Elucidation of the rotary mechanism of serine recombinases

每个细胞的遗传信息都以碱基对序列的形式储存在极长而薄的双螺旋DNA分子中。细胞中含有一种叫做重组酶的酶，这种酶可以通过切断DNA链并将其末端重新连接到新的伙伴上来改变DNA序列。这些酶的作用必须非常精确，因为它们有可能对DNA造成损害，并伴随遗传信息的丢失。丝氨酸重组酶是这些“DNA剪切和粘贴”酶中的一组，来源于细菌和古细菌。重组酶的分子识别并结合到称为位点的特定DNA序列上。然后两个重组酶-DNA复合物结合在一起，重组酶在每个位点的中心破坏DNA链。随后，一个非同寻常的过程发生了，这个巨大的蛋白质加DNA复合体的一半相对于另一半旋转，交换了一对断裂DNA末端的位置。然后交换的末端与它们的新伙伴连接，完成DNA序列的编辑。这种旋转机制起初是有争议的，因为没有其他酶有类似的功能，尽管我们现在有强有力的间接证据支持旋转，但我们对丝氨酸重组酶如何实现这一非凡的壮举知之甚少。在这个项目中，我们将采用先进的方法，使我们能够观察正在进行DNA重排的单个酶-DNA复合物，因此我们可以看到它发生时的旋转。我们将制作每个包含两个荧光染料分子的复合物，使它们之间的距离随着旋转的发生而变化。这些染料吸收的光量和它们发出的荧光的亮度将告诉我们它们之间的距离。因此，我们可以知道重组酶旋转DNA末端的速度有多快，它是否在旋转过程中随时停顿，以及旋转过程如何受到实验因素、DNA序列变化或酶突变的影响。我们之前的研究表明，在重组酶-DNA复合体的中心有一个较小的模块，它可以结合DNA位点并将它们聚集在一起，就像完整的酶一样。我们将使用类似的单分子实验来测试这个非常简单的模块是否也能引起旋转。丝氨酸重组酶对DNA分子的操纵在生物技术、合成生物学和纳米技术方面具有巨大的潜力，例如用于疾病治疗的特定缺陷基因的编辑，或利用纳米级分子马达的内在旋转机制。我们的项目将为这些酶的机制提供新的见解，从而可能导致其独特性质的增强和新应用的开发。

Every cell's genetic information is stored as sequences of basepairs in immensely long, thin double-helical DNA molecules. Cells contain enzymes called recombinases that can alter DNA sequences by cutting strands and rejoining the ends to new partners. The actions of these enzymes must be very precise, as they have the potential to cause damage to the DNA and concomitant loss of genetic information. The serine recombinases are one group of these "DNA cut and paste" enzymes, derived from bacteria and archaea. Molecules of the recombinase recognize and bind to specific DNA sequences called sites. Two recombinase-DNA complexes then come together, and the recombinase breaks the DNA strands at the centres of each site. An extraordinary process then takes place where one half of this large protein-plus-DNA complex rotates relative to the other half, swapping the positions of a pair of broken DNA ends. The swapped ends are then joined to their new partners, completing the editing of the DNA sequence. This rotation mechanism was controversial at first as no other enzymes do anything like it, and although we now have strong indirect evidence supporting rotation, we still know very little about how the serine recombinase enzyme achieves this remarkable feat. In this project, we will apply advanced methods that allow us to observe single enzyme-DNA complexes that are undergoing DNA rearrangement, so we can see rotation as it happens. We will make complexes that each contain two fluorescent dye molecules, placed so that the distance between them changes as rotation takes place. The amount of light absorbed by these dyes and the brightness of the light they give out as fluorescence will tell us how far apart they are. We can thus tell how fast the recombinase can rotate the DNA ends, whether it pauses at any times during rotation, and how the rotation process can be affected by experimental factors, changes in the DNA sequence, or mutations of the enzyme. Our previous studies have revealed that there is a smaller module at the heart of the recombinase-DNA complex which can bind DNA sites and bring them together just like the complete enzymes. We will use similar single-molecule experiments to test whether this very simple module can also cause rotation.The manipulation of DNA molecules by serine recombinases has enormous potential in biotechnology, synthetic biology, and nanotechnology such as for the editing of specific faulty genes for disease treatment, or exploiting the intrinsic rotary mechanism in nanoscale molecular motors. Our project will provide new insights into the mechanisms of these enzymes that might lead to enhancement of their unique properties and development for new applications.