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Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos
Published on: June 23, 2023
Dynamic behavior of DNA topoisomerase IIβ in response to DNA double-strand breaks
Keiko Morotomi-Yano1, Shinta Saito2, Noritaka Adachi2,3
1Department of Bioelectrics, Institute of Pulsed Power Science, Kumamoto University, Kumamoto, 860-8555, Japan.
Abstract:
DNA topoisomerase II (Topo II) is crucial for resolving topological problems of DNA and plays important roles in various cellular processes, such as replication, transcription, and chromosome segregation. Although DNA topology problems may also occur during DNA repair, the possible involvement of Topo II in this process remains to be fully investigated. Here, we show the dynamic behavior of human Topo IIβ in response to DNA double-strand breaks (DSBs), which is the most harmful form of DNA damage. Live cell imaging coupled with site-directed DSB induction by laser microirradiation demonstrated rapid recruitment of EGFP-tagged Topo IIβ to the DSB site. Detergent extraction followed by immunofluorescence showed the tight association of endogenous Topo IIβ with DSB sites. Photobleaching analysis revealed that Topo IIβ is highly mobile in the nucleus. The Topo II catalytic inhibitors ICRF-187 and ICRF-193 reduced the Topo IIβ mobility and thereby prevented Topo IIβ recruitment to DSBs. Furthermore, Topo IIβ knockout cells exhibited increased sensitivity to bleomycin and decreased DSB repair mediated by homologous recombination (HR), implicating the role of Topo IIβ in HR-mediated DSB repair. Taken together, these results highlight a novel aspect of Topo IIβ functions in the cellular response to DSBs.
Insights
DNA topoisomerase IIβ (Topo IIβ) dynamically responds to DNA double-strand breaks (DSBs), recruiting to damage sites and influencing homologous recombination repair. Inhibiting Topo IIβ impairs this recruitment and DNA repair efficiency.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA topoisomerase II (Topo II) resolves DNA topological issues during replication, transcription, and segregation.
- The role of Topo II in DNA repair, particularly in response to DNA double-strand breaks (DSBs), is not fully understood.
Purpose of the Study:
- To investigate the dynamic behavior and function of human Topo IIβ at DNA double-strand break sites.
- To determine the involvement of Topo IIβ in DNA double-strand break repair pathways.
Main Methods:
- Live cell imaging with laser microirradiation to induce site-specific DSBs.
- Immunofluorescence and detergent extraction to assess endogenous Topo IIβ association with DSBs.
- Photobleaching analysis to evaluate Topo IIβ mobility.
- Assessment of DSB repair in Topo IIβ knockout cells using DNA damaging agents and homologous recombination assays.
Main Results:
- EGFP-tagged Topo IIβ rapidly recruited to laser-induced DSBs.
- Endogenous Topo IIβ tightly associated with DSB sites.
- Topo IIβ exhibited high mobility in the nucleus, which was reduced by catalytic inhibitors (ICRF-187, ICRF-193), preventing recruitment to DSBs.
- Topo IIβ knockout cells showed increased sensitivity to bleomycin and impaired homologous recombination (HR) repair of DSBs.
Conclusions:
- Human Topo IIβ dynamically participates in the cellular response to DNA double-strand breaks.
- Topo IIβ plays a crucial role in homologous recombination-mediated DNA double-strand break repair.
- These findings reveal a novel function of Topo IIβ in DNA damage response pathways.
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