In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2 +/+ and MSH2 À/À mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells. Finally, MSH2 À/À cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.

Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence and distribution of T-lymphocytes expressing TcRbeta in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology, distribution and number of T-cells and TcRbeta+ cells in the various sampled lymphoid organs. The highest TcRbeta expression was revealed by real-time PCR in the thymus, with high levels also being found in the gut. In the thymus, DLT15+ and TcRbeta+ cell populations were concentrated in the cortex and TcRbeta+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection. The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRbeta+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRbeta+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular immunity. The large population of TcRbeta(-)/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRgammadelta phenotype fraction.

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DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

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