Faulty DNA repair can promote mutations, cancer and cell death. The process by which protein components of repair detect damaged or modified bases within DNA is an important but poorly understood type of protein-DNA interaction. The bacterial UvrA, UvrB, and UvrC proteins work together to identify and remove DNA damage in a process called nucleotide excision repair (NER). One of the most remarkable aspects of nucleotide excision repair is that it can remove a wide range of DNA lesions that differ in chemistry and structure. The UvrA and UvrB proteins are believed to recognize damage-induced distortion in the DNA helix rather than the lesion per se. However, detailed studies of the kinetics, thermodynamics and structures of the Uvr proteins have been limited due to their instability. To overcome this problem we recently cloned and overexpressed UvrA, UvrB and UvrC from the thermophilic bacteria, Bacillus caldotenax and Thermotoga maritima. The proteins maintain optimal activity at 65 ?C and are amenable to both structural and biophysical studies. Work during the last four years has led to a clearer understanding of how UvrA hands off damaged DNA to UvrB and the specific residues in UvrB responsible for DNA damage processing. In collaboration with Dr. Caroline Kisker?s group, we have obtained a new crystal structure of UvrB allowing high resolution of domain 2 for structure-function studies. Furthermore, we recently have obtained a co-crystal structure of UvrB bound to single-stranded DNA. In addition, both the N-terminal and C-terminal endonuclease domains of UvrC have been crystallized and a series of over 40 mutants in the full-length protein have been characterized by a series of biochemical assays. These structure-function studies of the UvrA, UvrB, and UvrC proteins form a basis for understanding the fundamental molecular processes of NER. Our long-term goal is to have a complete understanding of how structural perturbations induced by specific DNA lesions are detected and removed by the NER machinery at the atomic level.
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