The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.

The C. elegans E3 ubiquitin ligase RPM-1 consists of 3,766 amino acids, with a RING finger domain at the C-terminus that functions to target the DLK-1 kinase for degradation for synapse development and axon termination. rpms-1 ( for rpm-1 short, aka F07B7.12 ) resides 35 kb away from rpm-1 on chromosome V, and is a near-perfect 12 kb duplication of rpm-1 , including the entire promoter region and coding sequences. RPMS-1 consists of 1,964 amino acids and is identical to the N-terminal half of RPM-1 , except the last 40 amino acids. Previous studies showed that transgenic overexpression of the duplicated region of rpm-1 (+) did not rescue synapse defects of rpm-1 loss of function mutants. Here, using CRISPR editing, we generated a double knockout of rpm-1 and rpms-1 . We find that axon and synapse defects in rpm-1rpms-1 double mutants resemble those in rpm-1 single mutants. Expression levels of endogenously tagged DLK-1 protein are increased to a comparable degree in rpm-1 and rpm-1rpms-1 mutants, compared to the control. These data, along with previous transgene expression analysis, support the idea that rpms-1 does not have a major role in RPM-1-mediated cellular processes.