(A) Representative images of G-ImuB exposed to 2× MIC mitomycin C (MMC) for 4 hr (top panel) or 2× MIC MMC plus griselimycin (GRS) for 4 hr (center panel), and the G-ImuBAAAAGG mutant exposed to 2× MIC MMC for 4 hr (bottom panel). Scale bars, 5 μm. (B) Interactions of M. smegmatis β clamp with (i) GRS, (ii) ImuB, or (iii) the replicative DNA PolIIIα subunit, DnaE1. The interaction of the β clamp with GRS is represented by the X-ray structure of the complex (PDB id: 5AGU). Predicted interactions with ImuB (S347-I359) and DnaE1 (M947-G954) are derived from the respective AlphaFold models. Interacting peptides are shown as smoothed traces with side chains. The brown β clamp region indicates residues in contact with corresponding peptides. Molecular contacts were derived from 3D structures using the VoroContacts web server. Detailed contact data are provided separately (Supplementary file 1 – DnaN Contact Data). (C) Cells aligned by mid-cell position, arranged according to cell length and colored (magenta – mCherry-DnaN foci, green – G-ImuB foci) according to fluorescence intensity, showing the presence of G-ImuB foci following MMC treatment and the lack of foci after GRS exposure. G-ImuBAAAAGG shows no foci after MMC treatment, similar to the G-ImuB strain following GRS exposure. (D) Proportions of cells containing either mCherry-DnaN or G-ImuB foci following exposure to GRS alone at 2× MIC or 5× MIC, or in combination with either single-dose ultra-violet (UV) or MMC at 2× MIC or 5× MIC. Additional Source data are available in Figure5.zip, accessible at http://doi.org/10.5061/dryad.76hdr7szc.