DNA-Stimulated Liquid-Liquid phase separation by eukaryotic topoisomerase ii modulates catalytic function
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eLife assessment
Type II topoisomerases are essential players in virtually every aspect of genome organization and function of all organisms. The in vitro data presented here clearly demonstrate that eukaryotic type II topoisomerases phase separate under physiological conditions, forming liquid-liquid condensates, and that the outcomes of type topoisomerase II activity on DNA are altered in these condensates. The experiments and methods are sound, clearly described, and fully support the insightful and carefully formulated interpretation of the data. This work has broad implications for dissecting and delineating the myriad fundamental roles of this centrally important molecule.
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Abstract
Type II topoisomerases modulate chromosome supercoiling, condensation, and catenation by moving one double-stranded DNA segment through a transient break in a second duplex. How DNA strands are chosen and selectively passed to yield appropriate topological outcomes – for example, decatenation vs. catenation – is poorly understood. Here, we show that at physiological enzyme concentrations, eukaryotic type IIA topoisomerases (topo IIs) readily coalesce into condensed bodies. DNA stimulates condensation and fluidizes these assemblies to impart liquid-like behavior. Condensation induces both budding yeast and human topo IIs to switch from DNA unlinking to active DNA catenation, and depends on an unstructured C-terminal region, the loss of which leads to high levels of knotting and reduced catenation. Our findings establish that local protein concentration and phase separation can regulate how topo II creates or dissolves DNA links, behaviors that can account for the varied roles of the enzyme in supporting transcription, replication, and chromosome compaction.
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eLife assessment
Type II topoisomerases are essential players in virtually every aspect of genome organization and function of all organisms. The in vitro data presented here clearly demonstrate that eukaryotic type II topoisomerases phase separate under physiological conditions, forming liquid-liquid condensates, and that the outcomes of type topoisomerase II activity on DNA are altered in these condensates. The experiments and methods are sound, clearly described, and fully support the insightful and carefully formulated interpretation of the data. This work has broad implications for dissecting and delineating the myriad fundamental roles of this centrally important molecule.
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Reviewer #1 (Public Review):
Jeong and coauthors demonstrate that eukaryotic type II topoisomerases undergo liquid-liquid phase separation (LLPS) under physiological conditions, and that the outcome of type II topoisomerase activity on supercoiled plasmid DNA is altered within condensates. The authors used budding yeast (Saccharomyces cerevisiae) topoisomerase II (scTopoII) to demonstrate LLPS and explore the dependence of LLPS on protein concentration, DNA concentration, and both the presence and phosphorylation sate of the unstructured C-terminal domains (CTDs) of scTopoII. Crucially, the authors verify the fluid-like behavior of the condensates, confirming coalescence of drops directly, and establishing exchange between condensed droplets and the aqueous phase via FRAP experiments. The condensates form under nominally physiological …
Reviewer #1 (Public Review):
Jeong and coauthors demonstrate that eukaryotic type II topoisomerases undergo liquid-liquid phase separation (LLPS) under physiological conditions, and that the outcome of type II topoisomerase activity on supercoiled plasmid DNA is altered within condensates. The authors used budding yeast (Saccharomyces cerevisiae) topoisomerase II (scTopoII) to demonstrate LLPS and explore the dependence of LLPS on protein concentration, DNA concentration, and both the presence and phosphorylation sate of the unstructured C-terminal domains (CTDs) of scTopoII. Crucially, the authors verify the fluid-like behavior of the condensates, confirming coalescence of drops directly, and establishing exchange between condensed droplets and the aqueous phase via FRAP experiments. The condensates form under nominally physiological conditions, but the critical concentration decreases significantly when DNA exceeding 100 base pairs is included. As expected, the condensates can be solubilized with increasing salt or DNA concentrations. Based on established phase condensation prediction algorithms, the authors identify the CTDs of the yeast and two human isoforms of topo II as the most likely protein elements driving LLPS. They expand on this prediction by performing a useful alignment of several representative eukaryotic topo II enzymes, which reveals low homology but conservation of disorder and high frequencies of charged amino acids, both of which contribute to LLPS. The authors confirm the importance of the CTDs in LLPS by demonstrating that isolated CTDs can form condensates under a more limited set of conditions than the WT protein, whereas removing the CTDs from scTopoII inhibited LLPS altogether. In contrast, phosphorylation of the CTDs altered the biophysical properties of the condensates (fluidity for example) but not affect the propensity to form condensates. By employing a 2.9 kb negatively supercoiled DNA as the condensation scaffold and adding ATP to the condensates, the authors could measure the effects of LLPS on topo II activity. They demonstrate convincingly that topo II activity is driven towards catenation of circular DNA in condensates with full length topo II and interestingly towards the formation of knotted substrates when comparable concentrations of scTopoII lacking the CTDs was used. The authors round our this elegant work by comparing the results obtained with scTopoII with the two human isoforms hsTopoIIα and hsTopoIIβ. Together these results indicate that eukaryotic topo II enzymes can phase separate with DNA under physiological conditions and that this process can change the outcome of the strand passage reaction catalyzed by the type II enzymes. These findings help explain previous results demonstrating linking and knotting of closed circular DNA by high concentrations of type II topoisomerases in vitro, and may help unravel the roles of these enzymes in both promoting and resolving chromosome entanglements in vivo.
The main thing that others may criticize is the lack of the demonstration of LLPS and its role in vivo, but I think their findings, especially the different activities under LLPS permissive and inhibitory conditions, stand on their own.
The experiments are clear and compelling and the results support the conclusions of the study. The finding of different morphological states with plasmid DNA under some conditions is interesting and should be more fully investigated to understand the nature of this different structure that may be more relevant in vivo than the more conventional condensates observed with short DNA substrates.
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Reviewer #2 (Public Review):
Despite the long history of the study of topo II, the role of its long CTD in vitro and in vivo has remained poorly understood. The current manuscript provides solid lines of evidence that the intrinsically disordered CTD modulates topo II's enzymatic activities through LLPS. The experiments reported here were properly performed, and the conclusions are largely supported by the data presented, thereby making them an excellent contribution to the field. The current manuscript contains some weaknesses, though. The phosphatase treatment experiments are weak (Figure 4), and the role of phosphorylation on topo II-mediated LLPS remains unclear. The experiments using human topo IIs are also weak (Figure 6): the potential differences between topo IIa and topo IIb have not been rigorously tested or properly …
Reviewer #2 (Public Review):
Despite the long history of the study of topo II, the role of its long CTD in vitro and in vivo has remained poorly understood. The current manuscript provides solid lines of evidence that the intrinsically disordered CTD modulates topo II's enzymatic activities through LLPS. The experiments reported here were properly performed, and the conclusions are largely supported by the data presented, thereby making them an excellent contribution to the field. The current manuscript contains some weaknesses, though. The phosphatase treatment experiments are weak (Figure 4), and the role of phosphorylation on topo II-mediated LLPS remains unclear. The experiments using human topo IIs are also weak (Figure 6): the potential differences between topo IIa and topo IIb have not been rigorously tested or properly discussed. Most importantly, the difference in the catalytic mode between the full-length and CTD-lacking topo II needs to be tested and described more convincingly along with quantitative data (Figure 5).
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Reviewer #3 (Public Review):
This is a remarkable paper which was a pleasure to read. It documents the ability of Type II Topoisomerases of yeast and human to undergo liquid-liquid phase separation, describes the basis for this process in protein structure, and reveals its modulation by DNA and post-translational modifications. Each finding is supported by rigorous, well-controlled and carefully executed and interpreted experiments. The conclusions are clear and unavoidable. The Discussion presents knowledgeable evaluations both of the mechanistic bases for the observed effects and the likely general (and some specific) implications of the findings for context-specific moduation of Topoisomerase II activity. I have no suggestions for improvement. This paper is a classic in this already sophisticated field. The authors present important …
Reviewer #3 (Public Review):
This is a remarkable paper which was a pleasure to read. It documents the ability of Type II Topoisomerases of yeast and human to undergo liquid-liquid phase separation, describes the basis for this process in protein structure, and reveals its modulation by DNA and post-translational modifications. Each finding is supported by rigorous, well-controlled and carefully executed and interpreted experiments. The conclusions are clear and unavoidable. The Discussion presents knowledgeable evaluations both of the mechanistic bases for the observed effects and the likely general (and some specific) implications of the findings for context-specific moduation of Topoisomerase II activity. I have no suggestions for improvement. This paper is a classic in this already sophisticated field. The authors present important new and interesting observations while, at the same time, providing the general reader with a beautiful, well-referenced overview of the intricacies of Type II Topoisomerases.
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