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泛素化促进损伤DNA模板的跨损伤合成，PCNA SUMO化防止异常重组。该建议的重点是了解蛋白质的相互作用调节的PCNA的翻译后修饰，以及这些相互作用如何调节translesion合成和重组。所提出的研究是可能的，因为我们的小组进行了高度创新的技术开发，允许使用分裂/融合策略生产大量的修饰的PCNA。利用这种方法，我们确定了泛素修饰的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及调控。将在单分子结合和重组酶测定中分析修饰的PCNA。这些研究将测试PCNA作用的工具带模型。目的2研究Ubi-PCNA和SUMO-PCNA的结构和动态变化。结构研究将确定修改后的PCNA的动力学和测试的假设，动态蛋白质-蛋白质伴侣复合物的监管是至关重要的。研究还将确定PCNA的翻译后修饰如何影响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。分裂/融合方法允许组成型修饰的PCNA在细胞中表达。这允许在体内分析泛素化和SUMO化的PCNA的作用。这些研究将提供一个清晰的理解，究竟如何PCNA招募蛋白质复制叉，如何与PCNA结合的不同蛋白质的活动进行调节和协调，如何在多步骤过程中发生在PCNA不同蛋白质之间的交接，以及如何翻译后修饰的PCNA有这些过程中的不同影响。

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.