The Escherichia coli DinD Protein Modulates RecA Activity by Inhibiting Postsynaptic RecA Filaments

Abstract

Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to singlestranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.The Escherichia coli SOS response is a coordinately regulated network of genes induced in reaction to heavy or persistent DNA damage (1). The SOS regulon is repressed by the LexA protein, which is inactivated as a repressor upon cellular stress such as heavy DNA damage. The cellular signal for SOS induction is the RecA protein bound to single-stranded DNA (ssDNA). 2RecA is the central DNA recombinase in bacteria and carries out several distinct functions when activated (2). The most appreciated function of RecA is the catalysis of homologous DNA recombination crucial to the generation of genetic diversity. However, based on frequency of use, the primary role of RecA lies in the multiple pathways for the recombinational repair of stalled DNA replication forks. Through much in vitro work, it has become increasingly apparent that the RecA protein is under considerable control by a network of proteins that function to modulate when and where RecA protein binds to DNA (3). The PsiB protein has recently been shown to bind to free RecA protein, effectively inhibiting RecA from nucleating onto ssDNA (4). RecA is also inhibited from nucleating onto ssDNA bound by the single-stranded DNA-binding protein (SSB) (5). The SSB-imposed inhibition is relieved by the action of the RecF, RecO, and RecR proteins (3). Following nucleation, RecA protein protomers assemble into a nucleoprotein filament, extending cooperatively in the 5Ј to 3Ј direction. The RdgC protein can inhibit RecA binding to ssDNA and can interfere with homologous DNA pairing by binding to duplex DNA (6, 7). Some proteins shown to dismantle RecA filaments are known DNA translocases, such as UvrD (8) and PcrA helicases (9). Filament extension can be blocked through the action of the RecX protein (10), whereas the DinI protein antagonizes the function of RecX by stabilizing RecA filaments, inhibiting filament end-dependent disassem...

Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli

“…These accessory proteins include those involved in RecA nucleation onto ssDNA (PsiB), in loading and unloading of RecA onto DNA coated with single-strand DNA binding protein (SSB) (RecFOR) and in regulating RecA filament stability (DinI and RecX) (reviewed in Cox (2007) ). Other proteins such as UvrD ( Petrova et al, 2015 ), PcrA ( Fagerburg et al, 2012 ; Park et al, 2010 ), and RuvAB ( Eggleston et al, 1997 ), and DinD dismantle RecA filaments ( Uranga et al, 2011 ).…”

Proteomic and metabolomic profiles demonstrate variation among free-living and symbiotic vibrio fischeri biofilms

“…Stress-inducible biofilm formation also produces DNA damage, which can trigger the bacterial SOS response initiated by the sensor protein RecA [ 49 , 50 ], which was overexpressed in the biofilm samples. In addition, these methods detected the protein RuvA (responsible for Holiday junction formation as well as initiation of the SOS response).…”

The draft genome of a new Verminephrobacter eiseniae strain: a nephridial symbiont of earthworms

“…The ability to uptake the free DNA from the environment may serve as a potential source of nutrition and facilitate the DNA repair of the symbiont bacteria to maintain their genome stability (Davidson et al 2014). The DinD (DNA damage-inducible protein D) gene plays a major role in recombinational DNA repair by modulating the Recombinase A (RecA) gene activity (Uranga et al 2011). Notably, the RecA gene was also identified in the genome dataset of V. eiseniae msu.…”