8224 Nucleic Acids Research, 2010, Vol. 38, No. 22 Figure 3. Effects of Rv1495 on the single-stranded DNA cleavage activity of MtbTopA. 50-end-labeled 32-mer oligonucleotide (50-CAGTGAGCGAGCTTCCGCTTGACATCCC AATA-30) (27) was used as a speciﬁc single-stranded DNA substrate. DNA-binding and cleavage assays of MtbTopA were performed as described under ‘Materials and Methods’ section. (A) DNA-binding activity assays for MtbTopA, Rv1495 and Rv0054 (a positive control). The protein concentrations were indicated on top of the panels. (B) ssDNA cleavage assays for MtbtopA (left panel) and the effects of Rv1495 (right panel). The 32-mer substrates and 19-mer cleavage products were indicated by arrows. (C) The effects of the denatured Rv1495 protein (left panel) or GST (right panel) on the cleavage activity of MtbTopA. band of lower molecular weight (Figure 4A, left panel, lanes 2–5), consistent with mRNA cleavage. When the concentration of Rv1495 was kept constant (0.75 mM), the level of cleavage products steadily decreased as MtbTopA content was increased (Figure 4A, middle panel, lanes 4–6), conﬁrming that the MtbTopA inhibited the mRNA cleavage activity of Rv1495. In contrast, Rv0054, used as a negative control, had no inhibitory effect on cleavage (Figure 4A, right panel, lanes 4–6). The inhibitory domain of MtbTopA was further characterized by purifying the NTD of MtbTopA, and its CTD (Figure 4B). The interactions of these two domains with Rv1495 were then characterized by a bac- terial two-hybrid assay. As shown in Figure 4B, CTD interacted with Rv1495, while NTD did not. CTD also showed an equivalent inhibitory effect on the mRNA cleavage by Rv1495 as was seen with the wild-type MtbTopA (Figure 4C, lanes 2–5). No signiﬁcant effect was observed for the NTD protein under similar reaction conditions (Figure 4C, lanes 9–12). Therefore, the mRNA cleavage activity of Rv1495 was inhibited through the CTD of MtbTopA. Rv1495 interacts with M. smegmatis topoisomerase I (MsmTopA) both in vitro and in vivo Mycobacterium smegmatis also contains an ortholog of the M. tuberculosis H37Rv topoisomerase I, MsmTopA (17). To detect if the interaction is conserved in both mycobacterial strains, we examined the interaction between Rv1495 with the M. smegmatis MsmTopA using a bacterial two-hybrid assay. As shown in Figure 5A, the co-transformant with Rv1495 and MsmTopA genes obtained a similar growth on the select- ive medium to that seen with the Rv1495 and MtbTopA cotransformant. No growth was observed for several negative controls (Figure 5A). This indicated that