Figure 1. Identification and characterization of M. smegmatis NucS.

(a) Schematic representation of the process for identifying the nucS transposon mutant. ∼11,000 clones from the M. smegmatis insertion mutant library were replicated onto plates with (Rif) or without (No Rif) rifampicin (step 1). One single clone (circled) produced a high number of Rif-R colonies. After isolation and purification (step 2), the frequency of spontaneous Rif-R mutants (bottom plate) was checked and compared with that of the wild-type (upper plate), demonstrating its hypermutable phenotype. (b) Multiple sequence alignment of NucS sequences. M. tuberculosis (NucS_Mtu), M. smegmatis (NucS_Msm) and P. abyssi (NucS_Pab) sequences are from Uniprot (identifiers are P9WIY4, A0R1Z0 and Q9V2E8, respectively). Solid lines over the alignment indicate protein domains as defined previously for P. abyssi NucS¹¹ (black, DNA-binding; grey, nuclease). Identical amino acid residues are shown in black. Catalytic residues required for nuclease activity in P. abyssi NucS¹¹ are labelled with asterisks. Nuclease motifs of P. abyssi NucS are also indicated¹¹. (c) DNA-binding activity of NucS. In a gel-based EMSA, purified NucS protein (1–16 μM) is capable of binding to 45-mer ssDNA (50 nM) (left side) but not to 45-bp dsDNA (50 nM) (right side). The arrow indicates the position of the DNA–NucS complex.