Exploration of the Role of the C-Terminal Domain of Human DNA Topoisomerase IIα in Catalytic Activity

[Image: see text] Human topoisomerase IIα (TOP2A) is a vital nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. TOP2A is a homodimer with a symmetrical, multidomain structure. While the N-terminal and core regions of the protein are well-studied, the C-terminal domain is poorly understood but is involved in enzyme regulation and is predicted to be intrinsically disordered. In addition, it appears to be a major region of post-translational modification and includes several Ser and Thr residues, many of which have not been studied for biochemical effects. Therefore, we generated a series of human TOP2A mutants where we changed specific Ser and Thr residues in the C-terminal domain to Ala, Gly, or Ile residues. We designed, purified, and examined 11 mutant TOP2A enzymes. The amino acid changes were made between positions 1272 and 1525 with 1–7 residues changed per mutant. Several mutants displayed increased levels of DNA cleavage without displaying any change in plasmid DNA relaxation or DNA binding. For example, mutations in the regions 1272–1279, 1324–1343, 1351–1365, and 1374–1377 produced 2–3 times more DNA cleavage in the presence of etoposide than wild-type TOP2A. Further, several mutants displayed changes in relaxation and/or decatenation activity. Together, these results support previous findings that the C-terminal domain of TOP2A influences catalytic activity and interacts with the substrate DNA. Furthermore, we hypothesize that it may be possible to regulate the enzyme by targeting positions in the C-terminal domain. Because the C-terminal domain differs between the two human TOP2 isoforms, this strategy may provide a means for selectively targeting TOP2A for therapeutic inhibition. Additional studies are warranted to explore these results in more detail.