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|Title:||Mutational and functional analysis of the conserved Pif1 DNA helicase in Saccharomyces cerevisiae|
|Authors:||Geronimo, Carly Lay Alfonso|
|Advisors:||Zakian, Virginia A|
|Contributors:||Molecular Biology Department|
|Publisher:||Princeton, NJ : Princeton University|
|Abstract:||Pif1 family helicases are ATP-dependent DNA helicases that are conserved from bacteria to humans. Pif1 family helicases perform many critical roles that promote the integrity of the genome. Saccharomyces cerevisiae Pif1, the founding member and best-studied helicase of this family, has multiple functions. Pif1 inhibits telomerase action at telomeres and double-strand breaks, promotes Okazaki fragment maturation and maintenance of mitochondrial DNA, suppresses genome instability at G-quadruplex (G4) motifs, contributes to fork arrest at the rDNA replication fork barrier, promotes break-induced replication, promotes fork progression at tRNA genes and centromeres, and resolves converged forks during DNA replication termination. In vitro, Pif1 exhibits weak unwinding activities on conventional 5’-tailed duplex DNA, but robustly unwinds G4 DNA and RNA/DNA hybrids. Pif1 also displaces proteins on DNA and promotes the processivity of DNA Polymerase . To elucidate the mechanism and regulation for its roles, I conducted a mutational and functional analysis of Pif1 to identify domains and residues that are essential for distinct Pif1 functions in vivo. This thesis describes four projects on S. cerevisiae Pif1. The first project is a published study about the Pif1 Signature Motif (SM). The SM is a 23-amino acid motif that is located in the helicase domain but is present in only Pif1 helicases. We showed that the SM is essential for mitochondrial and nuclear functions and for ATPase activity, but not DNA substrate binding. The second project describes the discovery of the Pif1 nuclear localization signal (NLS). Prior to this thesis work, the sequence that targeted Pif1 to the nucleus was unknown. Here, we identified four residues (781KKRK784) as the putative Pif1 NLS motif. The final projects are ongoing and require further investigation. The third project describes the analysis of the Pif1 amino-terminus, which was largely uncharacterized at the start of this work. Here, I identified a region of 43 amino acids that is important for Pif1’s role during DNA replication. The fourth project describes the early stages of analysis of Pif1 alleles that disrupted G4 DNA binding/unwinding in vitro. The goal of this study is to determine which functions in vivo may involve a G4 substrate.|
|Alternate format:||The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu|
|Type of Material:||Academic dissertations (Ph.D.)|
|Appears in Collections:||Molecular Biology|
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