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

地球上的生物将它们的遗传信息编码在长串状的DNA分子上。这些DNA“基因组”包含构建它们所在的有机体所有部分的指令。每个人类细胞都太小，肉眼看不见，但每个细胞都含有超过两米长的DNA，分为23对“染色体”。这种DNA以一种特殊的方式被包裹起来，这样它就可以装入细胞核(保存基因组的地方)。更糟糕的是，基因组并不是坐在那里无所事事。在整个细胞周期中需要发生许多动态过程，包括“转录”(读取遗传指令的一部分，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。想想解开耳机或仙女灯--然后倍增吧！如果你有一把神奇的剪刀，可以剪断电线并重新密封，而不会留下任何痕迹，事情就会容易得多。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拓扑异构酶是一种蛋白质，存在于地球上的每一个生物体中，从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特点：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工作，有时拓扑异构酶无法重新密封它所做的切割，这意味着我们只剩下附着在拓扑异构酶上的DNA断裂。这些DNA损伤的例子可以杀死细胞，或者导致突变，从而对生命的遗传指令产生负面影响。对人类来说，这可能会导致神经紊乱或癌症。幸运的是，还有其他特殊的蛋白质已经进化来修复这些拓扑异构酶连接的DNA断裂。人类和酵母是一个更广泛的被称为真核生物的相关生物家族的一部分。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，我们收集了许多关于其中两个：拓扑异构酶I(Top1)和拓扑异构酶II(TOP2)的信息，但关于第三个：拓扑异构酶III(Top3)的信息要少得多。这在一定程度上是因为我们已经发现了可以增加Top1和TOP2失败并造成DNA损伤的可能性的化学物质，这使得在实验室进行研究变得更容易。我们还没有发现任何对top3起作用的化学物质，所以我们不太清楚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了这种DNA损伤。最近我找到了一种略微修改Top3的方法，这样它就更有可能失败并造成损害。在我的项目的第一部分，我将用这个来找出更多关于酵母细胞TOP3损伤和修复的信息。这将是非常有用的，因为它将告诉我们一个未被探索的关于真核DNA损伤的话题。这也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损伤可能会导致人类疾病。我最近还帮助开发了一种方法，使我们能够非常准确地找出拓扑异构酶切割DNA基因组的位置。最近，我用这一点来找出TOP2在哪里切断了DNA。在我的项目的第二部分，我将进一步开发这种方法，使其适用于top3，并使用它来准确地找出top3在基因组中何时何地活跃。这将告诉我们许多Top3参与的基因组过程，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。

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.