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

Kanika Jain,Michael M Cox,Elizabeth A Wood,Michael Lichten

doi:10.1371/journal.pgen.1009972

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.

Highlights

DNA replication, indispensable to the survival and reproduction of all living organisms, is a highly coordinated and complex process
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

Summary

Introduction

DNA replication, indispensable to the survival and reproduction of all living organisms, is a highly coordinated and complex process. The progress of replication forks is regularly challenged by barriers extrinsic and intrinsic These include DNA lesions produced by reactive oxygen species (ROS) or other DNA damaging agents and protein-DNA complexes. Encounters with such barriers can result in replication fork stalling or collapse. In some unknown fraction of encounters, a fork engages in “lesion-skipping”, disengaging and re-initiating downstream and leaving the lesion behind in a post-replication gap [1,2,3,4,5,6,7,8,9,10] These events represent a major source of mutagenesis and, if unresolved, can result in cell death. If post-replication gaps are not processed and closed prior to the replication cycle, subsequent fork encounters will generate a double strand break

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