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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. Author summary: The RarA protein is part of a widespread protein family that is highly conserved from bacteria to humans. While the family clearly plays an important role in genome stability in all organisms, its molecular function remains undefined. Part of the reason for the lack of progress is genetic redundancy, where overlapping molecular functions render it difficult to discern the genetic effects of a gene when it is absent. In this study, we pinpoint a series of enzymes that must be absent to observe a strong effect of a deletion of the gene encoding RarA. In particular, a loss of RuvB function renders a rarA deletion highly sensitive to DNA damage. An additional loss of recQ or recG makes the cell inviable. Suppression of these defects by inactivation of proteins that load RecA protein indicate that RarA is somehow involved in the processing or reversal of branched DNA structures created by RecA and that RarA is part of an expanded version of the classic RecFOR pathway for repair. [ABSTRACT FROM AUTHOR] |
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