ATPase activity of B. subtilis RecA affects the dynamic formation of RecA filaments at DNA double strand breaks

Abstract

RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed “threads”, which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, and still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduces formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. While lowered ATP-binding activity only moderately affected RecA dynamics, loss of ATPase activity lead to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and non-specifically DNA-bound molecules into filaments upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Our data suggest that RecA/ssDNA filaments change in subcellular localization and length involving ATP-driven homology search.

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in liveEscherichia coliwith single-molecule sensitivity

“…These also provide evidence for stable trimeric, tetrameric, hexameric and the filamentous forms when ssDNA is present [63], consistent with our findings post-treatment. Our snapshot observation of filament stoichiometry cannot shed light directly on models of dynamic nucleation or stepwise growth, as explored in [64][65][66]. Rather, it explores molecular details of the characteristic protein subunits within the mature filament at steady state.…”