Mechanism of Disease-causing mutations in PCNA

摘要 PCNA是许多细胞过程的关键调节剂和促进剂， DNA复制，DNA修复，重组，染色质结构和凋亡。 PCNA是一种环状复合物，在DNA上充当滑动平台， 数十种不同蛋白质的排列组合并作用于染色质。 PCNA活性异常与肿瘤的发生、转移有关。 因此，PCNA是开发化学治疗剂的靶标。 PCNA中两个独立的点突变（Ser 228 Ile和Cys 148 Ser）导致了PCNA的突变。 常染色体隐性遗传病（PCNA相关DNA修复障碍或PARD） 是由DNA修复缺陷引起的我们假设这些突变 破坏PCNA的稳定性和/或与伴侣蛋白结合的能力。我们进一步 假设PCNA在折叠稳定性方面的缺陷导致寿命缩短 在DNA上，从而优先抑制DNA修复蛋白， PCNA在DNA上的寿命结束。我们的初步研究表明，S228 I 突变破坏了PCNA伴侣的结合位点，但一些伴侣 克服这种破坏以结合PCNA。我们假设S228 I突变 破坏了结合位点的结构和动力学， 路径不成比例地受到干扰。我们进一步发现， 突变破坏了PCNA的稳定性，这可能会减少PCNA在细胞内的寿命。 DNA.为了支持这一假设，我们发现染色质上的PCNA水平是 患者来源的成纤维细胞异常低。 我们将讨论这些假设与三个具体目标：（1）以确定如何 PARD突变改变PCNA结构、稳定性和动力学，（2） 确定PARD变异体对PCNA寿命的生化影响， 伴侣结合和活性，以及（3）鉴定哪些细胞因子和 路径被打乱。我们的研究将确定 PCNA介导的途径，如DNA修复和DNA复制， PCNA如何影响癌症我们的工作也将揭示PCNA结构如何 和动态控制合作伙伴的绑定，这将指导努力发展小 破坏特定PCNA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.