The RecA-NT homology motif in ImuB mediates the interaction with ImuA', which is essential for DNA damage-induced mutagenesis

Abstract

The mycobacterial mutasome-comprising ImuA', ImuB, and DnaE2-has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, which is predicted to enable mutasome function via its interaction with the β clamp, is a catalytically inactive Y-family DNA polymerase. Like some other members of the Y-family, ImuB features a recently identified amino acid motif with homology to the RecA N terminus (RecA-NT). Given the role of RecA-NT in RecA oligomerization, we hypothesized that ImuB RecA-NT mediates the interaction with ImuA', an RecA homolog of unknown function. Here, we constructed a panel of imuB alleles in which the RecA-NT was removed or mutated. Our results indicate that RecA-NT is critical for the interaction of ImuB with ImuA'. A region downstream of RecA-NT, ImuB-C, appears to stabilize the ImuB-ImuA' interaction, but its removal does not prevent complex formation. In contrast, replacing two hydrophobic residues of RecA-NT, L378 and V383, disrupts the ImuA'-ImuB interaction. To our knowledge, this is the first experimental evidence suggesting a role for RecA-NT in mediating the interaction between a Y-family member and an RecA homolog.

Keywords: DNA repair; Mycobacterium tuberculosis; antibiotic resistance; mutagenesis; mutasome; protein–protein interaction.

Figure 1

Computational model of mutasome interactions.A, AlphaFold model of the ImuAʹ–ImuB complex. The ImuAʹ structure is shown as Connolly (solvent excluded) surface. For ImuB, only the C-terminal region is shown in the cartoon representation. The ImuAˈ surface in contact with ImuB is colored in yellow. Green represents the RecA-NT motif in ImuB (residues 360–387), pink represents the ImuB-C (residues 422–525), and orange indicates the linker between the two (residues 388–421). Spheres mark the residues at which C-terminal truncations of ImuB were made. ImuB residues predicted to be necessary for the ImuAʹ–ImuB interaction are labeled and represented in sticks (V374, L378, and V383). B, comparison of ImuAʹ–ImuB model (left) with X-ray structure of the RecA–RecA complex (right). Superposition of the two structures is shown in the middle. For ImuB and the second RecA, only the RecA-NT motif (shades of green) is shown. RecA dimer was obtained from the structure of RecA filament (Protein Data Bank ID: 3CMW, (31)). C, conservation logo of RecA-NT derived from the aligned mycobacterial ImuB homologs with the corresponding Mycobacterium smegmatis ImuB sequence added below (in black). Numbering corresponds to M. smegmatis sequence. Individual residues selected for site-directed mutagenesis are indicated with a star above the logo. D, M. smegmatis imuAʹimuB operon with predicted interaction sites. In ImuAʹ, the predicted ImuB interaction site stretches from G97 to M144. In ImuB, the β clamp-binding motif (352-QLPLW-356) is followed by RecA-NT (G360-V387) and ImuB-C (P422-E525). The panels below indicate the site-directed mutations and ImuB C-terminal deletion mutants constructed in this study.

Figure 2

The C terminus of ImuB is essential for ImuAʹ–ImuB complex formation in vitro.A, SDS-PAGE analysis of elution samples from two consecutive pull-downs. Following coexpression of Strep-ImuAʹ (★) with either His-ImuB (WT, •) or His-ImuB-ΔC433 (▲), the cell-lysate supernatant was loaded onto a HisTrap column (IMAC) and eluted with an imidazole gradient. Eluted fractions were subsequently loaded on a StrepTrap column, and Strep-ImuAʹ was eluted with desthiobiotin. B, SDS-PAGE analysis of coexpression of Strep-ImuA′-His-ImuB, Strep-ImuAʹ, and His-ImuB-ΔC380. Expression samples correspond to the total expression after growth for 3 h following IPTG induction. Bands corresponding to individual induced proteins are marked His-ImuB (•), Strep-ImuAʹ (★), and His-ImuBΔC380 ($∎$). C, SEC profiles of His-ImuB-ΔC433 and His-ImuB-ΔC380 following an IMAC. The inset shows the SDS-PAGE analysis of the peak fractions of both His-ImuB-ΔC433 (∗) and His-ImuB-Δ3C80 (∗∗). D, melting curves of His-ImuB-ΔC433 and His-ImuB-ΔC380 following SEC. His-ImuB-ΔC433 has a melting temperature of 55.1 °C and His-ImuB-ΔC380 of 55.6 °C. IMAC, immobilized metal affinity chromatography; SEC, size-exclusion chromatography.

Figure 3

Conserved hydrophobic residues of the Rec-NT motif are important for ImuA′–ImuB binding.A, SDS-PAGE analysis of the purification of His-ImuB mutants (ImuB-L378A, ImuB-V383A, and ImuB-L378A + V383A) with Strep-ImuAʹ after two sequential pull-downs with HisTag and StrepTag, respectively. Clarified extracts were loaded onto a HisTrap (IMAC) column and eluded with an imidazole gradient. Fractions containing His-ImuB were pooled, loaded into a StrepTrap column, and eluted with 5 mM desthiobiotin. Expression samples correspond to the total expression after growth for 3 h following induction with IPTG, and bands of individual induced proteins are marked His-ImuB (•) and Strep-ImuAʹ (★). B, SEC profiles of samples of His-ImuB and His-ImuB mutants (ImuB-L378A, ImuB-V383A, and ImuB-L378A + V383A) coexpressed with Strep-VFP–ImuAʹ following IMAC purification. The elution volumes for His-ImuBAʹ complexes and His-ImuB are highlighted with dotted lines. C, SDS-PAGE analysis of His-ImuB mutants coexpressed with VFP-Strep-ImuAʹ after IMAC and subsequent SEC. Clarified extracts were incubated with nickel beads and eluted with 500 mM imidazole. The fraction containing most of ImuB was injected into a S200 increase 2.4 ml column for SEC. Expression samples correspond to the total expression after growth for 3 h following induction with IPTG and are marked with (•) for His-ImuB and underlined (−−) for Strep-VFP–ImuAʹ. The top panel is the SDS-PAGE gel stained with Coomassie, and below is the same gel imaged at 532 nm for detection of the VFP fluorescent tag in ImuAʹ. The bottom panel corresponds to a crop of the gel and did not eliminate any bands. The marker lane added in the right-hand side of the bottom panel is the same as the one in the top panel and was properly aligned by superposition of the two images of SDS-PAGE gel. D, melting curves of His-ImuB and His-ImuB mutants (ImuB-L378A, ImuB-V383A, and ImuB-L378A + V383A) coexpressed with Strep-VFP–ImuAʹ following IMAC purification. The inflection point corresponding to the melting temperatures is marked with a dotted line. The melting temperatures measured were 45.1 °C for His-ImuB, 45.2 °C for His-ImuB-L378A, 45.4 °C for His-ImuB-V383A, and 45.9 °C for His-ImuB-L378A + V383A. IMAC, immobilized metal affinity chromatography; SEC, ssize-exclusion chromatography.

Figure 4

Evaluation of functional complementation of mutant alleles by means of mitomycin C (MMC) sensitivity.A, MMC damage sensitivity assays in which a twofold dilution series of a 10-fold dilution of each Mycobacterium smegmatis culture (indicated by 10⁻¹) was spotted on standard solid media (7H10) alone or supplemented with 0.06 μg/ml MMC. Images are representative of at least three biological repeats. Individual rows of spots were imaged from a single plate per treatment but represented as single rows for ease of presentation. B, MMC-kill curve showing survival (%) of the different strains exposed to 0.64 μg/ml MMC over a 30 h period. Survival was calculated based on the colony-forming units (CFUs)/ml at each time point divided by the CFU/ml at 0 h (prior to the start of treatment). The plot above represents the mean from three independent experimental repeats with the error bars representing the SD. ∗∗∗ indicate statistical significance at 6 h (p < 0.0001) compared with ImuA–ʹImuB using a two-way ANOVA with Tukey correction for multiple comparisons.

Figure 5

Evaluation of functional complementation of mutant alleles by means of UV sensitivity and UV-induced mutagenesis.A, UV damage sensitivity assays were performed by spotting twofold dilutions of a 10-fold diluted (10⁻¹) culture onto standard media. The spotted plates were then left unexposed (7H10) or exposed to 15 μJ/cm² UV. Images are representative of three biological repeats. Individual rows of spots were imaged from a single plate per treatment but represented as single rows for ease of presentation. B, UV-induced mutagenesis assays were performed to calculate the mutation frequency of the different strains upon exposure to UV as measured by the appearance of rifampicin-resistant mutants. (i) Graph represents the mean mutation frequency of each strain from at least three experimental repeats with the error bars representing SD. A Kruskal–Wallis analysis with Dunn’s correction for multiple comparisons was done to determine statistical significance. (ii) A subset of rifampicin containing plates used to calculate the rifampicin-resistant colonies as a proxy for mutation frequency in (i) of each strain.