Nuclear protein quality control mechanisms during genotoxic stress

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Cellular stress responses can alter cell cycle, gene expression, and protein homeostasis in an orchestrated way that promotes cell survival and future stress tolerance. This is true of DNA damaging stress in which proteins relocalize to different subcellular compartments as part of a multi-level DNA damage response including DNA repair, transcriptional changes and proteome remodeling. Yeast cells have been a useful model for understanding dynamic changes of proteins during the DNA damage response. Genotoxic stress can elicit several spatially and functionally distinct protein compartments, including DNA repair foci, stress granules, and a peri-nucleolar aggregate structure called the intranuclear quality control site (INQ). The function of the INQ compartment in genotoxic stress responses is not known. INQ formation is regulated by molecular chaperones and the proteasome, suggesting it is a site of protein quality control (PQC). Additionally, the INQ sits adjacent to the nucleolus, and human nucleoli have been identified as phase-separated protein quality control compartments. Thus, elucidating INQ formation and function in the DNA damage response will inform our understanding of spatial nuclear PQC and its role in stress responses across species. Robust stress responses contribute to healthy aging, industrial biological processes, and in organisms of agricultural importance. Therefore, understanding fundamental principles of stress biology will ultimately impact many sectors of research and industry. Our goal is to understand the regulation of protein sequestration at the INQ and develop Rpd3 INQ localization as a new model. We will address this goal with three specific aims: Aim 1. Identify regulatory post-translational modifications on proteins that regulate INQ sequestration: This will initially focus on SUMO or ubiquitin modifications of INQ resident proteins. Aim 2. Dissect the function of INQ resolving molecular chaperones: We will directly test the role of Cdc48 and its cofactors, or the Apj1-Hsp70-Hsp110 system in turnover of INQ substrate proteins. Aim 3. Define the function of Rpd3 sequestration at INQ: We hypothesize that the Rpd3 lysine deacetylase is sequestered at INQ to control the chromatin state of late firing DNA replication origins. Overall, this project will reveal the mechanisms of nuclear protein aggregate formation during the DNA damage response from the perspective of post-translational modifications and molecular chaperones. It will also directly test the functional consequences of INQ protein sequestration starting with direct tests of Rpd3 histone deacetylase function in the DNA damage response. These data will reveal how proteome remodeling and quality control contribute to DNA damage tolerance and recovery. Together these outcomes will elucidate a novel nuclear PQC pathway that is integrated with the DNA repair and transcriptional remodeling activities known to accompany cellular responses to genotoxic stress.