Skip to main content
. 2010 Apr 2;5(4):e9994. doi: 10.1371/journal.pone.0009994

Figure 2. Recombinant M2- and M3-APC each enhance topo IIα activity in vitro.

Figure 2

(A) Purified recombinant human topo IIα (0.12 µM) could slightly decatenate catenated DNA (catDNA) (lane 4). Addition of increasing amounts (0.12, 0.24, and 0.6 µM) of purified recombinant M2-APC (amino acid 1000–1326, lanes 5–7) or non-overlapping M3-APC (amino acid 1330–2058, lanes 8–10) resulted in progressively enhanced topo IIα DNA decatenation activity. M2-, or M3-APC (0.6 µM) alone did not display decatenation activity in the absence of topo IIα(lanes 2 and 3, respectively). (B) Using a higher concentration of topo IIα (0.18 µM) that displays slightly more activity in the absence of other proteins, the addition of M2- and M3-APC (0.18 µM) enhances topo IIα activity (lanes 7 and 8, respectively). In contrast, BSA (0.18 µM) did not enhance the DNA decatenation activity of topo IIα (lane 6). cat DNA, catenated kinetoplast DNA (kDNA); decat DNA, decatenated kDNA. (C) Purified recombinant human topo IIα (0.35 µM) could slightly relax supercoiled pBR322 plasmid DNA (lane 4). Addition of increasing amounts (0.35, 0.70, and 1.35 µM) of purified recombinant M2-APC (lanes 5–7) or M3-APC (lanes 8–10) resulted in progressively relaxed plasmids as indicated by slower migrating bands. M2-, or M3-APC (0.70 µM) did not display relaxation activity in the absence of topo IIα (lanes 2 and 3, respectively). (D) Under conditions where topo IIα displayed moderate plasmid relaxation activity, even in the absence of other proteins (lane 2), addition of BSA (0.35 µM) did not enhance this activity (lane 3), whereas addition of either M2- or M3-APC did (note reduction in faster migrating, highly supercoiled forms of DNA in lanes 5 and 7 compared to lane 2). (A–D) Representative assays from at least four independent experiments are shown.