Mechanisms of fork restart in response to genotoxic stress

描述（由申请人提供）：基因组完整性不断受到环境暴露于基因毒素的威胁。许多基因毒素诱导复制应激并导致复制叉在对复制应激高度敏感的基因组区域停滞。停滞的复制会导致DNA损伤或重排，这对基因组的稳定性是有害的。因此，修复途径已经进化到对抗复制应激诱导的基因组不稳定性。不足为奇的是，复制应激反应蛋白的缺陷会导致癌症以外的疾病。有效的重新启动和修复停滞的复制最近被认为是保持基因组稳定性的最重要的机制之一。因此，了解停滞的复制是如何重新启动/修复的，对于理解细胞如何修复基因毒素引起的DNA损伤以及分叉停滞在调节癌变早期事件中的作用至关重要。然而，fork重启的机制定义不清，许多对fork重启至关重要的因素仍未确定。本应用程序的重点是了解新识别的参与者在对抗复制压力中的作用。CST复合物由Ctc1、Stn1、Ten1组成，是一种保守的高亲和力单链DNA结合蛋白复合物。最近的研究表明，缺乏CST会损害停滞的复制在全基因组范围内的重新启动，并诱导基因组区域的DNA损伤。但是，它在重新启动停滞的复制中的作用尚未被描述。本研究的目的是了解CST在复制胁迫下保持基因组稳定性中的作用，目的是为细胞如何抵抗基因毒素诱导的复制胁迫引起的DNA损伤提供新的见解。我们将绘制受CST保护的全基因组序列图，描述在复制胁迫下CST缺陷引起的染色体不稳定性，确定CST如何与已知的分叉重启因子相互作用以挽救停滞的复制，并确定Stn1的翻译后磷酸化在重启停滞的复制中的作用。这将通过整合ChIP-seq，质谱，荧光DNA纤维分析，分子和生化方法来完成。拟议的研究结果将揭示挽救停滞复制和保持基因组稳定性的新信息。

DESCRIPTION (provided by applicant): Genome integrity is constantly threatened by environmental exposure to genotoxins. Many genotoxins induce replication stress and cause replication fork stalling at genomic regions that are highly sensitive to replication stress. Stalld replication can lead to DNA lesions or rearrangements that are detrimental to genome stability. Therefore, repair pathways have evolved to counter replication-stress-induced genome instability. Not surprisingly, defects in replication-stress-response proteins give rise to disease that extend beyond cancer. Efficient restart and repair of stalled replication has recently been recognized as one of the most important mechanisms for preserving genome stability. Therefore, understanding how stalled replication is restarted/repaired is fundamentally important for understanding how cells repair DNA damages caused by genotoxins and the role of fork stalling in modulating early events in carcinogenesis. However, the mechanism underlying fork restart is poorly defined, and many factors critical for fork restart remain unidentified. This application focuses on understanding the role of a newly-identified player in countering replication stress. The CST complex, consisting of Ctc1, Stn1, Ten1, is a conserved high-affinity single-stranded DNA binding protein complex. Recent studies have shown that deficiency in CST impairs the restart of stalled replication genome- wide and induces DNA damage in the genomic region. However, its role in restarting stalled replication has not been characterized. The objective of this proposal is to understand the role of CST in preserving genome stability under replication stress, with the goal to provide novel insights into how cells counteract DNA damage caused by genotoxin-induced replication stress. We will map sequences protected by CST genome-wide, characterize chromosome instabilities caused by CST deficiency under replication stress, ascertain how CST interplays with the known fork-restart players to rescue stalled replication, and determine the role of post- translational phosphorylation of Stn1 in restarting stalled replication. This will be accomplished by integration of ChIP-seq, mass spectrometry, fluorescent DNA fiber analysis, molecular and biochemical methods. Findings from the proposed research will reveal novel information of rescuing stalled replication and preserving genome stability.