Protein Interactions Underlying Mutagenic Translesion Synthesis in Yeast

酵母中诱变跨损伤合成的蛋白质相互作用

• 批准号: 1615866
• 负责人: Dmitry Korzhnev
• 金额: $ 65万
• 依托单位: University of Connecticut Health Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1615866&HistoricalAwards=false
• 关键词: Protein; Interactions; Underlying; Mutagenic; Translesion

This project aims to elucidate tightly controlled mechanisms that allow eukaryotic cells to faithfully reproduce their genetic material despite the presence of DNA damage. DNA lesions can block replication, triggering a cascade of events leading to cell death. To prevent this, cells utilize translesion synthesis (TLS) DNA polymerases that can copy over the DNA lesions while temporarily leaving them unrepaired. These TLS enzymes are central to cell survival after DNA damage; however, they are also error-prone and often introduce mutations in genomic DNA. The project will employ an interdisciplinary approach that combines structural biology, biochemical methods and in vivo assays to decipher key protein-protein interactions (PPIs) underlying mutagenic TLS in a yeast model system. This research will advance our understanding of the structural organization of the multi-protein TLS complexes and also provide new insights into mechanistic questions about TLS: How do mutagenic TLS enzymes gain access to DNA replication forks? How do active multi-polymerase complexes assemble? What molecular events drive TLS DNA polymerase switching? These questions are central to our understanding of events leading to mutagenesis in eukaryotes and the ability of cells to cope with DNA damage and faithfully transfer genetic information from one generation to the next. This project creates a solid platform for integration of research and education at multiple levels, from advanced post-PhD training to K-12 education. The project will provide plentiful material for graduate and undergraduate teaching, including advanced training courses such as the Connecticut NMR Workshop, laboratory projects for graduate students, and mini-projects for the diverse undergraduate students from the Undergraduate Summer Research Internship program. Finally, it will also engage students from the nearby Farmington High School in "real world" scientific research under the umbrella of the Cutting Edge Bioresearch Internship program. In S. cerevisiae, replicative bypass of most DNA lesions requires the coordinated action of TLS polymerases Rev1, pol eta and pol zeta that replace replicative polymerases at replication forks stalled by DNA damage or fill damage-containing single-stranded DNA gaps left after replication. This process is initiated by mono-ubiquitination of the processivity factor PCNA, which plays a key role in the assembly of the multi-polymerase TLS complex at DNA damage sites. The mechanism by which this TLS complex, often called the Rev1/pol zeta mutasome, is assembled on ubiquitinated PCNA is poorly understood. Important questions remain unanswered about how TLS enzymes gain access to sites of DNA damage and how DNA polymerases switch during TLS. The main goal of this project is to understand the mechanisms by which the TLS mutasome achieves efficient lesion bypass by analyzing its organization at a structural level. Our central hypothesis is that mutasome activity is regulated by protein-protein interactions (PPIs) mediated by accessory domains and regulatory subunits of the TLS DNA polymerases. We will test this hypothesis by using a structural analysis to precisely map PPIs that influence the assembly of the S. cerevisiae Rev1/pol zeta mutasome, combined with in vivo assays to probe the significance of these PPIs for mutagenic TLS. To date, the structure and PPIs of accessory modules of yeast TLS enzymes remain largely uncharacterized. To gain insights into the structural organization and regulation of the yeast TLS machinery, we will undertake the following specific aims. In Aim 1, we will examine key PPIs that control the Rev1/pol zeta mutasome interactions with the sliding clamp PCNA. In Aim 2, we will determine structures and probe PPIs of the essential accessory modules that mediate assembly of the Rev1/pol zeta complex. In Aim 3, we will explore additional PPIs that stabilize multi-subunit Rev1/pol zeta assembly. The project will provide new insights into how individual components of the TLS mutasome work together to achieve efficient lesion bypass.The project is funded jointly by the Genetic Mechanisms Cluster and the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.

该项目旨在阐明严密控制的机制，使真核细胞在DNA损伤的情况下仍能忠实地复制其遗传物质。DNA损伤可以阻止复制，触发一系列导致细胞死亡的事件。为了防止这种情况，细胞利用跨损伤合成(TLS)DNA聚合酶，它可以复制DNA损伤，但暂时不修复它们。这些TLS酶是DNA损伤后细胞生存的中心；然而，它们也容易出错，经常在基因组DNA中引入突变。该项目将采用一种结合结构生物学、生化方法和体内分析的跨学科方法，以破译酵母模型系统中潜在的突变TLS的关键蛋白质-蛋白质相互作用(PPI)。这项研究将促进我们对多蛋白TLS复合体的结构组织的理解，并为TLS的机制问题提供新的见解：突变的TLS酶如何获得DNA复制分叉？活性多聚合酶复合体是如何组装的？是什么分子事件驱动了TLS DNA聚合酶的转换？这些问题对于我们理解导致真核细胞突变的事件以及细胞应对DNA损伤并将遗传信息从一代忠实地转移到下一代的能力是至关重要的。该项目为从高级博士后培训到K-12教育的多个层面的研究和教育整合创造了坚实的平台。该项目将为研究生和本科教学提供丰富的材料，包括高级培训课程，如康涅狄格州核磁共振研讨会，研究生的实验室项目，以及为本科生暑期研究实习计划的不同本科生提供的微型项目。最后，它还将在尖端生物研究实习计划的保护伞下，让附近法明顿高中的学生参与“真实世界”的科学研究。在酿酒酵母中，大多数DNA损伤的复制旁路需要TLS聚合酶Rev1、PolEta和PolZeta的协调作用，这些聚合酶取代复制叉处的复制聚合酶，这些复制聚合酶因DNA损伤而停滞不前，或者填补复制后留下的含有损伤的单链DNA缺口。这一过程是由过程性因子增殖细胞核抗原的单一泛素化启动的，该因子在DNA损伤部位组装多聚合酶TLS复合体中起关键作用。这种TLS复合体，通常被称为Rev1/polZeta突变酶，组装在泛素化的增殖细胞核抗原上的机制尚不清楚。关于TLS酶如何进入DNA损伤部位以及DNA聚合酶在TLS过程中如何转换等重要问题仍未得到解答。这个项目的主要目标是通过在结构水平上分析TLS突变酶体的组织结构，了解TLS突变酶体实现有效病变旁路的机制。我们的中心假设是，突变酶的活性是由TLS DNA聚合酶的辅助结构域和调节亚基介导的蛋白质-蛋白质相互作用(PPI)调节的。我们将通过结构分析来验证这一假设，以精确地定位影响酿酒酵母Rev1/PolZeta突变酶组装的PPI，并结合体内试验来探索这些PPI对诱变TLS的意义。到目前为止，酵母TLS酶的辅助模块的结构和PPI在很大程度上仍未确定。为了深入了解酵母菌TLS机制的结构组织和调控，我们将承担以下具体目标。在目标1中，我们将检查控制Rev1/polZeta突变体与滑动钳制增殖细胞核抗原相互作用的关键PPI。在目标2中，我们将确定介导Rev1/PolZeta复合体组装的关键辅助模块的结构和探测PPI。在目标3中，我们将探索其他PPI，以稳定多亚基Rev1/PolZeta组装。该项目将为TLS突变体单个组件如何共同工作以实现有效的损伤绕过提供新的见解。该项目由遗传机制组和生物科学局分子和细胞生物科学司的分子生物物理学组联合资助。