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Title: Functional Analysis of the hPif1 P109S, S223T, L319P, and R592C Mutations in S. cerevisiae, Four Polymorphs Linked to the Emergence of BRCA1-Associated Breast Cancer in Humans
Authors: Kazarian, Gregory
Advisors: Zakian, Virginia
Department: Molecular Biology
Class Year: 2014
Abstract: Pif1, a 5’ ->3’ directed ATP-dependent nucleic acid helicase, is the founding member of the Pif1 Family of helicases, a family that has homologues in a diverse range of organisms from bacteria to humans. Studies in S. cerevisiae have revealed that Pif1 is a multi- functional protein, with roles in both the nucleus and mitochondria. Both isoforms are involved in a variety of nucleic acid interactions including the regulation of telomere length, nuclear Okazaki fragment processing and maturation, the regulation of replication fork movement, and the maintenance of nuclear and mitochondrial DNA. In the absence of Pif1 in S. cerevisiae, cells rapidly accumulate mitochondrial and nuclear DNA damage, exhibiting deletions, double-strand breaks, and rearrangements. Though the mechanism of DNA damage is debated, favored models implicate the failure to resolve stable guanine tetramer (G4) secondary structures in the absence of the Pif1 helicase as the major cause of DNA damage. Less is known about the function of the human Pif1 protein (hPif1). The hPif1 protein, like ScPif1, plays a role in both the nucleus and mitochondria, where it is thought to play a similar role in DNA maintenance. In humans, mtDNA is thought to contribute to a plethora of diseases, from various cancers, to aging and neurodegeneration. Additionally, recent studies have linked several hPif1 mutations to the emergence of breast cancer, highlighting the need for further studies of this protein. Our study attempted to establish a model in which to study the function of the hPif1 protein. The ability of hPif1 to rescue the respiratory function of haploid scpif-m1 S. cerevisiae lacking the mitochondrial ScPif1 protein was assessed by petite induction assays, demonstrating the result that hPif1 partially complements the mtDNA maintaining function of ScPif1 in an N- and C- terminal independent, helicase dependent manner. The P109S, S223T, L319P, and R592C breast-cancer linked mutations were chosen for study, and the S223T, L319P, and R592C mutations were shown to significantly diminish the function of the hPif1 protein. This study highlights the need for the further study of the hPif1 protein and its potential role in human disease pathogenesis.
Extent: 100 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Molecular Biology, 1954-2020

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