Abstract
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.
Highlights
Wild type ∆recO recF15 ∆recD2 ∆recX ∆addAB ∆recJ ∆recQ ∆recS ∆recU ∆recG ∆ruvAB ∆radA/sms ∆polY1 ∆polY2 DnaX-CFP D static dyna −drug static dyn +H2O2 static+Mitomycin C (MMC) static dyn calculated by step-size distributions and Gaussian fits for RarA-mVenus in the different backgrounds studied in exponential growth, and after 60 min induction with 0.5 mM H2O2 or 50 ng/ml MMC
We sought to investigate the intracellular dynamics of RarA in response to different kinds of DNA damage, using RarA-YFP or RarA-mVenus constructs, and analysed if its mobility is altered in different genetic backgrounds
RarAmVenus expressing cells did not show sensitivity to Mitomycin C (MMC) (Fig. S2B), and RarA-YFP expressing cells were insensitive to exposure of methyl methanesulfonate (MMS) (Fig. 1A), nor were they temperature sensitive (Fig. 1B), unlike dnaBts or dnaCts mutant cells
Summary
Wild type ∆recO recF15 ∆recD2 ∆recX ∆addAB ∆recJ ∆recQ ∆recS ∆recU ∆recG ∆ruvAB ∆radA/sms ∆polY1 ∆polY2 DnaX-CFP D (μm[2] s−1) static dyna −drug static dyn +H2O2 static+MMC static dyn calculated by step-size distributions and Gaussian fits for RarA-mVenus in the different backgrounds studied in exponential growth, and after 60 min induction with 0.5 mM H2O2 or 50 ng/ml MMC. RarA tracks in dnaB37ts cells (Fig. 6F) were mostly confined (~70%), and specially merged with the replication machinery (54%) (Fig. 6G), while tracks in dnaC30ts cells (Fig. 6E), mostly presented random movement (~60%), and confined tracks were located preferentially in “far” positions relative to replication forks (Fig. 6G). Taken together, these data suggest that DnaC contributes to RarA binding to the replication machinery while a putative DnaB interaction relates to the removal of RarA from a newly assembled replication fork, which does not occur in the absence of DnaB
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