细胞应对R-loop所造成复制压力的分子机制研究

Preservation of genome integrity is a fundamental requirement for cell survival and organism development. In the past three decades, a considerable body of evidence has point to that transcription, a fundamental and ineluctable process of life, can affect genome stability. One mechanism underlying the impact of transcription on genome integrity is through the formation of R-loop, a structure in which a nascent transcript is hybridized with the template DNA strand, leaving the non-template strand unpaired. While such RNA:DNA hybrids have been detected in organisms from bacteria to human, our previous work in E. coli and vertebrate cells, together with others' studies in yeast, has established that R-loop formation is an intrinsic threat to genome stability throughout evolution. Despite these advances in understanding the catastrophic effects of R-loop formation on preserving genome integrity, relatively little was known about the underlying mechanism and this indeed decelerates follow-up studies to understanding the molecular determinants and pathological consequences of transcriptional R-loop, a potent source for the acquisition of an enabling characteristic of cancer. Recently, a work from my lab shed new insights into the evolutionarily conserved phenomenon of transcription-induced R-loops, and into the mechanism by which these structures lead to genomic instability. Our findings provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability in E. coli and human cells as well. In this grant, I propose to address a critical question derived from our findings: how do higher eukaryotic cells respond to R loop-induced replication stress? All of the proposed experiments will be extensions of our previous work, and many of them are ongoing. These studies will cast new light on the molecular bases of genomic instability in tumor cells, and implicate an alternative avenue for developing new prognostic and diagnostic strategies for cancer.

维持基因组稳定是生物体生存和繁衍的根本。然而，过去三十多年的研究使我们认识到细胞中无时不刻进行着的RNA转录可以对基因组稳定性产生负面影响。原因之一是形成R-loop结构,一种由新生RNA与其模板DNA重配对形成的异常RNA:DNA杂交分子。我们实验室前期工作揭示干扰DNA复制叉运行是R-loop破坏基因组稳定性的重要机制，广泛存在于从大肠杆菌到人等各种生物细胞中(Genes & Dev.,2011)。该项工作确立了DNA复制损伤与R-loop介导的基因组非稳性之间的因果联系，提示R-loop是一种进化保守的内源性复制压力。本申请中，我们希望在上述原创性研究工作基础上，进一步解析细胞应对R-loop所造成复制压力的分子机制。这一研究将加深对于高等真核细胞基因组不稳定性形成原因、应对机制及可能产生的病理后果的理解，有可能为癌症的早期诊断、治疗和药物开发提供新的理论基础。

复制叉运行受阻是真核细胞中破坏基因组稳定性的重要内因。在本项目开展过程中，我们意外地发现在缓解肿瘤细胞复制压力中扮演重要角色的A蛋白存在一种之前并不了解的位置特异性蛋白切割事件。这一新的翻译后加工事件的发现促使我们进一步对该切割事件的分子机理以及其生物学意义等方面开展探索性研究，最终揭示这一翻译后加工事件可以避免A蛋白发生K-48连接的多聚泛素化和蛋白酶体介导的降解，进而参与调控人细胞内A蛋白的丰度及细胞应对DNA损伤引起的复制压力的能力。这一工作为解析A蛋白在肿瘤细胞中的表达调控机制和生物学功能提供了一个全新的视角。