Mapping transient interactions with and on genomic DNA

New York University, New York NY

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Abstract

PROJECT SUMMARY Collisions between the DNA replication and transcription machineries – known as Transcription-replication conflicts (TRCs) appear to occur in all eukaryotes. Although these conflicts have been extensively investigated as a source of genome instability, we currently lack tools to determine how frequently these conflicts occur, where they are located in the genome, and whether the two complexes meet in a head-on or a co-oriented disposition. In addition to unwinding DNA to generate two single strands to serve as the template for replication, DNA helicases play diverse roles in DNA replication and repair. Mutations in genes encoding helicases are implicated in a number of human diseases including but not limited to cancer. A major limiting factor in our understanding of helicase function in cells is an inability to precisely determine the sites at which they are active. The proposed work encompasses two ongoing projects: Existing methods to map protein-DNA interactions do not distinguish sites at which a helicase is close to DNA from those at which it is actively unwinding. The first project takes advantage of a novel strategy to map helicase activity on genomic DNA via fusion to a single-strand-specific cytidine deaminase. Using this strategy, we will define the targets of Pif1-family helicases at high resolution, investigate the degree to which partially redundant helicases can compensate for one another, and determine which sites each helicase targets during each phase of the cell cycle. This work will also establish pipelines that will allow future investigation of other helicases and single-stranded DNA binding proteins in unicellular and multicellular eukaryotes. The second project will advantage of an optimized split-enzyme proximity biotinylation system to map genomic loci at which replication and transcription complexes physically interact. Among other experiments, we will test a long-standing model wherein replication forks can bypass transcribing RNA polymerases without evicting them, allowing for the resumption of transcription without loss of the nascent RNA transcript. The core DNA replication and transcription machineries, as well as most helicases, are highly conserved throughout eukaryotes. Both projects will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome, rapid replication, and genetic manipulability of S. cerevisiae make this an ideal model in which to study the intersection of fundamental biological processes. Therefore, the results of this work will provide molecular insights into genome instability in humans; these results will be directly applicable to our understanding of the etiology and progression of cancer, as well as diseases associated with impaired helicase function.