The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ

Abstract

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

The Escherichia coli serS gene promoter region overlaps with the rarA gene

“…The absence of a complete rarA sequence itself does not increase the cell sensitivity to DNA damaging agents like UV or NFZ. Removal of a RecA-loading system like RecF or RecO, however, increases cells' sensitivity to UV irradiation, as observed previously [66][67][68][69][70]. Interestingly, deletion of rarA in a ΔrecF or ΔrecO background rescues their damage sensitivity.…”

“…The Escherichia coli RarA protein is required for most of this RecA-independent recombination [41]. More prominently, the RarA protein is involved in the resolution of recombination intermediates as part of an expanded RecFOR pathway for the amelioration of post-replication gaps [66].…”

Spatiotemporal Dynamics of Single-stranded DNA Intermediates inEscherichia coli

“…There is no information available to estimate the size of post-replication gaps. The results of two recent studies [61,62] indicate that the RecJ nuclease [63] acts within the early stages of postreplication gap repair. If RecJ were to expand post-replication gaps by degrading the 5′ gap end of the dsDNA downstream, this would create a binding site for large amounts of SSB.…”

ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks

“…Bacteria have evolved an extensive regulatory network called the SOS response to control the synthesis of factors that protect and repair the genome. Processes coordinately regulated within the SOS response include error-free DNA repair ( 1 ), error-prone lesion bypass ( 2 , 3 ), inhibition of cell division ( 4 ), and homologous recombination (HR) ( 5 , 6 ).…”