SINGLE MOLECULE STUDIES OF RAD4 RAD23 REVEAL A DYNAMIC DNA DAMAGE RECOGNITION PROCESS
Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix destabilizing and/or distorting DNA lesions, such as UV induced photoproducts. As the first step towards productive repair, the human NER damage sensor XPC RAD23B needs to efficiently locate sites of damage among billons of base pairs of undamaged DNA. In this dissertation, we investigated the dynamic protein DNA interactions during the damage recognition step using a combination of fluorescence based single molecule DNA tightrope assays, atomic force microscopy, as well as cell survival and in vivo repair kinetics assays. We observed that quantum dot labeled Rad4 Rad23, the yeast homolog of human XPC RAD23B, formed nonmotile complexes on DNA or conducted a one dimensional search via either random diffusion or constrained motion along DNA. Using atomic force microscopy, we studied binding of Rad4 lacking the b hairpin domain 3 (BHD3) to damage containing DNA and found that this structural motif is non essential for damage specific binding or DNA bending. Furthermore, we demonstrated that deletion of seven residues in the tip of b hairpin in BHD3 increased Rad4 Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance. Finally, we explore existing physical models and examples of subdiffusive motion, and discuss a model in which constrained motion by Rad4 Rad23 on DNA may be driven by conformational changes of the protein.