Human Topoisomerase IIα Rapidly Relaxes Positively Supercoiled DNA

Abstract

Movement of the DNA replication machinery through the double helix induces acute positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single-and double-stranded DNA breaks, respectively, it has been assumed that type I enzymes relax the positive supercoils that precede the replication fork. Conversely, type II enzymes primarily resolve the precatenanes and untangle catenated daughter chromosomes. However, studies on yeast and bacteria suggest that type II topoisomerases may also function ahead of the replication machinery. If this is the case, then positive DNA supercoils should be the preferred relaxation substrate for topoisomerase II␣, the enzyme isoform involved in replicative processes in humans. Results indicate that human topoisomerase II␣ relaxes positively supercoiled plasmids >10-fold faster than negatively supercoiled molecules. In contrast, topoisomerase II␤, which is not required for DNA replication, displays no such preference. In addition to its high rates of relaxation, topoisomerase II␣ maintains lower levels of DNA cleavage complexes with positively supercoiled molecules. These properties suggest that human topoisomerase II␣ has the potential to alleviate torsional stress ahead of replication forks in an efficient and safe manner.

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

“…30 In addition, human topo IIα and β, which differ markedly in their C-terminal domains, show significant differences in their substrate preferences. 31 Furthermore, topo IIs from Chlorella virus (PBCV-1 and CVM-1), which naturally lack the C-terminal domain, show no preference for relaxing positively or negatively supercoiled substrates. 32 Since models for topology simplification depend on the selection of appropriate T segments, it is plausible that the phenomenon might be influenced by the presence of the C-terminal domain.…”

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.…”

“…They share a high degree of overall sequence homology with 68% identity and 86% similarity (15,16). So far, the only major in vitro difference between the two isoforms is a preferential relaxation of positive supercoils by the IIα isoform (17), whereas other basic catalytic aspects are very similar (18–21). Moreover, they have the same capacity for complementing essential topoisomerase II functions in temperature-sensitive Δtop2 yeast mutants (22,23).…”

The dynamic interplay between DNA topoisomerases and DNA topology

“…Both human Topo IIα and E coli Topo IV preferentially relax positive supercoils (Charvin et al 2003;Crisona et al 2000;McClendon et al 2005McClendon et al , 2008Neuman et al 2009;Seol et al 2013a;Stone et al 2003). The underlying mechanism for this chiral discrimination is embedded in differential interactions between positive and negative writhe and the C-terminal domains of the topoisomerases (Corbett et al 2005;McClendon et al 2008;Neuman et al 2009;Seol et al 2013a).…”