ImuA is a key protein involved in the mutagenesis of Mycobacterium species. It forms a functional mutasome with DnaE2 (an error-prone polymerase), ImuB (a Y-family polymerase), and sliding clamp. ImuA shares weak homology with RecA, but its role within the mutasome remains unknown. ImuA from mycobacterial species has been difficult to characterize because its overexpression leads to solubility problems in E.coli and its native host M. smegmatis. Here, we characterize ImuA from M. thermoresistible, which expresses as a soluble protein when fused to an N-terminal SUMO tag. During the purification of ImuA we found that the majority of the protein is tightly bound to DNA, but size-exclusion chromatography provides a minor amount of DNA-free protein. Protein that co-purifies with DNA has 2-fold more ATP hydrolysis activity compared to the DNA-free protein, but addition of ssDNA or dsDNA results in an equivalent activity. Addition of ImuB in the reaction mixture doubled the ATPase activity, suggesting ImuA and ImuB form a complex. ImuA bound to DNA can be visualized using negative stain electron microscopy. Incubation of ImuA with single-stranded circular M13mp8 DNA results in nucleoprotein filaments with an average thickness of 11.86 ± 0.25 nm. EM images also indicate that addition of ATP to DNA-bound ImuA causes oligomerization. A collection of 130 EM images of the ImuA oligomer enabled us to generate a low-resolution model that depicts 6-8 residues of ImuA are arranged in a ring with a diameter of 120 Å. The central hole of the ring is 25 Å wide which can accommodate dsDNA. Altogether, we demonstrate that ImuA binds to DNA, has ATPase activity, and oligomerizes on DNA. By analogy to the role RecA plays in the E.coli Pol V mutasome, we expect that ImuA alone or in complex with ImuB facilitates the recruitment of DnaE2 to sites of DNA damage.

Characterization of ImuA , a protein involved in the mutagenesis of Mycobacterial species

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