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.

Keywords: C-terminal domain; DNA binding; RNA; subnuclear localization; topoisomerase IIα.