The role of Rad50, Mre11 and Nbs1 complex in DNA double strand break repair

The ability of cells to repair DNA double strand breaks (DSBs) is of utmost importance for survival and maintenance of genomic integrity. Without the aptitude to mediate efficient repair, cells run the risk of acquiring mutations that can lead to tumorigenesis. Mammalian cells predominantly utilize the non homologous end joining (NHEJ) pathway to repair DSBs. NHEJ has been postulated to depend on the activity of the evolutionarily conserved complex containing Rad50, Mre11 and Nbs1 (RMN). Studies in mammalian cells have revealed that mutations in the human Mre11 and Nbs1 gene products result in Ataxia telagiectasia like disorder (ATLD) and Nijmegen breakage syndrome (NBS) respectively. These disorders, like Fanconi anemia (FA), are characterized by hypersensitivity to genotoxic agents and cancer predisposition. I hypothesized that the RMN complex plays a direct catalytic role in end joining of DNA substrates in nuclear extracts and that this involves the complex binding to and stabilizing broken chromosome ends. DNA end-joining in extracts from normal cells was found to be reduced 5-fold when Rad50 protein levels were decreased. Conversely, targeting Rad50 levels in extracts from FA cells had no further effect on residual end-joining activity. Patient derived NBS fibroblasts were observed to be deficient in the repair of both extrachromosomal and chromosomal DSBs. End joining activity was also significantly impaired in extracts from NBS cells as compared to normal cells. Genetic correction of the NBS cells with Nbs1 cDNA restored resistance to death by DNA alkylating agents and also corrected the DNA end joining defect. Furthermore, an analysis of RMN dependent DNA tethering activity revealed robust tethering in normal cell extracts, while tethering activity was significantly impaired in extracts from cells expressing a dominant negative FANC allele. As compared to normal cells, tethering was almost non existent in NBS extracts. The tethering activity in extracts from the genetically corrected NBS cells was restored to levels similar to those observed in normal cell extracts. Taken together, these data strongly suggest that functional nibrin is specifically needed for the end-joining repair mechanism to work properly and that the pathogenesis of FA and NBS is directly related to aberrant RMN function.