Ability of Viral Topoisomerase II To Discern the Handedness of Supercoiled DNA:  Bimodal Recognition of DNA Geometry by Type II Enzymes

Abstract

Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIα and Eschericia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIα and β to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II, but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under-and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domains during DNA cleavage.Although the classic structure of the Watson-Crick double helix is free from either torsional or axial stress, DNA in living systems is subject to these topological challenges. Globally, the DNA of eukaryotes and eubacteria is maintained in an underwound (i.e., negatively supercoiled) state (1-4). This underwinding makes it easier to separate the two strands of the genetic material, and thereby facilitates critical processes such as DNA replication and transcription. In contrast, the actions of DNA tracking systems overwind (i.e, positively supercoil) the genetic material immediately preceding replication forks and transcription complexes (1,(3)(4)(5). If this overwinding is not alleviated, the ensuing torsional stress rapidly halts the movement of tracking systems along the double helix (1,3,5-7).

Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

“…Consistently, in yeast, Top1 deficiency increases negative supercoiling without producing detectable changes in nucleosomal spacing (37). The increase in Top2a in both siTop1 cell lines might correspond to a compensatory mechanism for Top1 deficiency as Top2a has recently been shown to relax positive supercoiling preferentially, whereas Top2h and Top3 are equally effective at removing positive and negative supercoiling (38). However, the compensatory increase of Top2a activity might contribute, together with the Top1 deficiency, to the chromosomal rearrangements observed in the siTop1 cells because Top2 can promote chromosomal translocations (39).…”

C-Terminal regions of topoisomerase II and II determine isoform-specific functioning of the enzymes in vivo

“…Recent work in yeast demonstrates that topoisomerase II relaxes chromatin even more efficiently than topoisomerase I (71). In mammals, topoisomerase IIα, but not IIβ, appears to be a key player in removal of this type of torsional stress during replication (17), and it was postulated that this isoform-specificity is determined by the divergent C-terminal regions (72). The residues that were suggested to play this role in replication are all within the α CTRs analyzed here.…”

DNA topoisomerase II, genotoxicity, and cancer

“…For many years, the C-terminal domain was thought to contribute little to the enzymatic activity of any type II topoisomerase. However, several recent studies suggest that this portion of the protein plays an intriguing and important role in the recognition of DNA geometry [59][60][61][62][63][64][65]. As such, it may impart unique attributes, such as the ability to supercoil DNA [59][60][61] or act in front of replication forks [62,64], to specific type II enzymes.…”

“…Other studies have utilized linear nucleic acid substrates, especially for mapping sites of drug-induced DNA scission. Recent reports have begun to describe interactions between type II topoisomerases and positively supercoiled substrates [61,62,64,65,187,188]. Several unexpected findings have emerged from these experiments.…”

“…In contrast, topoisomerase IIβ, which is not believed to play a role in DNA replication, relaxes positive and negative superhelical twists at similar rates [62]. On the basis of amino acid sequence comparisons between the two human topoisomerase II isoforms, as well as studies on bacterial and viral type II enzymes [59][60][61]65], it has been proposed that the ability to discern the geometry of DNA supercoils during relaxation resides in the C-terminal domain of human topoisomerase IIα.…”