PROJECT SUMMARY Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated by the replisome with auxiliary mechanisms, collectively called DNA damage tolerance pathways, that ensure genomic integrity via on-the-fly remodeling. The aberrance of these pathways can lead to chromosome instability and a broad range of diseases including cancer. The Schauer Lab’s long-term goal is to thus understand the molecular basis for genetic and epigenetic fidelity, with the goal of improving the treatment and/or prevention of diseases. In this proposal, the Schauer Lab will use a fully functional replisome reconstituted from over 30 pure polypeptides to study how replisomes bypass obstacles that regularly occur in the genome while enforcing genetic and epigenetic integrity across generations. They also propose to develop whole cell lysate systems to establish active replication forks on double-stranded DNA at natural origins of replication without the need for replication initiation on synthetic forks. DNA damage tolerance mechanisms will be studied using biochemistry, single-molecule biophysics, and structural biology. The structural dynamics of the S-phase damage response will be characterized, with a focus on the mediator kinase Mrc1 and the multiple ways it regulates the elongating replisome. The Schauer Lab also proposes to study the spatiotemporal mechanisms of rescue of lesion-stalled replisomes by translesion synthesis polymerases, and how both Mrc1 and ubiquitination of DNA sliding clamps regulates this response. When replicating chromatin, nucleosomes present a strong block to replication fork progression in the absence of histone chaperones. The Schauer Lab will study histone dynamics at the replication fork in reconstituted chromatin, with a focus on regulation of histone deposition symmetry by histone chaperones and in the molecular mechanisms of various replication- coupled histone chaperones themselves. Tools will be developed to track histone fate and dynamics at the single-molecule level. The goal is to get a better understanding of the processes that control epigenetic inheritance, important for maintaining cellularity during cell division. Finally, the Schauer Lab proposes to study collisions between the replication machinery and an actively elongating transcription complex, since these conflicts can be highly mutagenic. Transcriptional regulation by the rpb4/7 heterodimer will also be studied. Transcription will be reconstituted from either purified proteins, or whole-cell extracts, or a combination of the two. The Schauer Lab is developing biochemical and single-molecule tools for these projects that will afford an unprecedented glimpse into the molecular mechanisms behind these critical processes. Single-molecule fluorescence resonance energy transfer (smFRET) will be employed to track intermolecular interactions, allowing a characterization of the structural dynamics of these systems. Further, t...