Model Genetic Systems to Study DNA Repair

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Using an approach known as RNA interference, we have specifically depleted human cells of the FANCJ DNA helicase and characterized the sensitivity of the cells to a small molecule compound that stabilizes G-quadruplex DNA structures. This work enabled us to elucidate a novel function of the FANCJ helicase in the manintenace of chromosomal stability. Since individuals carrying homozygous mutations in the FANCJ helicase gene have a genetic disorder known as Fanconi Anemia characterized by genomic instability and cancer, we believe our results shed new insights to the cellular pathways of FANCJ that serve to counter replciational stress due to alterante DNA structures such as G-quadruplexes that arise in vivo. We have used isogenic pairs of mutant and corrected chicken DT40 cells as well as human cells for genetic complementation studies. The mutant cell lines were used for structure-function studies of patient derived helicase-inactivating mutations. This work enabled us to define genotype-phenotype relationships between clinically relevant mutations and human genetic diseases. Another aspect of this work was to used genetically defined cancer and normal cell lines and small molecules to probed the molecualr and cellular functions of the Werner syndrome helicase. These efforts enabled us to determine that inhibition of WRN helicase activity inhibits cell proliferation and its DNA repair function in vivo. These efforts in the basic sciences help to advance the idea that DNA repair proteins may be targeted in anti-cancer therapy to increase the sensitivity of tumors to DNA damaging chemotherapy drugs or radiation. We have also employed yeast as a model genetic system to study the role of the Werner syndrome helicase in DNA replciation and repair. This work has enabled us to characterize the catalytic requirements of WRN in a defined genetic DNA repair pathway that operates to provide cellular resistance to alkylating agents which impose replicational stress. Lastly, we have utilized mouse as a model genetic system to characterize the role of a helicase ortholog (RECQ1) found in humans whose biological significance is not well understood. Characterization of th eprimary mouse embryonic fibroblasts from RECQ1 knockout mice has revealed that the RECQ1 helicase has unique and important roles in genomic stability maintenance.