Characterizing a role for Drosophila melanogaster PIF1 helicase in DNA metabolism.
Abstract: ABSTRACT Non-homologous end joining (NHEJ) has classically been designated the main culprit of error-prone repair of DNA double-stranded breaks. However, emerging evidence is illuminating error-prone characteristics of homologous recombination repair. The highly conserved PIF1 helicase has been shown to perform a multitude of tasks in the budding yeast Saccaromyces cerevisiae including i... read morenvolvement in homologous recombination repair-associated mutagenesis during break-induced replication. Here, we have identified a PIF1 ortholog in Drosophila melanogaster and created a null mutant. We find that pif1 mutants exhibit a significantly lower hatching frequency compared to wildtype flies, likely due to defects in embryonic DNA replication. Absence of PIF1 results in multiple defects in early embryos, including; nuclear fallout, nuclear fragmentation/micronuclei, and asynchronous mitotic divisions. Utilizing a site-specific DNA gap repair assay, we show that PIF1 is required for extensive synthesis during homologous recombination. Furthermore, mutation frequencies measured during gap repair within a lacZ reporter gene was significantly decreased compared to wildtype suggesting a contribution of PIF1 during repair-associated mutagenesis. Together, our phenotypic characterizations of the pif1 mutant show that Drosophila PIF1 likely maintains a conserved function in break-induced replication, and plays a crucial role in DNA replication during embryogenesis.
Thesis (M.S.)--Tufts University, 2015.
Submitted to the Dept. of Biology.
Advisor: Mitch McVey.
Committee: Catherine Freudenreich, Sergei Mirkin, and Stephen Fuchs.
Keywords: Biology, Genetics, and Molecular biology.read less