RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity
arXiv:2110.01573 · doi:10.1098/rsif.2022.0437
Abstract
The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA, RecBCD a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks and also facilitating decoration of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA and the RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting changes in stoichiometry, copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA filamental stoichiometries equivalent to several hundred molecules that act largely in RecA tetramers before DNA damage, but switch to approximately hexameric subunits when mature filaments are formed. Unexpectedly, we find that the physiologically predominant form of RecB is a dimer.
References in corpus (5)
- Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness
- Inferring diffusion in single live cells at the single molecule level
- RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity
- Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes
- Single-molecular quantification of flowering control proteins within nuclear condensates in live whole Arabidopsis root