The ATPase activity of E. coli RecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double stranded DNA

Abstract

The Escherichia coli RecA protein catalyzes the central step of homologous recombination using its homology search and strand exchange activity. RecA is a DNA-dependent ATPase, but its homology search and strand exchange activities are largely independent of its ATPase activity. ATP hydrolysis converts a high affinity DNA binding form, RecA-ATP, to a low affinity form RecA-ADP, thereby supporting an ATP hydrolysis-dependent dynamic cycle of DNA binding and dissociation. We provide evidence for a novel function of RecA’s dynamic behavior; RecA’s ATPase activity prevents accumulation of toxic complexes caused by direct binding of RecA to undamaged regions of dsDNA. We show that a mutant form of RecA, RecA-K250N, previously shown to be toxic to E. coli, is a loss-of-function ATPase-defective mutant. We use a new method for detecting RecA complexes involving nucleoid surface spreading and immunostaining. The method allows detection of damage-induced RecA foci; STED microscopy revealed these to typically be between 50 and 200 nm in length. RecA-K250N, and other toxic variants of RecA, form spontaneous DNA-bound complexes that are independent of replication and of accessory proteins required to load RecA onto tracts of ssDNA in vivo, supporting the hypothesis that RecA’s expenditure of ATP serves an error correction function.

A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

“…Because the Dmc1-E157D mutant is modeled after RecA-E96D, which has been shown to form foci on undamaged DNA, we wanted to ask whether the same was true of the corresponding Dmc1 mutant [35]. To determine whether any of the foci that we observed in the dmc1-E157D background resulted from binding to chromosomes independent of DSBs, we introduced the spo11 mutation into our dmc1-E157D strains to block DSB formation.…”

“…Under normal circumstances in vivo , RecA family proteins form nucleoprotein filaments that are shorter than the resolution limit of conventional light microscopy (~200 nanometers). This is true for RecA, and for both eukaryotic RecA homologs, Rad51 and Dmc1 [32–35]. Super-resolution microscopy imaging of Dmc1 filaments formed during meiosis indicates that Dmc1 filaments are typically about 120 nanometers (nm) long, a length that corresponds to roughly 100 nucleotides when taking into account the fact that RecA family proteins stretch the DNA ~1.5 fold when assembled into a filament [36,37].…”

“…The ADP bound form of the protein has lower affinity for DNA than the ATP-bound form, and is inactive in homology search and strand exchange. In prokaryotes, RecA ATP hydrolysis is required for filament disassembly following strand exchange, or when the protein inappropriately assembles on dsDNA [35,42]. In contrast to RecA, the eukaryotic recombinases Rad51 and Dmc1 display relatively weak intrinsic ATPase activity and rely on Rad54 family ATP-dependent dsDNA translocases to promote their dissociation [44–48].…”

Omnipresent Maxwell's demons orchestrate information management in living cells

“…The relationship between RecA's ATPase activity and its DNA-binding affinity, together with the demonstration that strand exchange can occur in the absence of ATP hydrolysis, led to the proposal that a function of RecA's ATPase activity is to release the protein from the products of strand exchange, thereby making the hybrid intermediate available to late-acting recombination proteins. With this view, the critical role of RecA's ATPase activity is to prevent accumulation of toxic dsDNA complexes caused by direct binding of RecA to undamaged regions of dsDNA (Gataulin et al, 2018). Here, we have an ATP hydrolysis-dependent error-correction function that allows cells to discard the dead-end and potentially toxic complexes that result from direct RecA binding to dsDNA.…”

RecA and DNA recombination: a review of molecular mechanisms

“…Additionally, ATP hydrolysis promotes the disassembly of RecA-DNA filaments after completion of strand exchange (68). Furthermore, nucleoid surface spreading, immunostaining and super resolution microscopy studies performed on ATPase-defective mutants RecAK250N and RecAE96D in vivo revealed that ATP hydrolysis also promotes disassembly from undamaged regions of dsDNA, preventing the accumulation of dead-end and potentially toxic complexes (69).…”