DNA repair pathways preserve cellular homeostasis

项目摘要 真核细胞使用几种主要的DNA修复途径来监控和维持 它们基因组的完整性。过去的大多数研究都集中在对 细胞暴露后的基本机制和参与这些途径的关键蛋白 外源化学物质或辐射。相比之下，这些修复系统在 对细胞内自发形成的数万个DNA损伤做出反应 白昼在很大程度上是未被开发的。在这项拟议的研究中，我们认为这是第一次，我们将 研究在正常状态下维持细胞内环境平衡的所有主要途径的作用 生长，即对DNA损伤的内源性而不是外源性做出反应。我们的 初步研究表明，酿酒酵母(芽殖酵母)突变株在 同源重组(HR)途径修复DNA双链断裂 表现出许多由未修复的损伤引起的表型。这些表型包括3倍 在G2阶段花费的时间增加，这一特征取决于功能性 DNA损伤检查点基因，以及强烈增加的G2 DNA含量揭示 FACS分析，细胞显著增大，其他形态和生理发生显著变化 属性。我们随后从酵母缺失菌株文库中筛选出突变株，以询问 其他主要的修复途径在正常生长过程中最为关键。只有一个变种人 碱基切除修复(BER)途径也表现出强烈的细胞应激反应。我们 建议进行一系列实验，这些实验将通过(1)扩展这些发现 用遗传学和核磁共振技术研究HR突变体中持续激活的DNA损伤反应 基于显微镜的技术，(2)执行持久检查点模型的关键测试 在HR突变体中的激活，(3)表征构成DNA损伤的信号反应 BER途径突变体，以及(4)进行实验，以关键地测试反应的作用 氧物种(ROS)和停滞的复制分叉产生内源性检查点- 激活DNA损伤。我们的主要假设是HR和BER方面的缺陷，而不是其他 通路，导致高水平的未修复损伤，刺激复发的检查点信号 细胞中的级联和许多可量化的生理变化。因为主要的DNA修复 这个项目中分析的通路在包括人类在内的所有真核生物中都是保守的，研究结果 这项工作的成果将与人类致癌和细胞学的相关领域有很强的相关性 衰老。

Project Summary Eukaryotic cells employ several major DNA repair pathways to monitor and maintain the integrity of their genomes. Most past research has focused on characterization of the fundamental mechanisms and key proteins involved in these pathways after exposure of cells to exogenous chemicals or radiation. By contrast, the roles played by these repair systems in responding to the tens of thousands of DNA lesions that form spontaneously within cells each day is largely unexplored. In this proposed study, which we believe is the first of its kind, we will investigate the roles of all major pathways in maintaining cellular homeostasis during normal growth, i.e., in response to endogenous rather than exogenous sources of DNA damage. Our preliminary studies revealed that Saccharomyces cerevisiae (budding yeast) mutants defective in repair of DNA double-strand breaks (DSBs) by the homologous recombination (HR) pathway exhibit many phenotypes caused by unrepaired lesions. These phenotypes include a 3-fold increase in time spent in G2 phase, a characteristic dependent on the presence of functional DNA damage checkpoint genes, as well as strongly increased G2 DNA content revealed by FACS analysis, greatly enlarged cells, and striking changes in other morphological and physical properties. We subsequently screened mutants from a yeast deletion strain library to ask what other major repair pathways are most critical during normal growth. Mutants of only one other pathway, base excision repair (BER), also exhibited strong cellular stress responses. We propose to perform a series of experiments that will expand upon these findings by (1) characterizing the persistently activated DNA damage response in HR mutants using genetic and microscopy-based techniques, (2) performing critical tests of a model for persistent checkpoint activation in HR mutants, (3) characterizing the constitutive DNA damage signaling response in BER pathway mutants, and (4) performing experiments that will critically test the roles of reactive oxygen species (ROS) and stalled replication forks in generation of endogenous checkpoint- activating DNA lesions. Our major hypothesis is that defects in HR and BER, but not other pathways, lead to high levels of unrepaired lesions that stimulate a recurring checkpoint signaling cascade and many quantifiable physiological changes in cells. Since the major DNA repair pathways analyzed in this project are conserved in all eukaryotes including humans, the findings of the work will have strong relevance to the related fields of human carcinogenesis and cellular aging.