Elucidation of the rotary mechanism of serine recombinases

丝氨酸重组酶旋转机制的阐明

• 批准号: BB/R008493/1
• 负责人: Marshall Stark
• 金额: $ 60.24万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR008493%2F1
• 关键词: Elucidation; rotary; mechanism; serine; recombinases

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.

每个细胞的遗传信息都被存储为底培的序列，在非常长，薄的双螺旋DNA分子中。细胞中含有称为重组酶的酶，可以通过切割链并将末端重新加入新伴侣来改变DNA序列。这些酶的作用必须非常精确，因为它们有可能损害DNA并随之损害遗传信息。丝氨酸重聚糖酶是源自细菌和古细菌的这些“ DNA切割和粘贴”酶的一组。重组酶的分子识别并结合了称为位点的特定DNA序列。然后，两个重组酶-DNA复合物聚集在一起，重组酶在每个位点的中心打破了DNA链。然后发生了一个非凡的过程，在该过程中，这种大蛋白质加-DNA复合物的一半相对于另一半旋转，交换了一对破碎的DNA末端的位置。然后将交换的末端连接到他们的新合作伙伴，完成DNA序列的编辑。这种旋转机制起初是有争议的，因为没有其他酶可以做任何类似的事情，尽管我们现在有强有力的间接证据支持旋转，但我们仍然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，我们将采用高级方法，使我们能够观察正在经历DNA重排的单个酶-DNA复合物，因此我们可以看到它发生的旋转。我们将制作复合物，每个复合物都包含两个荧光染料分子，以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以及它们散发出的光的亮度，因为荧光会告诉我们它们相距多远。因此，我们可以分辨出重组酶可以旋转DNA末端的速度，无论它在旋转过程中的任何时间都会暂停，以及旋转过程如何受到实验因素，DNA序列的变化或酶突变的影响。我们以前的研究表明，重组酶-DNA复合物的中心有一个较小的模块，可以将DNA位点结合并将它们像完整的酶一样结合在一起。我们将使用类似的单分子实验来测试这种非常简单的模块是否也会引起旋转。丝氨酸重物组织酶对DNA分子的操纵在生物技术，合成生物学和纳米技术中具有巨大的潜力，例如用于疾病处理特定错误基因的疾病治疗，或利用内在旋转机制的特定缺陷基因的编辑。我们的项目将为这些酶的机制提供新的见解，这些酶可能会增强其新应用的独特性能和开发。