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.
Highlights
In mammals, there are two topoisomerase II (Topo II) isozymes, Topo IIα and Topo IIβ, which share striking sequence homology with one another in their N-terminal ATPase and central catalytic domains but differ in their C-terminal domains[7]
Laser-irradiated cells were subjected to fixation followed by immunostaining for endogenous Topo IIβ and phosphorylated DNA-PKcs, which served as a marker for induced DNA damage[30]
In contrast to the increasing knowledge regarding the biological and clinical relevance of Topo IIβ, limited information is available on the dynamic behavior of Topo IIβ, in relation to double-strand breaks (DSBs) induction
Summary
There are two Topo II isozymes, Topo IIα and Topo IIβ, which share striking sequence homology with one another in their N-terminal ATPase and central catalytic domains but differ in their C-terminal domains[7]. Previous studies have demonstrated that Topo II participates in various cellular processes associated with DNA topology problems, such as replication and mitosis, limited information is available regarding the involvement of Topo II in DSB repair, which may require the control of DNA topology. Topo IIβ recruitment to DSB sites was partially abrogated by inhibitors for PARP-1 and HDAC, but, contrary to our assumption, it did not require DNA-PKcs or ATM. Topo IIβ knockout cells exhibited increased sensitivity to bleomycin and reduced HR activity These observations provide novel insight into the participation of Topo IIβ in the cellular response to DSB induction
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