Protein Interactions Underlying Mutagenic Translesion Synthesis in Yeast

该项目旨在阐明严格控制的机制，使真核细胞忠实地复制其遗传物质，尽管存在DNA损伤。DNA损伤会阻止复制，引发一系列导致细胞死亡的事件。为了防止这种情况，细胞利用翻译合成（TLS） DNA聚合酶，它可以复制DNA损伤，同时暂时使它们无法修复。这些TLS酶是DNA损伤后细胞存活的核心；然而，它们也容易出错，并经常在基因组DNA中引入突变。该项目将采用跨学科的方法，结合结构生物学，生化方法和体内分析来破译酵母模型系统中诱变TLS的关键蛋白质-蛋白质相互作用（PPIs）。这项研究将促进我们对多蛋白TLS复合物的结构组织的理解，并为有关TLS的机制问题提供新的见解：诱变的TLS酶如何获得DNA复制叉？活性多聚合酶复合物如何组装？哪些分子事件驱动TLS DNA聚合酶转换？这些问题对于我们理解真核生物中导致突变的事件以及细胞应对DNA损伤和忠实地将遗传信息从一代传递到下一代的能力至关重要。该项目为从高级博士后培训到K-12教育等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生的教学提供丰富的材料，包括高级培训课程，如康涅狄格核磁共振研讨会，研究生的实验室项目，以及本科生暑期研究实习项目中不同本科生的小型项目。最后，它还将吸引附近法明顿高中（Farmington High School）的学生在“前沿生物研究实习项目”的保护下，参与“现实世界”的科学研究。在酿酒酵母中，大多数DNA损伤的复制绕过需要TLS聚合酶Rev1、pol eta和pol zeta的协同作用，这些聚合酶取代因DNA损伤而停滞的复制叉上的复制聚合酶，或填补复制后留下的含有损伤的单链DNA间隙。这个过程是由加工因子PCNA的单泛素化引发的，PCNA在DNA损伤位点的多聚合酶TLS复合物的组装中起着关键作用。这种TLS复合体（通常称为Rev1/pol zeta mutasome）在泛素化的PCNA上组装的机制尚不清楚。关于TLS酶如何进入DNA损伤位点以及DNA聚合酶在TLS过程中如何切换的重要问题仍未得到解答。本项目的主要目标是通过分析其结构水平上的组织，了解TLS突变体实现有效病变旁路的机制。我们的中心假设是，突变体的活性是由TLS DNA聚合酶的辅助结构域和调控亚基介导的蛋白-蛋白相互作用（PPIs）调节的。我们将通过结构分析来精确定位影响酿酒葡萄球菌Rev1/pol zeta突变体组装的ppi，并结合体内试验来探索这些ppi对诱变TLS的意义，从而验证这一假设。迄今为止，酵母TLS酶附属模块的结构和PPIs在很大程度上仍未被表征。为了深入了解酵母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/pol zeta突变体与滑动钳形PCNA相互作用的关键ppi。在目标2中，我们将确定介导Rev1/pol zeta复合物组装的基本附属模块的结构和探针ppi。在Aim 3中，我们将探索稳定多亚基Rev1/pol zeta组装的其他ppi。该项目将为TLS突变体的各个组成部分如何协同工作以实现有效的病变旁路提供新的见解。该项目由生物科学理事会分子和细胞生物科学司的遗传机制组和分子生物物理学组共同资助。

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.