DNA damaging agents in a single copy gene in isolated genomic DNA or in drug treated cells. Single-strand ligation PCR (sslig-PCR) demonstrated that cisplatin and nitrogen mustards reacted with guanine in an N-ras fragment with varying sequence specificity similar to that observed previously in plasmid DNA. In cisplatin-treated cells sslig-PCR demonstrated all the adducts found in isolated DNA and with the same sequence selectivity showing a preference for GG and AG sites. However, in cells an additional site of DNA binding of cisplatin was observed at the two occurrences of the sequence 5'-TACT-3' on the transcribed and non-transcribed strands. This sequence is not a recognised target for cisplatin and represents a novel adduct formed in cells.