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.

Topoisomerase IIα is an essential nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. The enzyme is a homodimer with a complex, multidomain structure. While the N‐terminal and core regions of the protein are well‐studied, the C‐terminal domain is poorly understood but appears to be involved in enzyme regulation. In addition, the C‐terminus varies widely among isoforms of topoisomerase II and appears to be a major region of post‐translational modification including several patches of Ser and Thr residues. Many of these Ser and Thr residues have not been studied for biochemical effects. Therefore, we generated a series of mutant versions of human topoisomerase IIα where we selectively changed patches of Ser and Thr residues in the C‐terminal domain to Ala and Gly residues. We designed, purified, and examined the activity of eleven mutant enzymes for changes in catalytic activity. The amino acid changes were made in the region of positions 1272 to 1525 with 1‐7 residues changed per mutant. Several mutants displayed varying 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 all had larger increases in DNA cleavage in the presence of etoposide when compared with wild‐type topoisomerase IIα. One mutant displayed decreased DNA cleavage below wild‐type levels. This mutant had changes between residues 1469‐1476 and displayed a decreased ability to both relax supercoiled DNA and to bind to plasmid DNA. Importantly, several residues in this region (Ser1469, Thr1470, Ser1471, and Ser1474) are known to have a role in mitosis indicating these are key regulators of enzyme function. Together, these results indicate that the C‐terminal domain of topoisomerase IIα influences catalytic activity and likely interacts with substrate DNA at various positions. Additional studies are warranted to explore these results in more detail.