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

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.

DNA topoisomerases are a double-edged sword. They facilitate topologically constrained genome processes via the transient formation of DNA strand breaks, but these covalent complex (CC) intermediates can become stabilised as permanent lesions that disrupt nuclear metabolism and cause genome instability. Significant research has focussed on eukaryotic Top1 and Top2, driven in part by the availability of small molecule poisons that trap their CCs. By comparison the function of Top3 has been comparatively obscured, and its contribution as an endogenous DNA damage source is unclear. Based on homology to bacterial type IA topoisomerases, I have identified "self-poisoning" mutations that trap eukaryotic Top3 CCs. To build a comprehensive picture of the cellular consequences of Top3-induced DNA, I will express these mutants in budding yeast, and analyse cellular Top3 CC levels, DNA damage signalling, cell cycle kinetics, cell growth/survival, and more. To reveal the genetics governing repair, I will compare phenotypes of mre11-, sae2-, tdp1-, and wss1- mutant strains, which are defective in the repair of other types of protein-linked breaks. To discover when and where Top3 acts in eukaryotes, and how this is influenced by intrinsic genome organisation, I will adapt the CC-seq methodology, which I recently developed, for the detection of Top3 CCs. I will use this technique to generate genome-wide maps of Top3 activity at different stages of the budding yeast cell cycle, with strand-specific nucleotide-resolution. I will draw mechanistic insights by correlating my data with publicly-available maps of genome features, and probe the involvement of Top3 in specific genome processes (DNA transcription, replication, homologous recombination and chromosome segregation) using chemical and genetic perturbation, and site-specific tools. This study will have a broad and timely impact, and be of great value to researchers investigating genome structure, function and stability.