Mechanisms of fork restart in response to genotoxic stress

响应基因毒性应激的分叉重启机制

• 批准号: 8800247
• 负责人: Weihang Chai
• 金额: $ 29.07万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8800247
• 关键词: Affinity; Aging; Attention; Binding; Biochemical; Cells; ChIP-seq; Chromosomal Instability; Chromosome Fragile Sites; Complex; DNA; DNA Damage; DNA Repair; DNA Sequence Rearrangement; DNA biosynthesis; DNA lesion; Data; Defect; Disease; Environmental Exposure; Event; Exposure to; Fiber; Gene Rearrangement; Genes; Genetic Variation; Genome; Genome Stability; Genomic Instability; Genomics; Genotoxic Stress; Goals; Knowledge; Lead; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Methods; Molecular Analysis; Mutagens; Nature; Pathway interactions; Phosphorylation; Proteins; Recruitment Activity; Research; Role; SS DNA BP; Side; Site; Stress; Technology; base; biological adaptation to stress; carcinogenesis; environmental agent; genome sequencing; genome-wide; human disease; improved; innovation; insight; nervous system disorder; novel; prevent; protein complex; public health relevance; repaired; response; telomere

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.

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。许多基因毒素会诱导复制应力，并在基因组区域降低了对复制应激高度敏感的复制叉。滞后复制会导致对基因组稳定性有害的DNA病变或重排。因此，修复途径已演变为应对重复应力诱导的基因组不稳定性。毫不奇怪，复制应力响应蛋白的缺陷会导致疾病超越癌症。有效的重新启动和修复停滞的复制已被认为是保存基因组稳定性的最重要机制之一。因此，了解停滞的复制是如何重新开始/修复的，对于了解细胞如何修复基因毒素引起的DNA损伤以及叉子停滞在调节致癌作用早期事件中的作用。但是，叉式重新启动的机制的定义较差，而对于叉子重新启动至关重要的许多因素仍然不明显。该应用程序的重点是了解新识别的玩家在反复复制压力中的作用。 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，CST缺乏会损害停滞的复制基因组的重新开始，并在基因组区域诱导DNA损伤。但是，其在重新启动停滞的复制中的作用尚未得到表征。该提案的目的是了解CST在复制应力下保存基因组稳定性中的作用，其目的是提供有关细胞如何抵消基因毒素诱导的复制应力引起的DNA损伤的新见解。我们将绘制受CST基因组保护的序列，表征由CST缺乏症在复制应力下引起的染色体不稳定性，确定CST与已知的Fork-Restart玩家的相互作用如何挽救停滞不前的复制，并确定STN1在重新重新重新重新重新重新重新重新重新重新重复失速复制中的转化后磷酸化的作用。这将通过整合芯片序列，质谱，荧光DNA纤维分析，分子和生化方法来实现。拟议研究的发现将揭示拯救停滞的复制和保存基因组稳定性的新信息。