Mechanism of Disease-causing mutations in PCNA

摘要 增殖细胞核抗原是许多细胞过程的关键调节器和促进器，例如 DNA复制、DNA修复、重组、染色质结构和细胞凋亡。 增殖细胞核抗原是一个环形的复合体，它作为DNA上的滑动平台 排列数十种不同的蛋白质来组装并作用于染色质。 增殖细胞核抗原活性异常与肺癌的发生和转移 因此，增殖细胞核抗原是化疗药物开发的靶点。 增殖细胞核抗原中两个独立的点突变(Ser228Ile和Cys148Ser)导致 常染色体隐性遗传病(增殖细胞核抗原相关DNA修复障碍或PARD) 这是由于DNA修复的缺陷造成的。我们假设这些突变 破坏增殖细胞核抗原的稳定性和/或结合配对蛋白的能力。我们进一步 假设增殖细胞核抗原折叠稳定性缺陷导致寿命缩短 在DNA上，从而优先抑制DNA修复蛋白 DNA上增殖细胞核抗原寿命的终结。我们的初步研究表明，S228I 突变扰乱了增殖细胞核抗原伙伴的结合位点，但一些伙伴 克服这种干扰以结合增殖细胞核抗原。我们推测S228I突变 破坏结合部位的结构和动态，从而使DNA修复 这条途径受到了不成比例的干扰。我们进一步发现，双方都 突变破坏了增殖细胞核抗原的稳定性，这可能会缩短增殖细胞核抗原的寿命。 DNA为了支持这一假设，我们发现染色质上的增殖细胞核抗原水平是 患者来源的成纤维细胞中异常低。 我们将以三个具体目标来解决这些假设：(1)确定如何 PARD突变改变了增殖细胞核抗原的结构、稳定性和动力学，(2)到 测定PARD变异体对增殖细胞核抗原寿命的生化影响 伙伴结合和活性，以及(3)确定哪些细胞因子和 道路被打乱了。我们的研究将确定该系统的健壮性或脆弱性 增殖细胞核抗原介导的DNA修复和DNA复制等途径，将 定义增殖细胞核抗原如何影响癌症。我们的工作还将揭示增殖细胞核抗原是如何结构的 和Dynamic控制合作伙伴约束，这将指导开发小型 破坏特定的增殖细胞核抗原介导的分子。

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.