Faulty DNA repair can promote mutations, aging, 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. One of the most remarkable aspects of nucleotide excision repair (NER) is that it can remove a wide range of DNA lesions that differ in chemistry and structure. During bacterial NER UvrA, UvrB, and UvrC proteins work together to identify and remove DNA damage. 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. We are collaborating with Dr. Bob London (NIEHS) for an analysis of the dynamics of UvrB upon ligand binding using NMR techniques. We are also collaborating with Dr. Caroline Kisker (Wurzburg, Germany) on solving protein and protein-DNA structures by X-ray crystallography. Combined with site-directed mutagenesis and biochemical analyses, 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. ? ? We are also performing a series of studies on the formation and repair of various genotoxic agents in an entire multi-celled eukaryotic organism, Caenorhabditis elegans. This organism contains the entire complement of NER proteins that are homologous to humans. In collaboration with Drs. Boyd and Freedman (NIEHS) analysis with specific mutations and RNAi knock-down technology allows a global assessment of the effect of DNA damage on the life and biology of the organism. Surprisingly BLAST analysis in C elegans has only found about 50% of the known base excision repair proteins of human cells. Therefore in collaboration with Drs. Asagoshi, Prasad, and Wilson, (NIEHS) we are investigating the presence of specific repair protein and their activities in cell-free extracts of L1 C elegans
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