Opening of a double stranded DNA replication fork by a hexameric helicase

哺乳动物细胞的内部工作涉及许多相互关联的小型“纳米机器”。单个机器通常由许多蛋白质组成，这些蛋白质共同完成细胞在生命中的一项功能，并决定其生存和复制的能力。了解单个机器如何工作是至关重要的，因为当这些机器发生故障时，后果可能是灾难性的。例如，一些癌症可以直接归因于处理细胞遗传信息（DNA）的机器故障。此外，入侵的微生物，如病毒，会带来它们自己的机器，当细胞被病原体劫持时，它们会破坏或摧毁细胞。因此，识别和理解这些机器如何运作可以成为对抗此类疾病状态的起点。生物科学可以激发纳米技术，其目的是开发基于蛋白质机器的小型机械装置，可以用来驱动或调节分子过程，从而使我们受益。在生物技术中发现了重要的例子，例如可以在单分子水平上操作的新兴纳米孔DNA测序设备。在原子水平上对细胞纳米机器的精确理解绝对依赖于精确结构信息的可用性。对于大分子组合，这些信息来自x射线结构分析，对于非常大的组合，它得到了强大的电子显微镜的极大帮助，可以对单个粒子成像。单独使用电子显微镜可以指导我们的理解，但在很大程度上缺乏提供关键细节的分辨率。E1，我们将要研究的蛋白质，来自于一大类病原体，乳头状瘤病毒，它会导致动物和人类的疣和癌症。乳头瘤病毒衍生的疾病具有重要的健康和经济意义。E1通常被称为“马达蛋白”，它形成一个六元（六聚体）环状组装体，一个在病毒DNA复制（繁殖）过程中解开DNA分子两条链的机器。它可以被看作是一个小型的放射状引擎，可以沿着DNA移动并分离DNA的两条链，因此它是一个原型纳米机器，也是抑制病毒复制和疾病的药物的重要靶标。在这里，我们的目标是了解E1机器如何解开DNA。目的是对E1进行详细的x射线结构研究，以确定其总体结构以及它如何与DNA相互作用。我们打算通过补充生化和生物物理研究来验证这些结果和测试功能模型。作为一种领先的易于处理的模型，它很有希望成为我们完全理解的第一台六聚体dna解绕机。我们工作的潜在应用包括通过基于结构的药物设计来制定靶向E1和相关解旋酶的策略。它们将有助于理解我们自己细胞中的复制过程，这些过程更复杂，更难以定义。在纳米生物技术和医学方面有潜在的应用。因此，我们设想我们的研究将产生广泛的影响，对健康有益，并改善国内和国际的生活和经济水平。

The inner workings of mammalian cells involve a number of interrelated, small, "nanomachines". Individual machines are usually composed of a number of proteins that work together to perform one of the cell's functions in life, and determine its ability to survive and replicate. Understanding how individual machines function is crucial because when these machines breakdown the consequences can be catastrophic. For example, some cancers can be attributed directly to the failure of machines that process the genetic information (DNA) of a cell. In addition, invading microorganisms such as viruses bring their own machines that subvert or destroy a cell as it is hijacked for the pathogen's own advantage. The identification and understanding of how these machines function can therefore be a starting point for combating such disease states. The biosciences can inspire nanotechnology, which aims to develop small mechanical devices based on protein machines that can be harnessed to drive or modulate molecular processes from which we benefit. Significant examples are found in biotechnology, such as emerging nanopore DNA sequencing devices that can operate at the single molecule level. The precise understanding of cellular nanomachines at the atomic level is absolutely dependent on the availability of precise structural information. For large molecular assemblies this information comes from X-ray structural analysis and, for very large assemblies, it is vastly assisted by powerful electron microscopes that can image single particles. Alone, electron microscopy can guide our understanding, but for the most part lacks the resolving power to provide critical detail. E1, the protein that we will study, is from a large group of pathogens, the papillomaviruses that cause warts and cancer in animals and man. Papillomavirus-derived diseases are of significant health and economic importance. E1 is often referred to as a "motor protein" that forms a six membered (hexameric) ring-like assembly, a machine that unwinds the two strands of the DNA molecule during replication (reproduction) of viral DNA. It can be viewed as a small radial engine that can move along DNA and separate its two strands and as such is a prototypic nanomachine and important target for drugs that could inhibit viral replication and disease.Here we aim to understand how the E1 machine unwinds DNA. The objectives are to carry out detailed X-ray structural studies on E1 to determine its overall structure and how it interacts with DNA. We intend to verify these results and test models of function with complementary biochemical and biophysical studies. As a leading tractable model, it has great promise for being the first hexameric DNA-unwinding machine that we fully understand. The potential applications of our work include formulating strategies to target E1 and related helicases through structure based drug design. They will help understand replication processes in our own cells that are more complex and challenging to define. There are potential applications in nanobiotechnology and in medicine. Consequently, we envision that our studies will have a wide impact, with health benefits and improved living and economic standards nationally and internationally.