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

Abstract

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.

Figure 1

Construct map for the pESC-URA hTOP2A plasmid. The map displays key features of the construct generated by GenScript. The map produced using SnapGene.

Figure 2

Location of mutants in the CTD of human TOP2A. Residues 1210–1531 are shown. Bars represent the region modified for each mutant. See Table 1 for specific amino acid changes made in each mutant. Positions shown in blue represent amino acids that are modified in association with mitosis according to the Phosphosite database (phosphosite.org). Positions in orange represent sites known to be involved in the stability of the protein.

Figure 3

Relaxation of plasmid DNA by human WT TOP2A and mutant TOP2A from the pESC-URA hTOP2A construct. Gel images show the migration of supercoiled plasmid (SC) in the absence of topoisomerase II (DNA) as well as in the presence of TOP2A at increasing time points (0.5–15 min on the left; 1–30 min on the right). The relaxed plasmid (Rel) migrates more slowly than SC plasmid. Wild type (WT) is shown at the top left followed by mutants (Mut) 1, 7, and 10. Mutants 4 and 9 are shown at the right on a 30 min time scale. Gel images are representative of four or more experiments.

Figure 4

Impact of mutations in the CTD on DNA cleavage and coordination of cleavage. Upper panel: Percent DNA cleavage is shown for reactions with WT or mutant hTOP2A and the plasmid DNA. Lower panel: DSB/SSB ratios were generated by taking the ratio of DNA in the linear/DSB band and dividing by the DNA in the nicked/SSB band. Error bars represent the standard deviation of the mean for three or more experiments. Asterisks (*) represent statistical significance: *p = 0.0127 when compared to WT.

Figure 5

Enhancement of TOP2-mediated DNA cleavage in the presence of the etoposide. Percent of DS-cleaved DNA in the presence of WT or mutant TOP2 with the100 μM (dark red) or 200 μM (blue) etoposide. Error bars represent the standard deviation of the mean for three or more experiments. Statistical significance was determined by analysis of variance (ANOVA) followed by multiple comparisons of means (*p = 0.0186; **p = 0.0061; ****p < 0.0001).

Figure 6

Plasmid DNA binding by TOP2A. DNA binding measured by the shifting of the SC DNA band in the presence of increasing concentrations of WT or mutant TOP2A. Left, a representative gel of WT TOP2A binding is shown. The unbound DNA is primarily in the supercoiled (SC) band, while the bound DNA shifts upward at increasing concentrations of TOP2A. At the highest concentrations, most of the DNA is retained in the origin of the gel. Right, quantification of the supercoiled DNA band for each mutant is shown. Error bars represent the standard deviation of the mean for three or more experiments.

Figure 7

Decatenation of kDNA. Decatenation of catenated kinetoplast DNA (kDNA) circles by wild-type (WT) TOP2A and TOP2A Mutants 1, 2, 3, 4, 7, and 9 is shown. Error bars represent the standard deviation of four or more independent experiments.

Figure 8

Sequence alignment of CTD of TOP2A and TOP2B showing locations of CTD TOP2A mutants and sites of consensus. Alignment generated from TOP2A sequence NP_001058.2 and TOP2B sequence NP_001059.2 using SnapGene (Needleman–Wunsch global alignment).