DNA replication and repair are crucial processes for maintaining genome stability. These processes are in part dependent on the activities of an emerging family of structure-specific endonucleases. These enzymes, typified by flap endonuclease-1 (FEN-1) are multi-functional. They possess both flap-specific endo- and nick-specific exo- (ribo-) dependent and DNA damage-inducible manner. FEN-1 nuclease is required both for the removal of RNA primers during lagging-strand DNA synthesis and for processing damage DNA fragments. Dysfunction of the FEN-1 gene results in a strong mutator phenotype, dramatic sensitivity to DNA alkylating agents, genomic instability, and conditional lethality of a yeast cell. However, little work has been done in a mammalian system. The broad and long-term goal of this study is to link the functional deficiency of this critical gene to the formation of genetic diseases such as cancers. Two major approaches to this goal are to detect mutations of FEN-1 in tumor tissues and to establish knockout mouse models. However, these approaches are not practical due to the FEN-1's dual functions both in DNA replication and in repair. Our previous biochemical studies with FEN-1 mutants indicated that the biochemical activities of FEN-1 nuclease are readily segregated in a way that eliminates one of the activities such as exonuclease activity of PCNA-interaction. Therefore, the hypothesis of the current study is that various deficiency or defects in the individual biochemical activities of FEN-1 lead to separation of multiple phenotypes observe din yeast, reflecting deficiency in a particular pathway. This will result in different susceptibilities in the human population to environmental stresses, which subsequently results in individual differences in the onset of a disease. These functional alterations, in the form of mutant FEN-1 proteins and by knock-ins rather than knockouts, will allow transgenic mice to survive but show phenotypes that may include early onset of diseases such as cancers.
The specific aims of this study are i) to further elucidate structural and functional relationship of FEN-1 nuclease and molecular enzymological mechanism; ii) to determine if specific biochemical activities of FEN-1 proteins are associated with specific DNA metabolic pathways and distinct phenotypes in yeast model system; iii) to establish the relationship among dysfunction of FEN-1 gene, genomic instability, and carcinogenesis by knock-in of the biochemically-characterized FEN-1 mutations in mice.
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