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The RecA protein and RecBCD complex are key bacterial components for themaintenance and repair of DNA, RecBCD a helicase-nuclease that uses homologousrecombination to resolve double-stranded DNA breaks and also facilitatingdecoration of single-stranded DNA with RecA to form RecA filaments, a vitalstep in the double-stranded break DNA repair pathway. However, questions remainabout the mechanistic roles of RecA and RecBCD in live cells. Here, we usemillisecond super-resolved fluorescence microscopy to pinpoint the spatiallocalization of fluorescent reporters of RecA and the RecB at physiologicallevels of expression in individual live Escherichia coli cells. By introducingthe DNA crosslinker mitomycin C, we induce DNA damage and quantify theresulting changes in stoichiometry, copy number and molecular mobilities ofRecA and RecB. We find that both proteins accumulate in molecular hotspots toeffect repair, resulting in RecA filamental stoichiometries equivalent toseveral hundred molecules that act largely in RecA tetramers before DNA damage,but switch to approximately hexameric subunits when mature filaments areformed. Unexpectedly, we find that the physiologically predominant form of RecBis a dimer.

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity