Single molecule tracking reveals functions for RarA at replication forks but also independently from replication...

RarA is a widely conserved protein proposed to be involved in recombination-dependent replication. We present a cell biological approach to identify functional connections between RarA and other proteins using single molecule tracking. We found that 50% of RarA molecules were static, mostly close to replication forks and likely DNA-bound, while the remaining fraction was highly dynamic throughout the cells. RarA alternated between static and dynamic states. Exposure to H₂O₂ increased the fraction of dynamic molecules, but not treatment with mitomycin C or with methyl methanesulfonate, which was exacerbated by the absence of RecJ, RecD2, RecS and RecU proteins. The ratio between static and dynamic RarA also changed in replication temperature-sensitive mutants, but in opposite manners, dependent upon inhibition of DnaB or of DnaC (pre)primosomal proteins, revealing an intricate function related to DNA replication restart. RarA likely acts in the context of collapsed replication forks, as well as in conjunction with a network of proteins that affect the activity of the RecA recombinase. Our novel approach reveals intricate interactions of RarA, and is widely applicable for in vivo protein studies, to underpin genetic or biochemical connections, and is especially helpful for investigating proteins whose absence does not lead to any detectable phenotype.