DNA Replication and the Intra-S Phase Checkpoint Response in the Model Eukaryote, Tetrahymena Thermophila.

模型真核生物嗜热四膜虫中的 DNA 复制和 S 相内检查点反应。

• 批准号: 0642157
• 负责人: Geoffrey Kapler
• 金额: $ 42万
• 依托单位: The Texas A&M University System HSC Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0642157&HistoricalAwards=false
• 关键词: DNA; Replication; Intra; Phase; Checkpoint

ABSTRACTDNA replication and the intra-S phase checkpoint response in the model eukaryote, Tetrahymena thermophila. The coordinated temporal regulation of eukaryotic replication origins assures that each segment of the genome is replicated once and only once per cell cycle. Chromosomes are under constant assault from intrinsic and extrinsic sources of genotoxic stress, generating lesions that promote the collapse of elongating replication forks or the formation of double-strand breaks. Eukaryotes have evolved elaborate checkpoint pathways that coordinate cell cycle progression with DNA repair. The master regulator of the intra-S phase DNA damage checkpoint, ATR, initiates a signaling cascade that stabilizes stalled replication forks and represses new origins from firing. Recent studies in the model eukaryote, Tetrahymena thermophila, have identified a sequence-specific DNA binding protein that represses origin activation early in S phase, and is further required for intra-S phase checkpoint activation. This factor, TIF1, lacks homology to known proteins in the checkpoint response, and is the only eukaryotic origin binding protein that has been shown to regulate replication timing. This project will exploit powerful reverse genetic approaches to study origin repression and checkpoint activation in Tetrahymena, with the goal of understanding how these two processes are integrated to promote genome stability during normal cell cycles and in response to genotoxic stress. Thus far, TIF1 has been shown to regulate initiation from the well-characterized ribosomal DNA (rDNA) replication origin. The mechanism of TIF1-mediated repression will be determined by assessing the effect of depleting TIF1 on the recruitment of conserved replication proteins to the rDNA replication initiation site. The role of TIF1 at non-rDNA origins will also be examined by studying replication timing at ~50 non-rDNA origins in wild type and TIF1-deficient strains, using a powerful single molecule approach, SMARD. The contributions of TIF1 and ATR to the repression of late firing origins during genotoxic stress will also be investigated. These studies will apply cutting edge technologies to address two important questions in chromosome biology: the poorly understood control of replication timing, and the coordinate regulation of DNA replication and repair. The early divergence of Tetrahymena from yeast and humans should serve as a reference point for understanding the evolution of the DNA damage checkpoint response. These studies will provide fundamental training in molecular biology and genomic-based approaches to graduate and undergraduate students, including the mentoring of under-represented minorities in science, including women. All reagents will be made readily available to colleagues, including members of the ciliate molecular biology community. The findings of these studies will be disseminated by the PI and students at local and national meetings, as well as be published in open access journals.

摘要：模型真核生物嗜热四膜虫中的 DNA 复制和 S 期内检查点反应。真核复制起点的协调时间调节确保基因组的每个片段在每个细胞周期复制一次且仅一次。 染色体不断受到内在和外在遗传毒性应激源的攻击，产生损伤，促进延长复制叉的崩溃或双链断裂的形成。 真核生物已经进化出了复杂的检查点通路，可以协调细胞周期进程与 DNA 修复。 S 期 DNA 损伤检查点的主调节器 ATR 启动信号级联，稳定停滞的复制叉并抑制新起点的激发。 最近对模型真核生物嗜热四膜虫的研究已经鉴定出一种序列特异性 DNA 结合蛋白，该蛋白可抑制 S 期早期的起源激活，并且是 S 期内检查点激活所必需的。 TIF1 因子与检查点反应中的已知蛋白质缺乏同源性，并且是唯一已被证明可以调节复制时间的真核起源结合蛋白。该项目将利用强大的反向遗传学方法来研究四膜虫的起源抑制和检查点激活，目的是了解这两个过程如何整合以促进正常细胞周期期间和响应基因毒性应激的基因组稳定性。迄今为止，TIF1 已被证明可以调节已明确表征的核糖体 DNA (rDNA) 复制起点的起始。 TIF1 介导的抑制机制将通过评估消耗 TIF1 对招募保守复制蛋白至 rDNA 复制起始位点的影响来确定。 TIF1 在非 rDNA 起源上的作用也将通过使用强大的单分子方法 SMARD 研究野生型和 TIF1 缺陷菌株中约 50 个非 rDNA 起源的复制时间来检查。 还将研究 TIF1 和 ATR 在基因毒性应激期间抑制晚期放电起源的贡献。 这些研究将应用尖端技术来解决染色体生物学中的两个重要问题：人们对复制时间的控制知之甚少，以及 DNA 复制和修复的协调调控。 四膜虫与酵母和人类的早期分化应该作为理解 DNA 损伤检查点反应进化的参考点。 这些研究将为研究生和本科生提供分子生物学和基于基因组的方法的基础培训，包括对科学领域代表性不足的少数群体（包括女性）的指导。 所有试剂都将随时提供给同事，包括纤毛虫分子生物学界的成员。 这些研究的结果将由 PI 和学生在地方和全国会议上传播，并在开放获取期刊上发表。