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: 9551636
• 负责人: Weihang Chai
• 金额: $ 23.87万
• 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 2019-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9551636
• 关键词: Affinity; Aging; Attention; Binding; Biochemical; Cells; ChIP-seq; Chromosomal Instability; Chromosome Fragile Sites; Complex; DNA; DNA Damage; DNA Repair; DNA biosynthesis; DNA lesion; DNA replication fork; Data; Defect; Disease; Environmental Exposure; Event; Exposure to; Fiber; Gene Rearrangement; Genes; Genetic Variation; Genome; Genome Stability; Genomic Instability; Genomic Segment; Genotoxic Stress; Goals; Impairment; Knowledge; Lead; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Methods; Molecular; Mutagens; Nature; Pathway interactions; Phosphorylation; Proteins; Research; Role; SS DNA BP; Side; Site; Stress; Technology; base; biological adaptation to stress; carcinogenesis; environmental agent; genome integrity; genome-wide; human disease; improved; innovation; insight; nervous system disorder; novel; prevent; protein complex; public health relevance; recruit; 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遗传毒素的威胁。许多基因毒素诱导复制应激，并在对复制应激高度敏感的基因组区域引起复制分叉停滞。Stalld复制会导致DNA损伤或重排，这对基因组稳定性是有害的。因此，修复途径已经进化为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。最近，有效地重新启动和修复停滞的复制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，对于了解细胞如何修复由基因毒素引起的DNA损伤以及分叉停滞在调节癌症发生的早期事件中的作用是至关重要的。然而，派生重启背后的机制定义不明确，许多对派生重启至关重要的因素仍然不确定。这个应用侧重于了解新发现的参与者在应对复制压力方面的作用。CST复合体由Ctc1、Stn1、Ten1组成，是一种保守的高亲和力单链DNA结合蛋白复合体。最近的研究表明，CST的缺乏会损害全基因组范围内停滞复制的重新开始，并导致基因组区域的DNA损伤。然而，它在重启停滞不前的复制方面的作用尚未得到证实。这项建议的目的是了解CST在复制压力下保持基因组稳定性方面的作用，目的是为细胞如何对抗遗传毒素诱导的复制应激引起的DNA损伤提供新的见解。我们将在全基因组范围内定位受CST保护的序列，表征CST缺乏在复制压力下引起的染色体不稳定性，确定CST如何与已知的分叉重新启动玩家相互作用来挽救停滞的复制，并确定Stn1的翻译后磷酸化在重新启动停滞复制中的作用。这将通过集成芯片序列、质谱学、荧光DNA纤维分析、分子和生化方法来实现。这项拟议研究的结果将揭示挽救停滞的复制和保持基因组稳定性的新信息。