A unique DinB residue permits elucidation of the DinB multi-protein complex formation in Escherichia coli

Cells rely on several pathways to repair DNA lesions, but without repair, cells tolerate them by using specialized DNA polymerases that replicate past lesions in a process known as translesion synthesis (TLS).  DinB is the most evolutionarily conserved TLS DNA polymerase and its ability to perform TLS is regulated by interactions with two other proteins. It has been shown in vitro that these three proteins form a multi-protein complex (MPC). We hypothesized that the MPC exists in vivo and that it can be isolated directly from cells. Here we show that this MPC can indeed be isolated directly from cells. Moreover, we have identified a surface interface unique to DinB-like proteins, which mediates MPC formation. We mutated a key residue on this interface, changing Cysteine 66 to an Alanine. Purification of the mutant protein, DinB(C66A), resulted in enhanced co-purification of the other MPC components, and of the putative intact MPC.  Notably, single deletion of genes encoding MPC components limits complex formation. Furthermore, cells that express the mutant protein exhibit phenotypes consistent with a stable MPC. Study of this unique DinB mutant has revealed a key interface, which seemingly modulates the strength of MPC binding, and has highlighted the requirement for contemporaneous binding of the MPC components.  Since TLS polymerases have been implicated in the evolution of antibiotic resistance in bacteria and of cancer in humans, it is critical to learn about the underlying mechanisms of regulation of these TLS DNA polymerases.