Coexpression and Reconstitution of Multiprotein Complexes in Eukaryotic Nucleotide Excision Repair
2018-11-27T00:00:00Z (GMT) by
Genetic transactions such as DNA replication, transcription, recombination, and repair are often initiated and controlled by dynamic interactions among intricate multiprotein complexes and chromatin DNA. While it is crucial to study the structural, biochemical and biophysical mechanisms of the interactions involving these molecules, such studies are often challenged by the difficulties in reconstituting large protein and protein-DNA complexes in sufficient purity and quantity. In this thesis, I focused on developing methods to prepare key such complexes that have seminal roles in several genetic transactions in eukaryotes, including transcription and nucleotide excision repair (NER). First, I describe the co-expression of histone octamer complex and the reconstitution of the nucleosome core particles (NCPs). NCPs are basic organization and packaging units of DNA in all eukaryotic cells, consisting of ~147 bp DNA wrapped around histone octamers. Nucleosomes are folded into compact, highly organized chromatin structures, which form the working platform for various DNA mechanisms including transcription and NER. Second, I discuss the co-expression and purification of the 5-subunit core of the general transcription factor II H complex (TFIIH) complex using MultiBac expression system. TFIIH is essential for trancription initiaton as well as NER DNA damage processing. Lastly, I describe preliminary studies on the interactions between TFIIH and Rad4-Rad23 complex, a key initial damage recognition factor in NER. The TFIIH-Rad4 interaction constitutes a key initiation step in NER that removes various environmentally induced DNA lesions and protect genomic integrity in cells.