The DNA mismatch repair system (MMR) plays a crucial role in maintaining genomic stability. It is capable of detection and correction of errors that arise during replication, for example base substitution mismatches or insertion-deletion loops. Hereditary nonpolyposis colon cancer is the most frequent form of hereditary colon cancer in humans and is caused by mutations of proteins in the MMR. Despite intensive investigations of several laboratories, there is still uncertainty about the composition of protein and DNA complexes and the role of the conformational changes involved. Therefore, the aim of this thesis was to establish fluorescence-based assays which allow the analysis of initial sub-steps in MMR. As the MMR is highly conserved and well-studied in Escherichia coli, these assays were developed for the Escherichia coli system.To observe complex formations in MMR the phenomenon of Förster Resonance Energy Transfer was used. By labeling one component of the MMR with a donor and another one with an acceptor fluorophore, it was possible to observe a complex formation between those two components. A similar setup was used to determine conformational changes of one MMR component that carried both, the donor and acceptor fluorophore. Both reactions could be followed by spectroscopic detection of the fluorescence signals. Several assays of this thesis required the generation and testing of suitable DNA constructs and fluorescence modification of different protein variants. For the fluorescent dye labeling of proteins, single-cysteine variants were selected from a set of variants which were originally generated for crosslinking studies. A possible disturbance of the protein activity by the fluorophores was excluded as the selected protein variants (MutS R449C D835R, MutL H297C, and MutH S85C) were not influenced in activity after fluorescent dye labeling.Changes in fluorescence intensity as well as FRET efficiency were monitored during each assay to visualize involved conformational changes and complex formations. Fluorescence spectra were always recorded as a quality control to ensure that the observed intensity changes were due to FRET. The analyzed processes in MMR were MutS mismatch recognition, MutS bending DNA, MutS sliding clamp formation, MutS-MutL complex formation, MutL interaction with DNA, MutL-MutH complex formation, and MutH forming and leaving the incision complex. Kinetic data sets for selected sub-steps were collected and used for the development of a kinetic model for the whole MMR system in frame of the European FP7 project mismatch2model. With this collection of fluorescence assays it is now possible to gain new insights into the sub-steps of MMR which helps to understand the intricate MMR process in detail. Currently, the fully active, fluorescent dye labeled proteins which were generated in frame of this thesis are used in single-molecule studies of the MMR, for example in a magnetic tweezers setup combined with fluorescence detection.
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