Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein

Abstract

Ubiquitous RarA AAA + ATPases play crucial roles in the cellular response to blocked replication forks in pro-and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of recA, recO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H 2 O 2-or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (addAB, recJ, recQ, recS) or branch migration (ruvAB, recG, radA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When rarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in rarA recU and rarA recX double mutant cells, and was blocked in rarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.

Bacillus subtilis PcrA Couples DNA Replication, Transcription, Recombination and Segregation

“…To elucidate whether PcrA prevents unscheduled RecA loading or dismantles its cognate recombinase assembled at or behind a stalled fork and which function(s) may counteract the PcrA antirecombinase activity, null mutants in presynaptic functions, namely end resection (Δ addAB , Δ recJ , Δ recQ ), RecA mediation ( recO 16), and positive (Δ rarA ) or negative (Δ recX , Δ recU ) modulators ( Table S1 ), were assessed in the pcrA - ssrA sspB ( pcrA T ) context ( Table 1 ) (Sanchez et al, 2006 ; Cárdenas et al, 2012 ; Romero et al, 2020 ).…”

“…The contribution of mutants in the modulation of RecA filament growth in the inviability of PcrA depleted cells was also assessed. RarA has at least two activities: to control the loading of pre-primosomal proteins at a stalled fork and to positively modulate RecA filament growth (Carrasco et al, 2018 ; Romero et al, 2019a , 2020 ). PcrA depletion lethality was not suppressed by rarA inactivation ( Figure 1B ).…”

Global diversity and distribution of prophages are lineage-specific within the Ralstonia solanacearum species complex

“…The ubiquity of these genes suggests that they may be under strong selection, and their presence in prophages from different phylotypes indicates that selection is phylotype-independent. As these genes have all been implicated in bacterial fitness [ 109 , 110 , 111 ], they may have been selected for by providing general selective advantage for their hosts. Alternatively, they may provide fitness benefits to the phages themselves by encouraging phage replication and transmission.…”

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

“…Surprisingly, purified ATPase-mutant RecA was defective in nucleating nucleofilaments in vitro , in contrast to E. coli RecA (52). Because we did observe some filamentous RecA-mVenus structures in vivo following DSB induction, we argue that its defect appears to be partially overcome in vivo , likely because of the action of RecA loading or accessory factors such as RecO and RarA (37,53). In any event, RecA from B. subtilis seems to differ in its molecular properties related to ssDNA binding compared with E. coli , so our study also strengthens the important idea that action of RecA at a molecular level, despite being generally conserved between organisms (including Rad51 from eukaryotes), must be viewed considering differing biochemical properties.…”

“…We therefore used super resolution fluorescence microscopy, to obtain a better resolution of filamentous structures, and an even higher temporal resolution than earlier. We used structured illumination microscopy (SIM) on a fully functional RecA-mNeonGreen fusion (37) and acquired images every 20 seconds, using 500 ms exposure time. Fig.…”