Analyse der biologischen Funktion des 'mammalian DNA mismatch repair system (MMR)' und wie Mutationen in MMR-Genen die DNA-Reparatur und Tumorempfänglichkeit beeinflussen?
Zusammenfassung der Projektergebnisse
Exonuclease 1 (Exo1), a 5’ to 3’ exonuclease, is involved in DSB repair, stalled replication fork processing, meiosis, telomere maintenance and remains the only known exonuclease in eukaryotic DNA mismatch repair (MMR). Genetic screens in yeast also suggest an important role of Exo1 in assembly of higher order MMR complexes in addition to the exonucleolytic function. Germ-line variations and mutations in human EXO1 have been identified in hereditary nonpolyposis colorectal cancer (HNPCC), however, the significance of these mutations for HNPCC remains uncertain. To delineate the roles of the enzymatic activity and the scaffold function of Exo1 in mammalian MMR and meiosis, we created two Exo1 mutant mouse lines. One line carries an Exo1-null mutation (Exo1null/null), leading to the complete loss of the Exo1 protein. The other mouse line carries the Exo1-E109K mutation (Exo1EK/EK), which inactivates the enzymatic function of the protein but does not interfere with protein stability, DNA binding capacity, or the ability of the protein to interact with other MMR proteins. The Exo1-E109K mutation was also found in several MSI positive human colorectal tumors of HNPCC patients. By carefully analyzing these two mouse models, we show that while the Exo1 protein is essential for all the cellular processes tested, the nuclease activity of Exo1 is surprisingly dispensable for mismatch repair, NHEJ, and meiosis. The nuclease activity is nonetheless required for the repair of DNA double strand breaks through DNA end resection and homologous recombination. We also show that both mouse models succumb to genomic instability, albeit with different tumor spectra, which suggests that various DNA repair processes could safeguard organs in a differential manner. Taken together, we present evidence that the Exo1-E109K mutation is a powerful separation of function mutant that allows the molecular dissection of the various functions of Exo1, be it nucleasedependent or nuclease-independent. In addition, our data also offers an explanation to the atypical nature of some hereditary HNPCC patients by attributing the phenotype to deficiencies in double strand break rather than mismatch repair.
