Figure 5.

Suppression of Topo IIβ recruitment to DSB sites by ICRF-187 and ICRF-193. (A) Immunofluorescent staining of endogenous Topo IIβ and phosphorylated DNA-PKcs in the presence and absence of Topo II inhibitors. HeLa cells were pretreated with DMSO, 20 µM ICRF-187, 20 µM ICRF-193, or 40 µM merbarone for 1 h and subsequently subjected to laser microirradiation. After 10 min incubation at 37 °C, cells were coimmunostained for Topo IIβ and pS2056 of DNA-PKcs. Representative images are shown. (B) Schematic diagram of domains and amino acid substitutions in human Topo IIβ. Topo IIβ is composed of an ATPase domain, a catalytic core domain, and a C-terminal domain (CTD). The amino acid substitutions G180I and L185F are considered to confer insensitivity of Topo IIβ to the ICRF inhibitors. The Y821S substitution is inferred to impair the topoisomerase activity. (C) Recruitment of EGFP-Topo IIβ mutants to DSB sites in the presence of ICRF-187. EGFP-Topo IIβ mutants were transiently expressed in HeLa cells. After pretreatment with DMSO or ICRF-187 for 1 h, cells were subjected to laser microirradiation followed by live cell imaging. Representative images at 30 sec after DSB induction are shown.