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, k on, for binding to the 1,2-d(GpG) adduct was 3.1 ؋ 10 4 M ؊1 s ؊1 and the specificity of binding was essentially dependent on k off . 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 MutYdependent 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.cis-Diamminedichloroplatinum(II) (cisplatin) 1 is among the most widely used anticancer chemotherapeutic agents in the treatment of many human tumors, particularly testicular and ovarian tumors (1). In the reaction between DNA and cisplatin, different types of bifunctional adducts are formed that are thought to be the key toxic lesions (2-4). Although these adducts are responsible for a variety of cellular responses including replication and/or transcription inhibition, there is not yet a clear understanding of the molecular mechanisms linking the formation of adducts and cisplatin-induced apoptosis (5, 6). Cisplatin reacts preferentially with the N-7 atoms of purine residues in DNA. The major adducts (90%) are 1,2-intrastrand cross-links at d(GpG) and d(ApG) sites and the minor adducts correspond to 1,3-intrastrand cross-links at d(GpNpG) (N being a nucleotide residue) and interstrand cross-links formed at d(GpC/GpC) sites (7-10). Once formed, cisplatin lesions such as the major 1,2-intrastrand cross-links can undergo replication bypass in cells treated with cisplatin (11,12). Be...

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