Disease-associated H58Y mutation affects the nuclear dynamics of human DNA topoisomerase IIβ

Abstract

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems and plays critical roles in various nuclear processes. Recently, a heterozygous H58Y substitution in the ATPase domain of human TOP2B was identified from patients with autism spectrum disorder, but its biological significance remains unclear. In this study, we analyzed the nuclear dynamics of TOP2B with H58Y (TOP2B H58Y). Although wild-type TOP2B was highly mobile in the nucleus of a living cell, the nuclear mobility of TOP2B H58Y was markedly reduced, suggesting that the impact of H58Y manifests as low protein mobility. We found that TOP2B H58Y is insensitive to ICRF-187, a TOP2 inhibitor that halts TOP2 as a closed clamp on DNA. When the ATPase activity of TOP2B was compromised, the nuclear mobility of TOP2B H58Y was restored to wild-type levels, indicating the contribution of the ATPase activity to the low nuclear mobility. Analysis of genome-edited cells harboring TOP2B H58Y showed that TOP2B H58Y retains sensitivity to the TOP2 poison etoposide, implying that TOP2B H58Y can undergo at least a part of its catalytic reactions. Collectively, TOP2 H58Y represents a unique example of the relationship between a disease-associated mutation and perturbed protein dynamics.

Introduction

DNA topoisomerase II (TOP2) is an ATP-dependent homodimeric enzyme that resolves DNA topological problems, such as torsional strain arising during transcription and DNA replication^1,2. To relieve DNA topological problems, TOP2 utilizes a mechanism of DNA strand passage through a DNA double-strand break: TOP2 binds to two segments of DNA, introduces a double-strand break in one DNA segment, translocates the second DNA segment through a DNA break, and ligates the cleaved DNA^3,4. A sequence of these reactions comprises a catalytic cycle, and various compounds have been developed to target specific steps of the cycle. TOP2 transiently forms a covalent complex with DNA ends during the catalytic cycle⁴. TOP2 poisons, such as etoposide, stabilize the covalent TOP2–DNA complex, which is readily converted into a DNA double-strand break in cells^4,5. Thus, TOP2 poisons exhibit anti-proliferating activity and are used for cancer therapy. TOP2 catalytic inhibitors, such as ICRF-187, have a different mechanism of action: they trap TOP2 into a non-covalent complex with DNA, which is called a closed clamp^4,6,7.

Eukaryotic TOP2 proteins have evolutionarily conserved domain structures: they consist of the N-terminal ATPase domain, the central catalytic core domain, and the C-terminal region^8,9,10 (Fig. 1a). Humans and other vertebrates possess two TOP2, termed TOP2A and TOP2B^8,11. The ATPase and core domains of human TOP2A and TOP2B share high sequence similarity, and the C-terminal regions are less conserved^9,10,12,13. Although their catalytic properties in vitro are nearly indistinguishable^14,15, TOP2A and TOP2B differentially participate in cellular functions^1,2. TOP2A is expressed in proliferating cells with peak expression at G2/M of the cell cycle^16,17. TOP2A plays critical roles in DNA replication and chromosome condensation/segregation and thus is essential for cell division^18,19. TOP2B is expressed throughout the cell cycle and is present in both proliferating and non-dividing cells¹⁷. Although TOP2B is dispensable for cell proliferation, its physiological role is particularly evident in differentiated, non-dividing cells, such as neurons^20,21. TOP2B is involved in the transcription of a distinct set of genes required for neural development^22,23,24. Knockout of the TOP2B gene in mice causes various defects in neural development, leading to breathing impairment and consequent neonatal death immediately after birth²⁰.

Although the complete loss of TOP2B functions leads to neonatal death in mice, individuals with partially dysfunctional TOP2B may remain viable. In general, perturbation of a particular activity of a multifunctional protein is more common than gross defects in its overall functions among human diseases caused by genetic disorders^25,26,27. As for TOP2B, a number of germline missense mutations were identified as causative for human diseases. In the catalytic domain of TOP2B, the missense mutations, S483L, A485P, EE587E, and G633S, were identified in patients with B-cell deficiency^28,29,30. Subsequent studies have demonstrated that these missense mutations in the catalytic domain lead to a significant decrease in the catalytic activity of TOP2B^28,29.

Another pathogenic germline mutation was reported in the N-terminal ATPase domain of human TOP2B. H58Y was a heterozygous de novo mutation identified in patients with autism spectrum disorder and global developmental delay^31,32. The patients carrying H58Y did not show any signs of B-cell deficiency, suggesting that the pathogenic impact of H58Y is different from those of the missense mutations in the central catalytic domains. H58Y is located in the N-terminal end of TOP2B and appears to have a relatively small impact on the TOP2B structure (Supplementary Fig. S1). We therefore imagined that H58Y exerts its deleterious effect on TOP2B functions other than the catalytic activity.

Previous studies have demonstrated that TOP2B is highly mobile in the nucleus of a living cell^{33,34,35,36,37}. It is widely accepted that nuclear proteins, except for core histones, are highly mobile^38,39,40,41. Dynamic nature is a common feature of most nuclear proteins, regardless of their biochemical properties or functional roles³⁹. The vast majority of molecules of nuclear proteins rapidly move by stochastic diffusion. Only a small fraction of the molecules transiently reside in their sites of action, typically on the order of seconds^38,39,40,42. For instance, more than 95% of molecules of transcription factors in the nucleus are estimated to be in diffusion or in transient nonspecific association with chromatin^40,42. High mobility is considered to serve as a part of the mechanism for targeting proteins to appropriate sites and is also inferred to ensure the regulatory plasticity of various nuclear functions^40,41,43. Importantly, alterations in the nuclear dynamics often reflect changes in the functional status of proteins^44,45,46,47. In the case of TOP2B, its nuclear behavior readily alters in response to various cues, such as reduced ATP levels, DNA damage, and TOP2 inhibitors^35,37, suggesting that the nuclear behavior of TOP2B reflects, at least in part, the status of TOP2B in living cells. In this viewpoint, we sought to examine whether the impact of H58Y may be reflected in the TOP2B dynamics. We found that H58Y confers a remarkable reduction in TOP2B mobility, representing an intriguing instance of the relationship between a disease-associated mutation and perturbed protein dynamics in living cells.

Results

Reduced nuclear mobility of TOP2B H58Y in living cells

High mobility is a general feature of many nonhistone proteins^38,39,40,41, and human TOP2B is also highly mobile in the nucleus^{33,34,35,36,37}. We therefore investigated whether the H58Y substitution may affect the nuclear dynamics of TOP2B. Using HeLa cells transiently expressing EGFP-TOP2B, we performed FRAP analysis: green fluorescence in a small area in the nucleus was photobleached, and the fluorescence recovery was monitored over time. As shown in Fig. 2a, we observed the fast recovery of the fluorescence of wild-type TOP2B tagged with EGFP (referred to hereafter as EGFP-TOP2B WT) after photobleaching. This result confirmed that EGFP-TOP2B WT is highly mobile as reported previously^{33,34,35,36,37}. Next, we carried out FRAP analysis on EGFP-TOP2B with the H58Y substitution (hereafter, EGFP-TOP2B H58Y). We observed that the fluorescence recovery was slow and partial as compared to EGFP-TOP2B WT (Fig. 2b). Quantification of fluorescence highlighted the marked difference between EGFP-TOP2B WT and H58Y (Fig. 2c). When compared at 30 s after photobleaching, the recovery of EGFP-TOP2B WT fluorescence reached approximately 90%, but that of EGFP-TOP2B H58Y was estimated to be less than 50% (Fig. 2c). These observations demonstrate that H58Y impacts the nuclear mobility of TOP2B in living cells.