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.