Mutagenesis of Intrinsically Disordered Domain Impacts Topoisomerase IIα Catalytic Activity

TL;DR

This study shows that changing specific amino acids in a disordered region of topoisomerase IIα can affect its DNA processing abilities, potentially guiding drug development.

Contribution

The study demonstrates that specific mutations in the C-terminal domain of TOP2A can selectively impact its catalytic functions.

Findings

The V1482D mutation increased DNA relaxation and decatenation activity.

The K1520I mutation also increased decatenation activity.

Mutations did not significantly affect DNA cleavage or binding.

Abstract

Human topoisomerase IIα and IIβ regulate DNA topology and knots in chromosomes during crucial cellular processes, making these enzymes common targets for anticancer drugs. However, selective inhibition of topoisomerase IIα (TOP2A) is desired to decrease adverse effects, which may be mediated by topoisomerase IIβ (TOP2B). The main region of difference between the two isoforms is the intrinsically disordered C-terminal domain (CTD), which is being studied as a target for selective inhibition. Our previous work examined several regions within the CTD to determine whether those regions impact biochemical function. In this current study, we designed and constructed four TOP2A mutants with amino acid substitutions in the CTD, which were then assessed for impact on biochemical activity. V1482D exhibited increased levels of relaxation, while both V1482D and K1520I exhibited increased levels of decatenation. No major impact on DNA cleavage or binding were observed with any of the mutants. The isolated impact of the changes on relaxation and decatenation supports the concept that the CTD can affect one aspect of the enzyme’s function in an isolated manner, which was seen in our previous study. Taken together, these results suggest that modification of specific positions within the CTD affects substrate selection. These results are mapped onto the CTD for consideration of potential regions to target for inhibition of TOP2A.

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