Escherichia coli DinB and Replication‐Transcription Collisions

DinB is an error prone DNA polymerase (DNAP) regulated by Escherichia coli 's DNA damage response. This enzyme is well known for its translesion synthesis activity, i.e. the ability to bypass potentially lethal lesions on template DNA. Curiously, cells in which DinB is expressed from an inducible promoter in a high copy number plasmid die upon induction of DinB expression. This loss of survival is rescued by a single amino acid substitution (DinB(V7G)), which does not affect DNA synthesis activity in vitro or translesion synthesis activity in vivo . Therefore, DinB(V7G) separates DinB's known function as a translesion synthesis DNAP and an additional unknown role that causes cell death at high intracellular concentrations. We have evidence that suggests DinB plays a role in the resolution of replication‐transcription collisions (RTCs). RTCs occur because DNAP III replicates rapidly (~1000 nt/s) while RNA polymerase (RNAP) transcribes relatively slowly (40–80 nt/s), and these two processes use the same template DNA at the same time. RTCs are most prevalent in genes encoded on the lagging strand, where DNAP and RNAP collide head on, and on highly expressed genes, regardless of orientation, due to longer RNAP occupancy of the template. Previous studies have identified the transcription factors NusA and DksA as key players in RTC resolution. NusA and DinB interact in E. coli . The nusAts11 allele is thought to cause a lethal frequency of RTCs at 42°C due to failure of RNAP to disengage from the template. The amino acid substitution in nusAts11 occurs at the DinB‐NusA binding interface. DinB is known to suppress the nusAts11 phenotype when expressed from a low copy number plasmid under its own promoter. Notably, we find that the DinB(V7G) variant fails to suppress the lethality of the nusAts11 strain at 42°C, though the interacting surface is unlikely changed as the V7 residue is located in the DinB active site. This finding suggests that DinB has an activity related to RTCs that is not performed by the DinB(V7G) variant. Previous studies have shown that DksA prevents RNAP pausing, and thus RTCs, especially during amino acid starvation. Therefore, ΔdksA cells have a notably slower growth during amino acid starvation due to the stalling of RNAP and thus RTCs. We find that this phenotype is exacerbated by DinB expression from its own promoter in a low‐copy number plasmid. In the presence of the rpoB2 allele, which encodes an RNAP variant that pauses less frequently and rescues dksA growth reduction in minimal medium, we find that dinB expressed from the low copy number no longer worsens growth rate reduction, suggesting that the DinB effect on growth rate is dependent on RNAP pausing. Interestingly, we found that the DinB(V7G) variant, which suppresses the dinB overproduction phenotype, also rescues the ΔdksA /pDinB + severe growth reduction phenotype. These data suggest that DinB plays a role in the RTCs. It is likely that the dinB overproduction phenotype is the result of an exacerbation of the RTCs as we uncovered at lower dinB concentrations in the Δ dksA strain. We hypothesize that DinB switches between DNA translesion synthesis and some yet undiscovered activity to help resolving RTCs and that in DinB(V7G) this equilibrium is altered. Support or Funding Information National Institute of General Medical Sciences [RO1GM088230 to V.G.G.]