SUMOylation and ubiquitylation of PCNA in recombination and translesion synthesis

描述(由申请人提供)：增殖细胞核抗原(增殖细胞核抗原)的翻译后修饰对于维持基因组的稳定性是必不可少的。增殖细胞核抗原泛素化促进受损DNA模板的跨损伤合成，而增殖细胞核抗原SUMO化可防止异常重组。这一建议侧重于了解由增殖细胞核抗原的翻译后修饰调节的蛋白质相互作用，以及这些相互作用如何调节跨病变的合成和重组。拟议的研究之所以成为可能，是因为我们团队进行了一项高度创新的技术开发，允许使用分裂/融合策略生产大量修饰的增殖细胞核抗原。利用这种方法，我们测定了泛素修饰的增殖细胞核抗原(Ubi-PCNA)和相扑修饰的增殖细胞核抗原(SUMO-PCNA)的X射线晶体结构。目的1研究Ubi-增殖细胞核抗原和SUMO-增殖细胞核抗原与靶蛋白的相互作用和调控。修饰后的增殖细胞核抗原将用于单分子结合和重组酶分析。这些研究将检验增殖细胞核抗原作用的工具带模型。目的2研究Ubi-增殖细胞核抗原和SUMO-增殖细胞核抗原的结构和动力学。结构研究将确定修饰的增殖细胞核抗原的动力学，并检验动态蛋白质-蛋白质伙伴复合体对调控至关重要的假设。研究还将确定翻译后修饰的增殖细胞核抗原如何影响与反重组解旋酶的相互作用，并调节重组。目的3研究Ubi-增殖细胞核抗原和相扑增殖细胞核抗原对DNA合成和重组的调节作用。分裂/融合方法允许在细胞中表达结构性修饰的增殖细胞核抗原。这使得体内分析泛素化和SuMO化的增殖细胞核抗原的效果成为可能。这些研究将提供一个明确的理解，确切地说，增殖细胞核抗原是如何将蛋白质招募到复制叉上的，不同蛋白质与增殖细胞核抗原结合的活性是如何调节和协调的，不同蛋白质之间的交接是如何在多步骤过程中发生的，以及增殖细胞核抗原的翻译后修饰如何对这些过程产生如此不同的影响。

DESCRIPTION (provided by applicant): Post-translational modifications of proliferating cell nuclear antigen (PCNA) are essential for maintaining genome stability. PCNA ubiquitylation facilitates translesion synthesis of damaged DNA templates, and PCNA SUMOylation prevents aberrant recombination. This proposal focuses on understanding the protein interactions regulated by post-translational modifications of PCNA and how these interactions modulate translesion synthesis and recombination. The proposed studies are possible because of a highly innovative technological development made by our group which allows for the production of large quantities of modified PCNA using a split/fusion strategy. Using this approach, we determined the X-ray crystal structures of both ubiquitin-modified PCNA (Ubi-PCNA) and SUMO-modified PCNA (SUMO-PCNA). Aim 1 is to study the interaction and regulation of target proteins by Ubi-PCNA and SUMO-PCNA. Modified PCNA will be analyzed in single-molecule binding and reconstituted enzyme assays. These studies will test the tool belt model of PCNA action. Aim 2 is to study the structure and dynamics of Ubi-PCNA and SUMO-PCNA. Structural studies will determine the dynamics of modified PCNA and test the hypothesis that dynamic protein-protein partner complexes are critical for regulation. Studies will also determine how post-translational modification of PCNA effects interactions with anti-recombinogenic helicases and modulates recombination. Aim 3 is to study the regulation of DNA synthesis and recombination by Ubi-PCNA and SUMO-PCNA. The split/fusion methodology allows expression of constitutively modified PCNA in cells. This allows in vivo analysis of the effects of ubiquitylated and SUMOylated PCNA. These studies will provide a clear understanding of exactly how PCNA recruits proteins to replication forks, how the activities of different proteins bound to PCNA are regulated and coordinated, how the hand-off between different proteins occurs on PCNA during multi-step processes, and how post-translational modifications of PCNA have such different effects on these processes.