DNA binding activity of the proximal C-terminal domain of rat DNA topoisomerase IIβ is involved in ICRF-193-induced closed-clamp formation.

Shinji Kawano,Kazushi Yasuda,Kunpei Fujimoto,Shogo Ikeda,Sergey Korolev

doi:10.1371/journal.pone.0239466

Abstract

DNA topoisomerase II (topo II) is an essential enzyme that regulates DNA topology by DNA cleavage and re-ligation. In vertebrates, there are two isozymes, α and β. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between α and β, is involved in each isozyme-specific intracellular behavior. The CTD of topo IIβ is supposedly involved in topo II regulation. Topo IIβ is maintained in an inactive state in the nucleoli by the binding of RNA to the 50-residue region termed C-terminal regulatory domain (CRD) present in the CTD. Although in vitro biochemical analysis indicates that the CTD of topo IIβ has DNA binding activity, it is unclear whether CTD influences catalytic reaction in the nucleoplasm. Here, we show that the proximal CTD (hereafter referred to as pCTD) of rat topo IIβ, including the CRD, is involved in the catalytic reaction in the nucleoplasm. We identified the pCTD as a domain with DNA binding activity by in vitro catenation assay and electrophoretic mobility shift assay. Fluorescence recovery after photo-bleaching (FRAP) analysis of pCTD-lacking mutant (ΔpCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo IIβ. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ΔpCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo IIβ to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.

Highlights

DNA topoisomerase II is an enzyme that catalyzes the cleavage and re-ligation of double-stranded DNA in the presence of ATP, thereby regulating DNA topology and resolving the DNA supercoiling that occurs during transcription, replication, and chromosome segregation [1]
We showed that the DNA binding activity of the C-terminal domain (CTD) of rat topo IIα is required for the efficient formation of DNA catenanes in vitro [22]
We hypothesized that the DNA binding activity of the CTD of rat topo IIβ could contribute to the efficient formation of DNA catenanes

Summary

Introduction

DNA topoisomerase II (topo II) is an enzyme that catalyzes the cleavage and re-ligation of double-stranded DNA in the presence of ATP, thereby regulating DNA topology and resolving the DNA supercoiling that occurs during transcription, replication, and chromosome segregation [1]. Topo II is conserved from prokaryotes to eukaryotes and consists of three structural domains: an N-terminal domain with ATPase activity (NTD), a catalytic core domain, and a C-terminal domain (CTD). Proximal C-terminal domain of topo IIβ assists catalytic reaction structures of the NTD and catalytic core domain of the topo II dimer excluding CTD have been revealed by x-ray crystallography [2,3,4]. A major role of the CTD in eukaryotic topo II is nuclear localization. Studies with CTD deletion mutants have shown that lack of CTD does not affect in vitro catalytic activity but lacks intracellular function [5,6,7]

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