Establishment and application of fluorescence recovery after photobleaching to study the nuclear dynamics of DNA repair proteins

[摘要] The genetic information is constantly exposed to physical and chemical DNA-damaging agents. To safeguard genome stability, nucleotide excision repair (NER) is a protective system that processes a wide diversity of structurally unrelated DNA lesions, including UV–induced photoproducts and cisplatin intrastrand crosslinks. The aim of this thesis was to establish novel fluorescent protein-based imaging techniques to study NER activity in living fibroblasts and to apply these methods to determine the mechanism by which low-dose formaldehyde, a widely used genotoxic chemical, inhibits DNA repair. The nuclear movements of NER factors were analyzed by measuring the kinetics of accumulation at lesion sites and by monitoring protein dynamics in fluorescence recovery after photobleaching experiments. In combination, this live-cell imaging approach reveals that formaldehyde, at noncytotoxic concentrations, impedes the intra-nuclear trafficking of the DNA damage recognition proteins DDB2 and XPC, thus retarding their recruitment to NER substrates. Further live-cell imaging experiments with fluorescently tagged ubiquitin suggest that DDB2 stimulates protein ubiquitylation in response to formaldehyde exposure. In conclusion, these findings indicate that formaldehyde-induced DNA-protein crosslinks interfere with the normal trafficking of NER subunits, thus distracting these repair factors from their physiologic function in recognizing and processing mutagenic DNA lesions.

[发布机构] University of Zurich

[效力级别] 570 Life sciences

[关键词] Institute of Veterinary Pharmacology and Toxicology;570 Life sciences;biology