Regulation of catalytic activity and nucleolar localization of rat DNA topoisomerase IIα through its C-terminal domain

Abstract

Type II DNA topoisomerase (topo II) catalyzes double-stranded DNA cleavage and re-ligation, thus solving problems in DNA topology. Vertebrates have two isozymes (α and β). Recently, the C-terminal regulatory domain (CRD), which regulates catalytic activity and subnuclear localization by associating with RNA, was identified within the C-terminal domain (CTD) of rat topo IIβ. In contrast, it is unclear whether a β CRD-like domain is present in the CTD of topo IIα. In this study, we aimed to identify an RNA-mediated regulatory domain in the rat topo IIα CTD. First, we exchanged the CTDs of rat topo IIα (amino acids 1,192–1,528) and β (1,201–1,614) and examined the two chimeras’ in vitro catalytic activities. Interestingly, the relaxation activities of topo IIα WT enzyme and both of the CTD-swapped mutants were inhibited in the presence of isolated cellular RNA, suggesting that the α CTD is involved in the RNA-mediated regulation of catalytic activity in topo IIα. The results of on-bead assays using a CTD-deleted mutant of rat topo IIα indicated that the RNA-mediated inhibition of the relaxation activity was caused by an interaction between the α CTD and RNA. Further, to identify the domain within the CTD that is associated with subnuclear localization of rat topo IIα, we transiently expressed EGFP-tagged CTD deletion mutants in human cells. The data indicated that the 1,192–1,289 region of rat topo IIα was required for targeting the enzyme to nucleoli. Finally, a relaxation assay using 1–1,289 and Δ1,192–1,289 truncated mutants indicated that the 1,192–1,289 region is involved in RNA-mediated inhibition. These results indicated that the CTD of rat topo IIα, containing the 1,192–1,289 region, is involved in the regulation of catalytic activity by associating with RNA, as well as in the localization to nucleoli in interphase cells.