Elucidating Eukaryotic Topoisomerase III Activity: An Enzymatic Double-edged Sword

阐明真核拓扑异构酶 III 活性：酶促双刃剑

• 批准号: BB/V005081/1
• 负责人: William Gittens
• 金额: $ 38.84万
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV005081%2F1
• 关键词: Elucidating; Eukaryotic; Topoisomerase; III; Activity

Organisms on Earth encode their genetic information on long string-like molecules of DNA. These DNA "genomes" contain the instructions to build all parts of the organism in which they exist. Each human cell is far too small to see by the naked eye, yet each one contains over two metres of DNA, divided up into 23 pairs of "chromosomes". This DNA is wrapped up in a special way so that it can fit inside the cell nucleus (where the genome is kept).To make matters worse, the genome doesn't just sit there doing nothing. There are many dynamic processes that need to occur throughout the cell cycle, including "transcription" (reading of one part of the genetic instructions, to build one part of the organism), "replication" (preparing for cell division, by copying all of the DNA), "chromosome segregation" (separating the copied DNA into two daughter cells).The chain-like nature of DNA makes these processes challenging. Think about untangling headphones or fairy-lights-then multiply it! It would be much easier if you had a magical pair of scissors that could cut the wire and re-seal it without leaving a trace. In the case of DNA genomes in nature, these magical scissors really exist. They're called DNA topoisomerases. DNA topoisomerases are proteins found in every organism on Earth, and have been around since an early time in evolution. There are different types of topoisomerases, which cut and re-seal the DNA in different ways. But they all have a common feature: they remain stuck to the DNA in the time between cutting and re-sealing. Very few things in nature work perfectly every time, and occasionally the topoisomerase will fail to re-seal the cut it has made, meaning that we are left with a break in the DNA that is stuck to a topoisomerase. These examples of DNA damage can kill the cell, or cause mutations which negatively alter the genetic instructions for life. In humans this can lead to neurological disorders, or cancer. Fortunately, there are other special proteins which have evolved to repair these topoisomerase-linked DNA breaks. Humans and yeast are part of a wider family of related organisms called eukaryotes. In eukaryotes there are three main types of topoisomerases. Over the last 40 years, we have gathered a lot of information about two of these: topoisomerase I (Top1) and topoisomerase II (Top2), but we have much less information about the third: topoisomerase III (Top3). This is partly because we have discovered chemicals which can increase the chance of Top1 and Top2 failing and making DNA damage, which makes it easier to study in the laboratory. We haven't discovered any chemicals that do this for Top3, so we don't know as much about which genome processes it is involved in, what the consequences are of the DNA damage it creates, and which other special proteins repair this DNA damage. Recently I have found a way to slightly modify Top3, so that it is much more likely to fail and create damage. In the first part of my project I will use this to find out more about Top3 damage and repair in yeast cells. This will be very useful, as it will tell us about an under-explored topic of eukaryotic DNA Damage. It will also lay the groundwork for future similar investigations in human cells, where this type of DNA damage might have consequences for human disease. I have also recently helped develop a method which allows us to very precisely find out where topoisomerases cut the DNA genome. Recently, I have used this to find out where Top2 cuts the DNA. In the second part of my project I will develop the method further so that it works for Top3, and use it to find out exactly when and where Top3 is active in the genome. This will tell us a lot about the genome processes that Top3 is involved in, which will be very useful for other researchers who work on topoisomerases, and other aspects of genome biology.

地球上的生物体将其遗传信息编码在DNA的长串状分子上。这些DNA“基因组”包含构建它们所存在的有机体的所有部分的指令。每个人类细胞都太小了，肉眼无法看到，但每个细胞都含有超过两米的DNA，分为23对“染色体”。这些DNA以一种特殊的方式被包裹起来，这样它就可以进入细胞核（基因组保存的地方）。更糟糕的是，基因组并不只是坐在那里什么也不做。有许多动态过程需要在整个细胞周期中发生，包括“转录”（阅读遗传指令的一部分，以构建生物体的一部分），“复制”（通过复制所有DNA为细胞分裂做准备），“染色体分离”（将复制的DNA分离成两个子细胞）。DNA的链状性质使这些过程具有挑战性。想想解开耳机或童话灯-然后乘以它！如果你有一把神奇的剪刀，可以剪断电线，然后不留痕迹地重新密封，那就容易多了。就自然界中的DNA基因组而言，这些神奇的剪刀确实存在。它们被称为DNA拓扑异构酶。DNA拓扑异构酶是存在于地球上每一种生物体中的蛋白质，在进化的早期就已经存在。有不同类型的拓扑异构酶，它们以不同的方式切割和重新密封DNA。但它们都有一个共同的功能：在切割和重新密封之间的时间里，它们仍然粘在DNA上。自然界中很少有东西每次都能完美地工作，偶尔拓扑异构酶会无法重新密封它所做的切割，这意味着我们在DNA中留下了一个被拓扑异构酶卡住的断裂。这些DNA损伤的例子可以杀死细胞，或导致突变，从而负面地改变生命的遗传指令。在人类中，这可能导致神经系统疾病或癌症。幸运的是，还有其他特殊的蛋白质已经进化到修复这些拓扑异构酶连接的DNA断裂。人类和酵母菌是一个更广泛的生物家族的一部分，这个家族被称为真核生物。在真核生物中有三种主要类型的拓扑异构酶。在过去的40年里，我们已经收集了大量关于其中两种的信息：拓扑异构酶I（Top1）和拓扑异构酶II（Top2），但我们对第三种的信息要少得多：拓扑异构酶III（Top3）。这部分是因为我们已经发现了可以增加Top1和Top2失败并造成DNA损伤的机会的化学物质，这使得在实验室中进行研究变得更容易。我们还没有发现任何化学物质对Top3有这种作用，所以我们不知道它参与了哪些基因组过程，它造成的DNA损伤的后果是什么，以及哪些其他特殊蛋白质修复这种DNA损伤。最近我找到了一种方法来稍微修改Top3，这样它就更容易失败并造成损害。在我的项目的第一部分，我将使用它来了解更多关于酵母细胞中Top3损伤和修复的信息。这将是非常有用的，因为它将告诉我们关于真核DNA损伤的一个未被探索的主题。它还将为未来在人类细胞中进行类似的研究奠定基础，这种类型的DNA损伤可能会对人类疾病产生影响。我最近还帮助开发了一种方法，使我们能够非常精确地找到拓扑异构酶切割DNA基因组的位置。最近，我用这个来找出Top2切割DNA的地方。在我的项目的第二部分，我将进一步开发该方法，使其适用于Top3，并使用它来找出Top3在基因组中活跃的确切时间和位置。这将告诉我们很多关于Top3参与的基因组过程的信息，这将对其他研究拓扑异构酶的研究人员以及基因组生物学的其他方面非常有用。