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，活细胞中DNA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回转酶是一种被称为拓扑异构酶（topo）的分子机器，在包括细菌在内的许多生物中都有发现，但在哺乳动物细胞中没有发现。它发挥了一项至关重要的功能，即放松DNA中被称为“超级线圈”的多余扭曲，否则这些扭曲会阻止DNA复制或基因密码被读取。DNA回转酶的这一重要作用导致了几种抗生素的发展，这些抗生素通过特异性靶向回转酶并干扰其功能来杀死细胞。尽管人们已经对DNA回转酶进行了广泛的研究，使用的方法可以分析它的结构、遗传学和生物化学，但目前对它在活细胞内的运作方式知之甚少。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gyrase的不同亚基上，以及与gyrase活动有关的细胞分子机制的其他部分上。我们将应用先进的生物成像方法，使我们能够观察这些单个荧光蛋白分子，并跟踪这些不同的分子成分，因为它们在活细菌细胞中以比分子本身扩散更快的速度移动，使我们能够不模糊地观察它们，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，以及它们中有多少参与了细胞活动。除了DNA回转酶，我们还将跟踪负责复制DNA的分子机制，以及在“转录”过程中使用的分子机制，在“转录”过程中，遗传密码被读出并转录成细胞中的不同蛋白质。这将使我们了解DNA回转酶如何在活跃的DNA复制和转录过程中发挥放松超螺旋DNA的重要作用。我们还将研究当已知的靶向gyrase的抗生素被添加到细菌中时，gyrase会发生什么。很多抗生素被认为是通过在酶与DNA结合的地方将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，这种荧光标记专门与断裂DNA的末端结合。然而，已知一些细胞能够在这种锁定状态下耐受DNA旋切酶分子。这样的细胞可以在靶向抗生素的DNA旋切酶的作用下存活，而这种对抗生素的耐受性随后会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。为了研究细胞对这种有毒的DNA旋切酶分子的耐受机制，我们将制造对特定类型的靶向抗生素的DNA旋切酶特别敏感的修饰细菌，并与正常细胞比较DNA旋切酶分子和DNA复制机制的反应。我们利用先进的光学显微镜对活细菌进行DNA旋转酶的单分子研究，将使我们能够探索一个长期存在的谜题，即分子如何利用ATP的水解，ATP是所有活细胞中通用的化学能货币，以发挥其放松DNA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是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.