Tackling tricky twists - how does DNA gyrase function inside living cells?

DNA是“生命分子”，为大多数生物体提供遗传密码。然而，DNA在活细胞中的作用不仅与遗传密码有关，还与DNA的动态形状或“拓扑结构”有关。DNA促旋酶是一种分子机器，称为拓扑异构酶（或简称“topo”），存在于包括细菌在内的多种生物体中，但在哺乳动物细胞中没有发现。它执行一个重要的功能，放松DNA中被称为“超螺旋”的多余扭曲，否则会阻止DNA被复制，或阻止其遗传密码被读出。DNA促旋酶的这种重要作用导致了几种抗生素的开发，这些抗生素通过特异性靶向促旋酶并干扰其功能来杀死细胞。虽然已经使用可以分析其结构，遗传学和生物化学的方法对DNA促旋酶进行了广泛的研究，但目前对它在活细胞内如何运作知之甚少。在这个项目中，我们将使用遗传学技术将称为荧光蛋白的特定单分子标签附着到促旋酶的不同亚基和参与促旋酶活动的细胞分子机制的其他部分。我们将应用先进的生物成像方法，使我们能够观察这些单一的荧光蛋白分子，并跟踪这些不同的分子成分，因为它们在活的细菌细胞中以比分子本身扩散更快的速度移动，使我们能够观察它们，并非常精确地确定它们的位置。这将使我们能够准确地测量这些分子在细胞中的作用位置，以及有多少分子参与了它们的细胞活动。沿着DNA促旋酶，我们还将追踪负责复制DNA的分子机制，以及在“转录”过程中使用的分子机制，其中遗传密码被读出并转录成细胞中的不同蛋白质。这将使我们能够了解DNA促旋酶如何在活跃的DNA复制和转录过程中发挥其松弛超螺旋DNA的重要作用。我们还将研究当已知靶向促旋酶的抗生素被添加到细菌中时，促旋酶会发生什么。许多抗生素被认为是通过在促旋酶与DNA结合时将DNA锁定在断裂状态来起作用的，为了研究这种锁定状态是如何发生的，我们将使用一种荧光标记，它特异性地结合到断裂DNA的末端。然而，已知一些细胞能够耐受处于这种锁定状态的DNA促旋酶分子。这些细胞可以在靶向抗生素的DNA促旋酶的作用下存活下来，并且这种抗生素的耐受性随后可能导致整个细菌群体对这些抗生素产生耐药性，使它们作为治疗人类细菌感染的药物无效。为了研究这种中毒的DNA促旋酶分子的细胞耐受性机制，我们将制备对特定类型的DNA促旋酶靶向抗生素特别敏感的修饰细菌，并比较DNA旋转酶分子和DNA复制机制与正常细胞相比的反应。我们的单-使用先进的光学显微镜对活细菌进行DNA促旋酶的分子研究，将使我们能够探索分子如何利用蛋白质水解的长期难题。ATP，所有活细胞中的通用化学能量货币，以执行其在DNA中放松扭转应力的重要作用。最重要的是，这样做可以帮助我们了解ATP如何被这类分子机器使用的基本细节。

DNA is the 'molecule of life' which provides the genetic code for most organisms. However, the action of DNA in living cells is not solely linked to the genetic code, but also to the dynamic shape, or 'topology', of the DNA. DNA gyrase is a type of molecular machine called a topoisomerase (or 'topo' for short), found in many types of organisms including bacteria, but not found in mammalian cells. It performs a vital function of relaxing excess twists known as 'supercoils' in the DNA that would otherwise prevent DNA from being replicated, or from its genetic code being read out. This essential role of DNA gyrase has resulted in the development of several antibiotics which kill the cell by specifically targeting gyrase and interfering with its function. Although there has been extensive research performed on DNA gyrase using methods which can analyse its structure, genetics and biochemistry, very little is currently known about how it operates inside living cells. In this project we will use genetics techniques to attach specific single molecule tags called fluorescent proteins to the different subunits of gyrase and to other parts of the cellular molecular machinery which are involved in the activities of gyrase. We will apply advanced methods of bioimaging which allow us to observe these single fluorescent protein molecules and to track these different molecular components as they move in live bacterial cells at a speed which is faster than the molecules themselves can diffuse, enabling us to observe them unblurred and to determine their location very precisely. This will allow us to measure accurately where in the cell these molecules act and how many of them are involved in their cellular activities. Along with DNA gyrase we will also track the molecular machinery responsible for replicating the DNA, and also molecular machinery used in the process of 'transcription' in which the genetic code is read out and transcribed into different proteins in the cell. This will allow us to understand how DNA gyrase performs its essential role of relaxing supercoiled DNA during active DNA replication and transcription.We will also study what happens to gyrase when antibiotics which are known to target gyrase are added to bacteria. Many of these antibiotics are believed to act by locking the DNA into a broken state at the point at which gyrase is bound to the DNA, and in order to study how this locked state occurs we will use a fluorescent label which binds specifically to the ends of broken DNA. However, some cells are known to be able to tolerate DNA gyrase molecules in this locked state. Such cells can survive the action of DNA gyrase targeting antibiotics, and this tolerance of antibiotics can subsequently lead to resistance against these drugs in a whole population of bacteria against these antibiotics, making them ineffective as a medicine to treat bacterial infections in humans. In order to study the mechanisms of cellular tolerance of such poisoned DNA gyrase molecules we will make modified bacteria which are particularly sensitive to a specific type of DNA gyrase targeting antibiotic, and compare how the DNA gyrase molecules and DNA replication machinery respond compared to normal cells.Our single-molecule investigations of DNA gyrase using advanced light microscopy on live bacteria will allow us to explore a longstanding puzzle of how the molecules use the hydrolysis of ATP, the universal chemical energy currency in all living cells, in order to perform its vital role of relaxing torsional stress in DNA. In doing this it may help us, most importantly, to understand fundamental details about how ATP is used by this general class of molecular machine.