Mechanism of Disease-causing mutations in PCNA

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Abstract PCNA is a critical regulator and facilitator of many cellular processes such as DNA replication, DNA repair, recombination, chromatin structure and apoptosis. PCNA is a ring-shaped complex that acts as a sliding platform on DNA for the arrangement of scores different proteins to assemble and act on chromatin. Abnormal PCNA activity is associated with the development and metastasis of cancer; consequently, PCNA is a target for development of chemotherapeutics. Two separate point mutations (Ser228Ile and Cys148Ser) in PCNA cause an autosomal recessive disorder (PCNA Associated DNA Repair Disorder or PARD) that results from defects in DNA repair. We hypothesize that these mutations disrupt PCNA’s stability and/or ability to bind to partner proteins. We further hypothesize that the PCNA defect in folding stability results in a shorter lifetime on DNA, thereby preferentially inhibiting DNA repair proteins that function near the end of PCNA’s lifetime on DNA. Our preliminary studies show that the S228I mutation disrupts the binding site for PCNA partners, yet some partners overcome this disruption to bind PCNA. We hypothesize that the S228I mutation disrupts the structure and dynamics of the binding site such that the DNA repair pathway is disproportionately perturbed. We further find that both PARD mutations disrupt PCNA stability, which could decrease the lifetime of PCNA on DNA. In support of this hypothesis, we find that PCNA levels on chromatin are abnormally low in patient-derived fibroblast cells. We will address these hypotheses with three specific aims: (1) To determine how the PARD mutations alter PCNA structure, stability, and dynamics, (2) to determine the biochemical effects of PARD variants on PCNA longevity and on partner binding and activity, and (3) to identify which cellular factors and pathways are disrupted. Our studies will determine the robustness or fragility of PCNA-mediated pathways such as DNA repair and DNA replication, which will define how PCNA affects cancer. Our work will also uncover how PCNA structure and dynamics controls partner binding, which will guide efforts to develop small molecules that disrupt specific PCNA-mediated.