Using nanofabricated DNA curtains to reveal protein during replication

描述(申请人提供)：所有生物必须准确地复制它们的基因组，才能将它们的遗传信息忠实地传递给后代。然而，在细胞内，DNA被大量的DNA结合蛋白所拥挤，这些蛋白可能会阻碍DNA的合成。众所周知，当DNA复制机制与包括转录RNA聚合酶在内的其他蛋白质碰撞时发生的碰撞会阻碍复制分叉，并导致重组事件，这是癌症的一个标志。尽管之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，但对复制和转录机制之间碰撞的机制细节知之甚少。这项建议的一个直接目标是直接观察模型病毒DNA聚合酶与单结合蛋白障碍之间的碰撞结果，如RNA聚合酶、乳糖抑制物和催化失活的EcoRI，这些都已被证明在体内阻碍复制分叉。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幕技术，直接可视化复制机制和蛋白质障碍之间的碰撞。这项建议的另一个方面是发展一种单分子分析方法，通过这种方法，可以用结合蛋白的串联阵列进行碰撞实验，因为它更能反映拥挤的生理环境。这些研究结果既有单链结合的蛋白质障碍，也有单链排列的蛋白质障碍，最终将导致与来自E.Coli和S.cerevisae的更复杂的多组分复制体的碰撞实验，这将提供更多关于碰撞诱导的高等生物体中复制分叉停滞和重组的细节。这 这项工作将有助于揭示复制机制处理蛋白质障碍的机制，并提供揭示这些碰撞的一些方面的可能性，这些方面不能通过其他方法更容易地获得。

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.