The long term goal of this grant is to identify genes and structures that promote genome integrity and determine their mechanism of action. In the past 3 funding periods, we focused on Pif1 family DNA helicases, multi-functional helicases with critical roles in maintenance of nuclear and mitochondrial (mt) DNA. Although most studies on replication fork progression study the effects of exogenous damage, even the genomes of undamaged cells present challenges for DNA replication. Protein complexes, highly transcribed genes, DNA secondary structures, and converged replication forks are natural replication barriers that are encountered in every S phase. Pif1 family helicases are the only identified enzymes that promote replisome movement past all of these structures.
Aim I presents genetic approaches to determine the unique &overlapping roles of ScPif1 and Rrm3, the two S. cerevisiae Pif1 family members, and their interactions with other genes. We will develop site specific assays to quantitate the impact of Rrm3 on hard to replicate DNA sequences by modifying the gross chromosomal rearrangement (GCR) assay;e.g., we will insert a tRNA gene that causes strong fork pausing in rrm3 cells into the GCR test interval and determine its impact on GCR rates in WT and mutant strains. We will identify genes that act in concert with/in place of Rrm3 to suppress GCR damage at this and other sites and determine the sequence of events that promote replication at the sites. Mutations in the Pif1 signature motif, a 21 amino acid motif that distinguishes Pif1 helicases from other proteins, as well as heterologous Pif1 helicases, will be tested for ability to suppress GCR events in site-specific GCR strains. We will use synthetic genetic analysis to identify which of the ~5000 non-essential yeast genes cause death or slow growth when absent in pif1-m2 cells. Similar screens will identify single or double mutants that are hyper- sensitive to G4-stabilizing drugs.
Aim II, which complements aim I, describes biochemical approaches to determine the unique and overlapping activities of Pif1 helicases. After years of being unable to purify eukaryotic Pif1 family helicases other than ScPif1, we purified six bacterial Pif1 helicases and showed that, like ScPif1, all robustly unwind G4 DNA. ScPif1 has three uncommon activities (e.g., preferential unwinding of RNA/DNA substrates). We will determine if these activities are conserved within the Pif1 family. Proteins with mutations in the Pif1 signature motif, including a mutation associated with increased risk of human breast cancer, will be tested to determine the molecular role of the motif. Collaborative structural and single molecule experiments are described.
Aim III presents experiments to determine molecular mechanisms of ScPif1 action at both telomeres and mtDNA. We will determine if Rif2 recruits ScPif1 preferentially to long telomeres and if direct interaction betwee ScPif1 &telomerase is required for its ability to evict telomerase from telomeres. We will ask if ScPif1 promotes mtDNA stability by suppressing G4-induced damage by determining if ScPif1 binds G4 motifs in WT mtDNA and if loss and/or rearrangement of mtDNA in pif1 cells initiates at G4 motifs.

Public Health Relevance

All parts of this proposal, which focuses on Pif1 family helicases and their unique functions in replication fork progression and telomerase regulation, are relevant to human health. Pif1 helicases have critical roles in replication of both mitochondrial and telomeric DNA, whose integrity impacts both life span and cancer in humans. We will use yeast as test tubes to determine the effects of breast cancer associated alleles of human PIF1 on replication fork progression and telomere maintenance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM026938-35
Application #
8637545
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Reddy, Michael K
Project Start
1979-07-01
Project End
2017-12-31
Budget Start
2014-02-01
Budget End
2014-12-31
Support Year
35
Fiscal Year
2014
Total Cost
$602,323
Indirect Cost
$226,401
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
McDonald, Karin R; Sabouri, Nasim; Webb, Christopher J et al. (2014) The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening. DNA Repair (Amst) 24:80-6
Willis, Nicholas A; Chandramouly, Gurushankar; Huang, Bin et al. (2014) BRCA1 controls homologous recombination at Tus/Ter-stalled mammalian replication forks. Nature 510:556-9
Bochman, Matthew L; Paeschke, Katrin; Chan, Angela et al. (2014) Hrq1, a homolog of the human RecQ4 helicase, acts catalytically and structurally to promote genome integrity. Cell Rep 6:346-56
Paeschke, Katrin; Bochman, Matthew L; Garcia, P Daniela et al. (2013) Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature 497:458-62
Webb, Christopher J; Wu, Yun; Zakian, Virginia A (2013) DNA repair at telomeres: keeping the ends intact. Cold Spring Harb Perspect Biol 5:
Bochman, Matthew L; Judge, Colleen P; Zakian, Virginia A (2011) The Pif1 family in prokaryotes: what are our helicases doing in your bacteria? Mol Biol Cell 22:1955-9
Paeschke, Katrin; Capra, John A; Zakian, Virginia A (2011) DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase. Cell 145:678-91
Capra, John A; Paeschke, Katrin; Singh, Mona et al. (2010) G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae. PLoS Comput Biol 6:e1000861
Bochman, Matthew L; Sabouri, Nasim; Zakian, Virginia A (2010) Unwinding the functions of the Pif1 family helicases. DNA Repair (Amst) 9:237-49
Boule, Jean-Baptiste; Zakian, Virginia A (2006) Roles of Pif1-like helicases in the maintenance of genomic stability. Nucleic Acids Res 34:4147-53

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