The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin.

Melissa Antoniou-Kourounioti,Christine Farr,Andrew Porter,Michael Mimmack

doi:10.3390/ijms20051238

Abstract

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme’s residence time on mitotic chromatin, as well as its subcellular localisation.

Highlights

Type II Topoisomerases (TOP2) can pass one segment of DNA duplex through a transient double-strand break in a second segment in an ATP-dependent catalytic process
Clarke and colleagues demonstrated that the most distal 31 amino acids, as well as encompassing the main nuclear localisation signal (NLS), are crucial for localisation to mitotic chromatin. They designated this component the chromatin tether (ChT). They concluded that, while important, the chromatin tether domain (ChT) does not function in isolation and that other parts of the C-Terminal Domain (CTD) contribute to the protein’s robust localisation to mitotic chromosomes [28]
Condensin II loads onto mitotic chromatin during prophase, while condensin I, which is cytoplasmic during interphase, only gains access after nuclear envelope breakdown (NEBD) [71]

Summary

Introduction

Type II Topoisomerases (TOP2) can pass one segment of DNA duplex through a transient double-strand break in a second segment in an ATP-dependent catalytic process. In eukaryotes the functional enzyme is formed through dimerization of TOP2 monomers, giving a structure that has three protein interfaces: the N-terminal ATPase gate (consisting of the ATPase and transducer domains); the DNA-binding gate (consisting of the TOPRIM domain, the Winged Helix Domain (with the active site tyrosine, Y805) and the Tower domain); and the C-gate (formed by the coiled-coil domain) [2,3,4] The structure of these parts of the TOP2 dimer has been extensively investigated through X-ray crystallography [5,6,7]. The eukaryotic CTD has not been crystallised and appears to be structurally disordered

Methods

Results

Conclusion