DNA损伤容忍关键蛋白质Rad5的结构和功能研究

Lesions in the DNA template cause replication stall and leave single strand DNA gaps in the replicated genome, which compromises its stability. Both prokaryotic and eukaryotic organisms have evolved complicated DNA Damage Tolerance (DDT) mechanisms, which resume the stalled replication and repair the single strand DNA gaps. The eukaryotic DDT is highly conserved. In humans its defects cause embryonic death or serious diseases. The dual-function protein Rad5 plays essential roles in the eukaryotic DDT. It is an ubiquitin ligase and catalyzes the poly-ubiquitination of the proliferating cell nuclear antigen, which initiates the error-free DDT branch. It also catalyzes the fork regression reaction, utilizing energy generated by ATP hydrolysis. At stalled replication forks, fork regression is an important mechanism for fork protection and provides lesion-free template for replication restart. The ATP hydrolase and ubiquitin ligase activities of Rad5 are regulated by double strand DNA binding. Our recent structure of the apo Rad5 provides insights into its function. However, to date the structural basis of Rad5’s interaction with its substrate DNA is not available, how it catalyzes fork regression and how DNA regulates its activities are poorly understood. In this project, we will carry out structural studies of the Rad5-DNA complex and structural-guided functional experiments to elucidate the molecular mechanism of the Rad5-catalyzed fork regression and the regulation of Rad5’s activities by DNA.

DNA模版的损伤造成DNA复制的停滞和复制后基因组的不稳定，DNA损伤容忍（DNA Damage Tolerance，DDT）是应对这种情况的关键机制。真核生物的DDT高度保守，其功能缺失导致严重的人体疾病。双功能酶Rad5在真核生物的DDT中发挥关键作用。它具有泛素连接酶功能，催化泛素链标记增殖细胞核抗原，启动无误的DDT分支。它也可通过水解ATP驱动复制叉退行，在保护停滞的复制叉和重启DNA复制中发挥关键作用。我们最近解析的apo Rad5的晶体结构为理解它的功能机理提供了初步线索。但是到目前为止，Rad5与其底物DNA相互作用的结构机制还不清楚，它如何催化复制叉退行及它的酶活性如何受到DNA结合的调控还缺乏理解。在本项目中，我们将从解析Rad5与其DNA底物形成的复合物的结构入手，结合生物化学/生物物理手段，揭示Rad5催化复制叉退行及其酶活性受DNA结合调控的分子机理。

DNA模版的损伤造成DNA复制的停滞和复制后基因组的不稳定，DNA损伤容忍（DNA Damage Tolerance，DDT）是应对这种情况的关键机制。真核生物的DDT高度保守，其功能缺失导致严重的人体疾病。双功能酶Rad5在真核生物的DDT中发挥关键作用。它具有泛素连接酶功能，催化泛素链标记增殖细胞核抗原（PCNA），启动无误的DDT分支。它也可通过水解ATP驱动复制叉退行，在保护停滞的复制叉和重启DNA复制中发挥关键作用。Rad5执行这些多种功能的分子机制还不清楚。在本项目中，我们解析Rad5的晶体结构并开展了结构指导的功能研究实验。我们的工作揭示了DNA调控Rad5的ATP水解酶活性的机制，以及DNA调控Rad5对PCNA泛素化标记反应的机理，并为深入理解Rad5催化的复制叉退行反应提供了线索。我们的工作增进了对Rad5及其在人体中的同源蛋白HLTF在DNA损伤容忍中工作机制的理解，可为深入理解相关疾病的发生和发展及开发新的防治手段提供新思路。