Genetic dissection of DNA damage tolerance in Bacillus subtilis : RecA and recombination functions regulate translesion synthesis

Mutagenesis is a fundamental, yet poorly understood, source of genetic variation that underpins microbial evolution and adaptation. When the Bacillus subtilis replicative DNA polymerase (DNAP) PolC encounters DNA lesions induced by endogenous or exogenous insults, it stalls and disassembles. If error-free DNA damage tolerance (DDT) sub-pathways fail to circumvent the lesion, bipartite translesion synthesis (TLS) DNAPs (PolY1 or PolY2 together with PolA) may bypass the damage to resume DNA synthesis. Mismatch repair subsequently removes misincorporated nucleotides. Here, we investigate which proteins loaded at stalled replication forks influence mutation dynamics mediated by TLS DNAPs. We demonstrate that ΔrecA, ΔpolA, or ΔpolY1 ΔpolY2 mutations strongly reduce cell survival following DNA damage and mutagenesis. The accessory proteins DisA, RarA, RecD2, DinG, and Mfd, which physically interact with PolA and/or RecA, differentially affect cell survival after DNA damage in the absence of TLS DNAPs and differentially modulate mutagenesis by regulating the activity of distinct TLS DNAPs, highlighting their roles in error-prone DDT. We also reveal that SOS-independent mutagenesis operates in the ΔpolA ΔrecA background. Elucidating the regulatory network underlying TLS provides a framework to understand bacterial speciation and may uncover new avenues to limit antibiotic resistance emergence.