The long-term research goal of the Zakian lab is to contribute to the understanding of mechanisms that promote the integrity of eukaryotic chromosomes. Proposed work falls into two areas. The first area is maintenance of telomeres, the ends of chromosomes, by telomerase, using budding and fission yeast as models. The second area is the progression of replication forks through hard-to-replicate sites and the roles of Pif1 family DNA helicases in this process. The proposed replication experiments will be carried out in budding yeast and human cultured cells. The two areas of research are linked by the role of Pif1 family helicases: budding yeast Pif1 is a negative regulator of telomerase while other Pif1 family helicases promote semi- conservative DNA replication of telomeres, as well as of other hard-to-replicate sites. There are two structural variants of DNA that also link the two areas. G-quadruplex (G4) structures, which can form in RNA or single- stranded DNA of the appropriate sequence, are held together by non-canonical G-G base pairs. R-loops are RNA-DNA hybrids that usually form during aberrant transcription. G4 DNA and r-loops are both stable, both slow replication forks and cause DNA damage, and both form at telomeres and other hard to replicate sites. Another link between the two areas is that the budding yeast Pif1 helicase unwinds G4 DNA and r-loops very efficiently. Experiments on regulation of telomerase will exploit our success in using mass spectrometry (MS) to identify novel regulators of budding yeast telomerase by their association with the telomerase holoenzyme. This approach identified the Cdc48-Npl4-Ufd1 complex as a regulator of Est1 abundance and activity. We also identified multiple subunits of RNAseP/MRP as being telomerase associated and will continue to explore how this association affects telomerase RNA. In the next funding period, we will extend the MS approach to fission yeast telomerase, which we anticipate will be equally fruitful. We will also use MS to identify post-translational modifications and protein-protein interactions of key telomere proteins in budding yeast as a function of telomere length using two genetic strategies to generate cultures where all cells have short or long telomeres. We are carrying out the function part of a structure-function analysis of budding yeast Pif1 with the goal of identifying substrate-specific residues that will enable us to connect the disparate roles of ScPif1 to its distinct biochemical activities. We are using a combination of recombinant adeno-associated viruses (rAAV) and CRISPR/Cas9 technology to construct human tissue culture lines that lack human PIF1 with the goal of determining its DNA targets and how its mutation is associated with familial breast cancer. Analysis will include cloning target sequences into a vector that allows us to monitor replication in mammalian cells by two- dimensional gels. We are using a novel approach to identify sites of G4 structures and r-loops in budding yeast genome-wide and in the ribosomal DNA in different mutants and growth conditions with the goal of providing physical evidence for the in vivo existence of both structures and genetic evidence for their interaction.
Every aspect of this grant, which focuses on regulation of telomerase, replication fork progression, and DNA helicases, is relevant to genome integrity and hence human health. Not only is telomerase up-regulated in almost all cancers, mutations in telomerase subunits, telomerase RNA binding proteins, telomere binding proteins or DNA helicases that affect telomere replication can all cause stem cell failure and premature death in humans. Likewise mutations in DNA helicases, including the human PIF1 helicase are linked to human diseases characterized by genome stability, including conditions as diverse as cancer and neurodegenerative disease, as are the DNA secondary structures studied in this grant.
