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（PCNA）的翻译后修饰对于维持基因组稳定性至关重要。PCNA泛素化促进了受损DNA模板的翻译合成，PCNA SUMOylation防止了异常重组。本研究的重点是了解由PCNA翻译后修饰调控的蛋白质相互作用，以及这些相互作用如何调节翻译合成和重组。这项研究之所以成为可能，是因为我们的团队开发了一项高度创新的技术，该技术允许使用分裂/融合策略生产大量改良的PCNA。利用这种方法，我们测定了泛素修饰的PCNA （Ubi-PCNA）和sumo修饰的PCNA （SUMO-PCNA）的x射线晶体结构。目的1：研究Ubi-PCNA和SUMO-PCNA对靶蛋白的相互作用和调控。修饰后的PCNA将在单分子结合和重组酶试验中进行分析。这些研究将检验PCNA作用的工具带模型。目的二是研究Ubi-PCNA和SUMO-PCNA的结构和动力学。结构研究将确定修饰的PCNA的动力学，并验证动态蛋白质-蛋白质伴侣复合物对调控至关重要的假设。研究还将确定翻译后修饰如何影响PCNA与抗重组解旋酶的相互作用并调节重组。目的3：研究Ubi-PCNA和SUMO-PCNA对DNA合成和重组的调控作用。分裂/融合方法允许在细胞中表达组成性修饰的PCNA。这允许在体内分析泛素化和summoylated的PCNA的影响。这些研究将使我们清楚地了解PCNA如何将蛋白质招募到复制叉上，与PCNA结合的不同蛋白质的活性如何被调节和协调，不同蛋白质在多步骤过程中如何在PCNA上发生交接，以及PCNA的翻译后修饰如何对这些过程产生不同的影响。

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.