SUMOylation and ubiquitylation of PCNA in recombination and translesion synthesis

PCNA 重组和跨损伤合成中的 SUMO 化和泛素化

• 批准号: 8580606
• 负责人: M. TODD WASHINGTON
• 金额: $ 35.77万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8580606
• 关键词: Back; Binding; Binding Sites; Biological Assay; Bypass; Cells; Complex; DNA; DNA Damage; DNA biosynthesis; DNA replication fork; Development; Enzymes; Event; Face; Failure; Genetic Recombination; Genome Stability; Genomic Instability; Hand; Human; In Vitro; Lead; Methodology; Methods; Modeling; Modification; Molecular Conformation; Mutagenesis; Pathway interactions; Polymerase; Positioning Attribute; Post-Translational Protein Processing; Process; Production; Proliferating Cell Nuclear Antigen; Protein Binding; Protein Dynamics; Proteins; Recruitment Activity; Regulation; Roentgen Rays; Shapes; Slide; Structure; Surface; Testing; Time; Ubiquitin; Work; base; carcinogenesis; coping; enzyme activity; flexibility; helicase; in vivo; innovation; insight; molecular dynamics; mutant; novel; prevent; public health relevance; recombinational repair; reconstitution; repaired; research study; single molecule; tool; yeast genetics

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.

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的泛素化促进了受损DNA模板的跨性别合成，PCNA sumoylation阻止了异常重组。该建议的重点是理解PCNA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跨性的合成和重组。由于我们的小组进行了高度创新的技术发展，因此提出的研究是可能的，该技术可以使用拆分/融合策略来生产大量改良的PCNA。使用这种方法，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。目标1是研究UBI-PCNA和SUMO-PCNA对靶蛋白的相互作用和调节。修改后的PCNA将在单分子结合和重构酶测定中进行分析。这些研究将测试PCNA作用的工具带模型。目标2是研究UBI-PCNA和SUMO-PCNA的结构和动力学。结构研究将确定修饰PCNA的动力学，并检验以下假设：动态蛋白质蛋白伴侣复合物对于调节至关重要。研究还将确定PCNA的翻译后修饰如何与抗染色型解旋酶相互作用并调节重组。目标3是研究UBI-PCNA和SUMO-PCNA对DNA合成和重组的调节。分裂/融合方法允许在细胞中表达组成型修饰的PCNA。这允许在体内分析泛素化和sumoymet PCNA的影响。这些研究将清楚地了解PCNA如何募集蛋白质以复制叉，如何调节和协调与PCNA结合的不同蛋白质的活性，在多步骤过程中不同蛋白质之间的交接方式以及PCNA在PCNA上发生的方式以及PCNA的PCNA变频后修饰如何对这些过程产生如此不同的影响。