Cancer research progress, having beneficially greatly from studies on powerful model systems, strongly supports the hypothesis that specific mutations leading to decreased genome stability are critical early events in tumori-genesis. Experiments in yeast show that eukaryotic genome integrity requires the action of the multi-functional enzyme Flap EndoNuclease (FEN-1), and that mutations in FEN-1 result in DNA duplication defects that occur in human tumors and inherited human diseases. FEN, which cleaves unpaired, over-hanging flaps in double- stranded DNA during repair and the terminal priming RNA base during DNA replication, is a structure-specific nuclease necessary for DNA repair and for processing the 5' ends of Okazaki fragments during lagging strand DNA synthesis. The proposed work aims to understand in atomic detain the structural metallobiochemistry responsible for FEN-1 catalytic activity, substrate specificity, and in vivo function. The structure of FEN-1 from the thermophilic archaebacterium Pyrococcus furiosus (pFEN-1) will be determined by X-ray crystallography and then used to solve pFEN-1 mutant structures as well as possible human, yeast, Archaeglobis fulgidus and Methanococcus jannaschii FEN-1 structures. The availability of multiple FEN-1 structures will identify structurally conserved, functionally important regions, and aim in the structure determinations and interpretations of FEN-1 complexes with DNA and with the processivity factor for DNA polymerase, termed proliferating cell nuclear antigen (PCNA). Based upon the high sequence homology among these enzymes, each FEN-1 structure should provide results applicable to the entire FEN-1 nuclease family. Thus, structures of any FEN-1 enzyme will be useful, and access to five FEN-1 enzymes will increase opportunities for successfully determining structures of FEN-1 complexes with DNA and PCNA. The high temperature optimum for the archael FEN-1 enzymes will assist the formation of stable complexes and allow possible trapping of DNA binding and cleavage intermediates via temperature control during mixing and crystallization steps. Competitive analyses of FEN-1 structures and complexes in the Tainer lab will be integrated with coupled biochemical and mutational analysis in the Shen lab, so mutant structures, biochemistry, and biology will experimentally test structure-based hypotheses and probe features key to FEN-1 structure- function relationships. Overall, this research will provide fundamental knowledge on FEN-1 structure and function relevant to defining its role in the regulation of genome fidelity and the mechanisms whereby loss of FEN-1 functions may lead to inheritable genetic defects and the initiation of cancer.
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