Using nanofabricated DNA curtains to reveal protein during replication

描述（由申请人提供）：所有生物体必须准确复制其基因组，以忠实地将其遗传信息传递给后代。然而，在细胞内，DNA被大量的DNA结合蛋白所拥挤，这些蛋白可以作为DNA合成的障碍。当DNA复制机器与其他蛋白质（包括转录RNA聚合酶）碰撞时发生的碰撞，已知会使复制叉停止，并导致重组事件，这是癌症的标志。尽管以前的生化研究已经探索了通过拥挤的DNA底物进行复制的问题，但对复制和转录机器之间的碰撞的机制细节知之甚少。该提议的直接目标是直接观察模型病毒DNA聚合酶与单链结合蛋白质障碍物（如RNA聚合酶、lac阻遏物和催化失活的EcoRI）之间的碰撞的结果，这些障碍物均已被证明阻碍体内的复制叉。这些研究将依靠格林实验室开发的单分子光学显微镜和“DNA帘幕”技术，直接可视化复制机器和蛋白质障碍之间的碰撞。该提议的另一个方面是开发单分子测定法，通过该测定法可以用结合蛋白质的串联阵列进行碰撞实验，因为其更能反映拥挤的生理环境。这些研究的结果与单一结合和串联排列的蛋白质障碍物将最终导致碰撞实验与更复杂的多组分复制体从大肠杆菌。coli和革兰氏阳性菌S.这将提供高等生物体中碰撞诱导的复制叉停滞和重组的进一步细节。这 这项工作将有助于揭示复制机器处理蛋白质障碍的机制，并提供揭示这些碰撞的某些方面的潜力，而这些方面是通过其他方法不能更容易地得到的。 公共卫生相关性：当DNA复制机器遇到其他DNA结合蛋白时发生的碰撞会导致染色体不稳定，这是恶性细胞的标志。尽管这些事件在癌症的发展中具有根本的重要性，但对这些碰撞的机制细节了解甚少。本提案中概述的实验旨在揭示DNA复制机制与DNA结合蛋白质障碍物之间碰撞的物理结果。

DESCRIPTION (provided by applicant): All organisms must accurately replicate their genomes to faithfully pass their genetic information onto subsequent generations. Within the cell, however, DNA is crowded by a large number of DNA-binding proteins that can act as obstacles to DNA synthesis. The collisions that occur when the DNA replication machinery collides with other proteins, including transcribing RNA polymerase, are known to stall the replication fork and result in recombination events that are a hallmark of cancer. Despite previous biochemical studies that have explored the problems of replicating through crowded DNA substrates, little is known about the mechanistic details of collisions between the replication and transcription machineries. An immediate goal of this proposal is to directly observe the outcomes of collisions between a model viral DNA polymerase and singly-bound protein roadblocks, such as RNA polymerase, lac repressor, and catalytically inactive EcoRI, which have all been demonstrated to impede the replication fork in vivo. These studies will rely on single molecule optical microscopy and "DNA curtain" technology, developed in the Greene lab, to directly visualize collisions between the replication machinery and protein obstacles. Another aspect of this proposal is the development of a single molecule assay by which collision experiments can be performed with tandem arrays of bound proteins as is more reflective of a crowded physiological setting. The results from these studies with both singly-bound and tandemly-arrayed protein obstacles will ultimately lead to collision experiments with the more complicated multicomponent replisomes from E. coli and S. cerevisae, which will provide further details of collision- induced replication fork stalling and recombination in higher organisms. This work will help reveal the mechanisms by which the replication machinery deals with protein obstacles, and offers the potential to reveal aspects of these collisions that cannot be more easily accessed through other methods. PUBLIC HEALTH RELEVANCE: Collisions that occur when the DNA replication machinery encounters other DNA-binding proteins can result in chromosomal instability, which is a hallmark of malignant cells. Despite the fundamental importance of these events in the development of cancer, the mechanistic details of such collisions are poorly understood. The experiments outlined in this proposal are designed to reveal the physical outcomes of collisions between the DNA replication machinery and DNA-bound protein obstacles.