The Escherichia coli RarA protein is an ATPase in the highly conserved AAA+ superfamily. Despite its presence in all organisms and consensus that it is essential for the maintenance of genomic stability, very little is currently known about its precise function and mechanism of action. RarA has strong sequence homology with clamp loader proteins that comprise part of the pol III holoenzyme complex. This suggests a possible role for RarA in polymerase loading during DNA replication. Furthermore, recent genetic experiments suggest that RarA may share some functional overlap with RecA, a protein essential for the non-mutagenic rescue of stalled replication forks in bacteria.
In this study, we use a combination of single-molecule time-lapse microscopy and traditional bulk assays to investigate the effects of rarA expression levels on the growth of E. coli cells. Strains lacking rarA exhibit slower growth, smaller cell size, and fewer replication foci as compared to wild-type. Conversely, over-expression of rarA is lethal, both in the presence and absence of recA. As RarA accumulates, cells filament and replication foci disappear before eventual cell death. Interestingly, deleting one or more genes encoding translesion (TLS) polymerases reduces this toxicity by up to three orders of magnitude. Despite this connection to TLS and our observation of pol III unloading, we find that the SOS response is not induced to appreciable levels by rarA over-expression. Our observations support a model in which RarA facilitates polymerase exchange at stalled replication forks, unloading pol III holoenzymes in favor of TLS polymerases.