Complex DNA lesions such DNA interstrand cross-links and triplehelix structures are functionally disruptive to the mammalian genome and pose unique challenges to the DNA repair system. It is most likely that repair of complex lesions involves cooperative actions of different DNA repair pathways such as homologous recombinational repair, excision repair, and mismatch repair. Our proposed program project will conduct comprehensive investigations on the processing of complex DNA lesions via molecular biology, genetics, and biochemical approaches. The Protein Factor and DNA Substrate (PPDS) Core will be responsible for the production of reagents used by each project and to establish procedures for generating these reagents. By streamlining the production of critical reagents such as protein factors, antibody, and DNA substrates, each project PI will gain addition time on experimentation and the results yielded from each project will be analogous and reliable. Specifically, the PPDS core will accomplish three service-related aims. 1. Preparation of recombinant DNA constructs for in vivo recombination studies, and for the purpose of producing recombinant proteins for biochemical studies and the generation of antibodies. 2. Fracfionafion of native proteins from mammalian extracts and purification of specific protein targets. 3. Production and quality control of site- and lesion-specific DNA substrates. 4. Construction of plasmid-based small-interference RNA vectors.
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