Research objectives and content
Nucleotide excision repair (NER) is the key pathway for the removal of UV-induced photoproducts and a variety of helix-distorting chemical adducts from DNA in mammalian cells. Nucleotide excision repair (NER) has recently been reconstituted in vitro and the research proposed will attempt to further elucidate the assembly and mechanism of the mammalian core NER reaction up to the excision of a lesion-containing oligomer. The experiments will be performed using defined DNA substrates bearing single lesions and purified proteins. To address where and how NER initiates, the association of damage recognition proteins XPA and RPA with different DNA lesions will examined and the likely resulting DNA structural changes analyzed. The association of transcription-repair factor TFIIH association with the damage recognition complex and subsequent DNA unwinding on both sides of the lesion will be investigated. The requirements for incision by the ERCC1XPF and XPG nucleases 5' and 3' to the lesion, respectively, will be examined and the possible synchronization of DNA unwinding with endonucleolytic incision explored. Finally, in addition to the assembly process, the disassembly of DNA repair complexes following DNA recognition will be a subject of inquiry. With so many factors (30) acting at the same small region of DNA, it is likely that a temporal order of both assembly and disassembly needs to occur.
Training content (objective, benefit and expected impact)
It is expected that the proposed research to be conducted in the Wood laboratory will continue to develop my knowledge and experimental expertise in the fields of nucleic acids enzymology, biochemistry, and molecular biology. Specifically, post-doctoral training utilizing techniques for the study of protein-DNA interaction and DNA structural change will be an important addition to my current experimental abilities. I also hope to further my protein purification skills and the large number of NER factors to be examined should assist that goal. Opportunity to apply techniques for the analysis of protein-protein interactions is also likely.
Links with industry / industrial relevance (22)