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以碱基对序列的形式存储在极长、极细的双螺旋 DNA 分子中。细胞含有称为重组酶的酶，可以通过切割链并将末端重新连接到新的伙伴来改变 DNA 序列。这些酶的作用必须非常精确，因为它们有可能对 DNA 造成损害并导致遗传信息丢失。丝氨酸重组酶是一组“DNA 剪切和粘贴”酶，源自细菌和古细菌。重组酶分子识别并结合称为位点的特定 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断裂 DNA 链。然后发生了一个非凡的过程，这个大的蛋白质加 DNA 复合物的一半相对于另一半旋转，交换了一对断裂的 DNA 末端的位置。然后交换的末端与新的伙伴连接起来，完成 DNA 序列的编辑。这种旋转机制最初是有争议的，因为没有其他酶能做到类似的事情，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，我们将应用先进的方法来观察正在进行 DNA 重排的单酶 DNA 复合物，这样我们就可以看到旋转的发生。我们将制作复合物，每个复合物包含两个荧光染料分子，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以荧光形式发出的光的亮度将告诉我们它们之间的距离有多远。因此，我们可以知道重组酶旋转 DNA 末端的速度、旋转过程中是否随时暂停，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，重组酶-DNA复合物的核心有一个更小的模块，它可以结合DNA位点并将它们结合在一起，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（例如编辑疾病的特定缺陷基因）方面具有巨大的潜力治疗，或利用纳米级分子马达的固有旋转机制。我们的项目将为这些酶的机制提供新的见解，这可能会增强其独特的特性并开发新的应用。