A comprehensive study of sequence variation effects on PCNA function.

Project Summary Mutations in genes encoding proteins that are essential for cell processes such as DNA replication can lead to cellular dysfunction and disease. Protein defects can perturb these cell processes by removing or surpassing cell cycle checkpoints, leading to errant growth and proliferation of cells, defining characteristics of cancer. As such, it is essential to investigate Proliferating Cell Nuclear Antigen (PCNA), the central player that coordinates DNA replication, DNA repair, and cell-cycle regulation. PCNA, also known as the sliding clamp, is a homotrimeric ring that slides along DNA to facilitate interactions of over 100 known proteins, many involved in cancer development and other important cellular processes. The sliding clamp is conserved across all life forms, providing insight into the evolution of DNA replication and cell-cycle machinery. Thus, PCNA is an ideal target to investigate mutational effects on protein function and the long-term impacts on the cell. In Aim 1, we will address an interesting paradox related to PCNA. Point mutations in PCNA that result in subtle biochemical effects cause severe disruption of organism fitness, suggesting that PCNA is especially sensitive to mutations. Conversely, PCNA genes across evolution are widely varying in sequence suggesting that PCNA is actually accepting of mutations. To investigate this contradiction, we will perform a mutational scan of all potential point mutations in the yeast PCNA protein. These mutants will then be exposed to DNA-damaging agents to assess the effects of the PCNA mutants on various PCNA functions. I predict that a mutational screen of PCNA will show mutational effects on cell viability and DNA damage response based on residue location in PCNA providing insight into the acceptability of point mutations in PCNA. This data will also provide insights into potential disease mutations that could impact human PCNA. The Kelch lab has previously investigated two disease-associated germline mutations in PCNA. These mutations lead to PCNA-associated DNA repair disorder (PARD), characterized by UV sensitivity, neurodegeneration, premature aging, and, most notably, the development of skin cancer. In Aim 2, we will investigate how patient-associated mutations in PCNA affect biochemical and cellular function. I selected variants based on association with cancer or PARD. I will establish mutant human retinal pigment epithelial (RPE1) cell lines using CRISPR/Cas9 techniques. Once these cell lines are established, I will assess the cellular impacts by using flow cytometry and DNA-damage assays. I will compare these results with tests of the biochemical functions using isothermal titration calorimetry and thermal shift assays. I predict that the mutations will exhibit defects in thermostability, cell regulation, and DNA repair. The impact of this study is two- fold. First, the study will enhance our understanding of how PCNA function and evolution are intertwined. Second, the study will investigate select human PCNA mutants that can inform cancer diagnosis and provide a framework for investigating other proteins.