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

模式真核生物嗜热四膜虫的DNA复制和S期检查点反应。真核生物复制起点的协调时间调控确保了基因组的每个片段在每个细胞周期只复制一次。染色体不断受到遗传毒性应激的内在和外在来源的攻击，产生损伤，促进细长复制叉子的崩溃或形成双链断裂。真核生物已经进化出复杂的检查点途径，以协调细胞周期进程和DNA修复。S期DNA损伤检查点的主调节器ATR启动了一个信号级联，稳定停滞的复制叉子，并抑制新的起始点的启动。最近在模式真核生物四膜虫的研究中发现了一种序列特异性的DNA结合蛋白，它在S期早期抑制起始激活，并在S期内检查点激活中进一步需要。这个名为TIF1的因子与检查点反应中的已知蛋白质缺乏同源性，是唯一被证明调节复制时机的真核来源结合蛋白。该项目将利用强大的反向遗传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是如何整合在一起的，以促进正常细胞周期中的基因组稳定性和对遗传毒性胁迫的反应。到目前为止，TIF1已被证明调控从已有的核糖体DNA(RDNA)复制起点开始。TIF1介导的抑制的机制将通过评估耗尽TIF1对保守的复制蛋白招募到rDNA复制起始点的影响来确定。TIF1在非rDNA起始处的作用也将通过使用一种强大的单分子方法SMARD研究野生型和TIF1缺陷型菌株在~50个非rDNA起始点的复制时间来检验。此外，还将研究TIF1和ATR在抑制遗传毒性应激过程中延迟放电起源中的作用。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：鲜为人知的复制时机控制，以及DNA复制和修复的协调调节。四膜虫与酵母和人类的早期差异可以作为理解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方面的基础培训，包括指导科学界代表性不足的少数群体，包括女性。所有试剂都将随时提供给同事，包括纤毛虫分子生物学社区的成员。这些研究的结果将由国际和平协会和学生在地方和国家会议上传播，并将在开放获取的期刊上发表。

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.