Nucleic Acids Research, 2010, Vol. 38, No. 22 8223 Figure 2. Effects of Rv1495 on topoisomerase activities of MtbTopA. Topoisomerase activity assays were performed as described under ‘Materials and Methods’ section. The plasmid pBluscript was used as DNA substrate in all reaction mixtures. The reaction was treated with 6% SDS and 4 mg/ ml proteinase K analyzed on a 0.8% agarose gel and then stained with ethidium bromide. Lanes 1, no topoisomerase as negative control. L, linearized plasmid; R, relaxed plasmid; S, supercoiled plasmid. (A) Different amounts of topoisomerase I (0–0.15 mM) were added to 200 ng of supercoiled pBluscript DNA. (B) Effects of different amounts of Rv1495 (0–6 mM) on the topoisomerase I (0.125 mM) activities on the supercoiled pBluscript DNA. (C) Effects of different amounts of heated-Rv1495 on the topoisomerase I activities. (D) Effects of different amounts of GST on the topoisomerase I activities. (E) EMSA assays for the plasmid DNA-binding activities of RV1495, Lsr2, or Rv0054. Lsr2 is a positive control. Rv0054 (MtbSSB) is a negative control. the inhibition of MtbTopA by Rv1495 was further analyzed by exploring the cleavage activity of MtbTopA. As shown in Figure 3A, a DNA-binding activity on the 32-mer single-stranded DNA substrate was demonstrated with a positive control, Rv0054 (MtbSSB) (Figure 3A, right panel, lanes 7–8). No DNA-binding activity was seen with Rv1495, even at a high protein concentration (0.96 mM) (Figure 3A, right panel, lane 2–5). However, a substantial shifted protein–DNA complex band, indicative of DNA-binding, was observed on the gel when an increasing concentration of MtbTopA (0.06–0.24 mM) was added to the reaction mixture (Figure 3A, left panel). This conﬁrmed that the MtbTopA had a good DNA-binding activity with this substrate. A complicated effect was observed with Rv1495 on the DNA-binding activity of MtbTopA (Supplementary Figure S3). The cleavage activity of MtbTopA was further characterized. As shown in Figure 3B (left panel), a stepwise increase in amounts of smaller DNA fragment (19-mer) was observed as 0.1–0.4 mM MtbTopA was added to the reaction mixture. When the concentration of MtbTopA was kept constant, the cleavage products steadily decreased in response to addition of increasing amounts of Rv1495 (3–9 mM) (Figure 3B, right panel), which indicated that Rv1495 inhibited the cleavage activity of MtbTopA. No effect was observed when either a heat-denatured Rv1495 protein or an unrelated protein, GST, was added (Figure 3C). MtbTopA inhibits mRNA cleavage by Rv1495 We characterized the effect of MtbTopA on the previously reported mRNA cleavage activity of Rv1495 (10). As shown in Figure 4A, when an increasing concentration of Rv1495 (0.5–2.0 mM) was added into the reaction mixture, the levels of the 11-mer mRNA substrate pro- gressively decreased, with concomitant appearance of a