The mechanism of DNA strand exchange by serine recombinases

所有生物体都含有极长的双螺旋DNA分子，这些分子携带着每个细胞生长和繁殖所需的信息。这些信息被编码在称为碱基的DNA构建块的序列中。细胞机器将代码翻译成有用的分子，如蛋白质。有时，细胞会改变它制造的蛋白质组，以获得新的特征。例如，感染人类的致病细菌可能会改变其表面上显示的蛋白质组，以避免被人类免疫系统检测和破坏。一种方法是通过将片段从一个地方移动到另一个地方，或者通过添加/删除片段来重新排列（或“编辑”）DNA序列。一类特殊的蛋白质称为位点特异性重组酶，专门负责这种“剪切和粘贴”的作用。为了正确地进行，重组酶必须确定DNA序列中必须切割的精确位置，然后在每个位置切割两条DNA链，并将切割的末端重新连接到所需的新排列中。一个位点特异性重组酶家族被提出通过一种显着的机制来进行这种反应，其中DNA链首先被切割，然后在“分子轮”上转动，使切割的末端重新排列，然后重新连接它们。这种机制在酶的世界中是前所未有的，并且对分子机器的设计和功能有影响，但尚未得到证实。因此，弄清楚这是否真的是DNA重排的方式非常重要。在这个项目中，我们将使用新的方法，我们可以看到正在进行反应的微小的单个重组酶“机器”发生了什么。我们将把成对的荧光分子附着在每台机器中的DNA部分上，我们可以用一种特殊的显微镜看到，当DNA片段靠近或远离时，荧光分子会闪烁。我们还将看到当我们将机器中的蛋白质片段连接到DNA上以限制它们彼此相对运动时会发生什么。这些实验将使我们能够找出反应的真实的机制，并分析其性质，例如反应速度有多快，以及什么会干扰它。

All living organisms contain immensely long double-helical DNA molecules that carry the information each cell needs to grow and multiply. This information is encoded in the sequence of the DNA building blocks called bases. Cellular machines translate the code into useful molecules such as proteins. Sometimes a cell changes the set of proteins that it makes in order to acquire new characteristics. For example, a disease-causing bacterium infecting a human being may switch the set of proteins displayed on its surface as a diguise to avoid detection and destruction by the human immune system. One way to do this is to rearrange (or 'edit') the DNA sequence by moving sections from place to place, or by adding/deleting sections. A special class of proteins called site-specific recombinases is dedicated to this 'cutting and pasting' role. To do it properly, the recombinase must identify the precise places in the DNA sequences that must be cut, then cut both DNA strands in each place and rejoin the cut ends in the required new arrangement. One family of site-specific recombinases has been proposed to do this reaction by a remarkable mechanism in which the DNA strands are first cut, then turned on a 'molecular wheel' to bring the cut ends into a new arrangement before rejoining them. This mechanism would be unprecedented in the world of enzymes, and has implications for the design and function of molecular machines, but it has not been proved. It is therefore very important to find out if this is really how the DNA is rearranged. In this project, we will use novel methods where we can see what is happening to the tiny individual recombinase 'machines' that are doing the reaction. We will attach pairs of fluorescent molecules to parts of the DNA in each machine, which we can see with a special type of microscope, and which will flash on and off as the bits of DNA move closer together or farther apart. We will also see what happens when we attach bits of the protein in the machine to the DNA to restrict their movement relative to each other. These experiments will allow us to find out the real mechanism of the reaction and analyse its properties, such as how fast it goes and what can interfere with it.