Probing the Impact of Serine and Threonine Patches in the C‐terminus on the Catalytic of Human Topoisomerase IIα

Abstract

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.