MSH2 missense mutations alter cisplatin cytotoxicity and promote cisplatin-induced genome instability

Defects in the mismatch repair protein MSH2 cause tolerance to DNA damage. We report how cancer-derived and polymorphic MSH2 missense mutations affect cisplatin cytotoxicity. The chemotolerance phenotype was compared with the mutator phenotype in a yeast model system. MSH2 missense mutations display a strikingly different effect on cell death and genome instability. A mutator phenotype does not predict chemotolerance or vice versa. MSH2 mutations that were identified in tumors (Y109C) or as genetic variations (L402F) promote tolerance to cisplatin, but leave the initial mutation rate of cells unaltered. A secondary increase in the mutation rate is observed after cisplatin exposure in these strains. The mutation spectrum of cisplatin-resistant mutators identifies persistent cisplatin adduction as the cause for this acquired genome instability. Our results demonstrate that MSH2 missense mutations that were identified in tumors or as polymorphic variations can cause increased cisplatin tolerance independent of an initial mutator phenotype. Cisplatin exposure promotes drug-induced genome instability. From a mechanistical standpoint, these data demonstrate functional separation between MSH2-dependent cisplatin cytotoxicity and repair. From a clinical standpoint, these data provide valuable information on the consequences of point mutations for the success of chemotherapy and the risk for secondary carcinogenesis

The molecular mechanism of DNA damage recognition by MutS homologs and its consequences for cell death response

We determined the molecular mechanism of cell death response by MutS homologs in distinction to the repair event. Key protein–DNA contacts differ in the interaction of MutS homologs with cisplatinated versus mismatched DNA. Mutational analyses of protein–DNA contacts, which were predicted by molecular dynamics (MD) simulations, were performed. Mutations in suggested interaction sites can affect repair and cell death response independently, and to different extents. A glutamate residue is identified as the key contact with cisplatin-DNA. Mutation of the residue increases cisplatin resistance due to increased non-specific DNA binding. In contrast, the conserved phenylalanine that is instrumental and indispensable for mismatch recognition during repair is not required for cisplatin cytotoxicity. These differences in protein–DNA interactions are translated into localized conformational changes that affect nucleotide requirements and inter-subunit interactions. Specifically, the ability for ATP binding/hydrolysis has little consequence for the MMR-dependent damage response. As a consequence, intersubunit contacts are altered that most likely affect the interaction with downstream proteins. We here describe the interaction of MutS homologs with DNA damage, as it differs from the interaction with a mismatch, and its structural translation into all other functional regions of the protein as a mechanism to initiate cell death response and concomitantly inhibit repair

Parameters of Reserpine Analogs That Induce MSH2/MSH6-Dependent Cytotoxic Response

Mismatch repair proteins modulate the cytotoxicity of several chemotherapeutic agents. We have recently proposed a “death conformation” of the MutS homologous proteins that is distinguishable from their “repair conformation.” This conformation can be induced by a small molecule, reserpine, leading to DNA-independent cell death. We investigated the parameters for a small reserpine-like molecule that are required to interact with MSH2/MSH6 to induce MSH2/MSH6-dependent cytotoxic response. A multidisciplinary approach involving structural modeling, chemical synthesis, and cell biology analyzed reserpine analogs and modifications. We demonstrate that the parameters controlling the induction of MSH2/MSH6-dependent cytotoxicity for reserpine-analogous molecules reside in the specific requirements for methoxy groups, the size of the molecule, and the orientation of molecules within the protein-binding pocket. Reserpine analog rescinnamine showed improved MSH2-dependent cytotoxicity. These results have important implications for the identification of compounds that require functional MMR proteins to exhibit their full cytotoxicity, which will avoid resistance in MMR-deficient cells

Cooperative Nuclear Localization Sequences Lend a Novel Role to the N-Terminal Region of MSH6

Human mismatch repair proteins MSH2-MSH6 play an essential role in maintaining genetic stability and preventing disease. While protein functions have been extensively studied, the substantial amino-terminal region (NTR*) of MSH6 that is unique to eukaryotic proteins, has mostly evaded functional characterization. We demonstrate that a cluster of three nuclear localization signals (NLS) in the NTR direct nuclear import. Individual NLSs are capable of partially directing cytoplasmic protein into the nucleus; however only cooperative effects between all three NLSs efficiently transport MSH6 into the nucleus. In striking contrast to yeast and previous assumptions on required heterodimerization, human MSH6 does not determine localization of its heterodimeric partner, MSH2. A cancer-derived mutation localized between two of the three NLS significantly decreases nuclear localization of MSH6, suggesting altered protein localization can contribute to carcinogenesis. These results clarify the pending speculations on the functional role of the NTR in human MSH6 and identif