8222 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 1. Physical interactions of Rv1495 with MtbTopA. (A) Bacterial two-hybrid assays (Stratagene) for the interaction of Rv1495 with MtbTopA, which were performed as described under ‘Materials and Methods’ section. Left panel, plate minus streptomycin (str) and 5 mM 3-amino-1,2,4-triazole (3-AT), Middle panel, plate plus str and 5 mM 3AT. Right panel, an outline of the plates. CK+, co-transformant containing pBT-LGF2 and pTRG-Gal11P as a positive control. CK , co-transformant containing pBT and pTRG as a negative control. (B) Pull-down assays for examining the interaction between Rv1495 and MtbTopA. Equimolar amounts of 6 His-MtbTopA combined with GST-Rv1495 were used for pull-down assays as described in the ‘Materials and Methods’ section. GST was used as the negative control. One predicted size of his-tagged MtbTopA protein, pulled down by GST-tagged Rv1495 protein, was further examined by a western blotting assay. Rv1495 inhibits the activities of MtbTopA The physical interactions between Rv1495 and MtbTopA suggested that the MazF protein was in some way regulating the activities of the MtbTopA topoisomerase. As shown in Figure 2, addition of increasing amounts of MtbTopA (0.05–0.15 mM) to a reaction mixture contain- ing plasmid pBluescript as a DNA substrate resulted in a progressive disappearance of the supercoiled plasmid DNA band, concomitant with the appearance of a relaxed plasmid band on the gel (Figure 2A). This con- ﬁrmed that the MtbTopA could relax supercoiling of this DNA substrate. This activity was again examined in the presence of Rv1495 under similar conditions. As shown in Figure 2, when the concentration of MtbTopA was kept constant (0.125 mM), the amount of supercoiled plasmid steadily increased with increasing additions of Rv1495 (Figure 2B). In contrast, heat-denatured Rv1495 (Figure 2C) or GST protein (Figure 2D) had no effect on supercoiling relaxation activity of MtbTopA. The in- hibitory effect was stronger if MtbTopA was preincubated with Rv1495 (Supplementary Figure S1). In further experiments, Rv1495 was found to form a protein complex with its antitoxin, Rv1494 (Supplementary Figure S2A and B), but the antitoxin could not block or reverse the effect of Rv1495 on the MtbTopA activity (Supplementary Figure S2C). A previous report showed that Lsr2 inhibited the activity of MtbTopA through its double-stranded DNA- binding ability (24). However, DNA-binding assays using pBluescript plasmid showed no similar activity for Rv1495 (Figure 2E, lanes 2–5), but conﬁrmed this activity for Lsr2 (Figure 2E, lane 7–9). When Rv0054 was used as a negative control, no double-stranded DNA-binding activity was observed (Figure 2E, lanes 11–13). Rv1495 inhibits single-stranded DNA cleavage by MtbTopA The function of the topoisomerase I depends on its binding to single-stranded DNA and subsequent cleavage activities (18,27). The mechanism underlying