Biology subjects

Brocas, C.

Publications and source records attributed to Brocas, C..

4 recordsLinked to original sources

bioRxiv2025

The CST complex mediates a post-resection non-homologous end-joining repair pathway and promotes local deletions

Repair of a DNA double-strand break (DSB) by non-homologous end-joining (NHEJ) generally leaves an intact or minimally modified DNA sequence. Resection initiation exposes single-stranded DNA and directs repair towards homology-dependent pathways and away from NHEJ. Therefore, NHEJ is not thought to be an available repair pathway once the DSB is resected. Here, we report that the Cdc13/Stn1/Ten1 (CST) complex, well characterized for its telomere-associated functions, acts after resection initiation to mediate a backup NHEJ repair. We found a CST-specific mutation signature after DSB repair, characterized by deletions of 5-85 bp, mostly dependent on NHEJ. In contrast, NHEJ-mediated small deletions of 1-4 bp and insertions are not affected in CST mutants. The interaction between CST and Pol-primase is critical for these intermediate size deletions, suggesting a role for fill-in synthesis. Consistently, in stn1{Delta} and Pol-primase mutant deficient for interaction with CST, resection is increased, leading to larger deletions of several kilobases mediated by microhomologies. Collectively, these results depict a more complex picture of repair pathway choice where CST allows a post-resection NHEJ repair, promoting local deletions but guarding against much larger and potentially more deleterious deletions and rearrangements.

molecular biology↗

bioRxiv2024

Rad52 Acts as an Assembly Chaperone to Form and Stabilize Rad51 Filaments Through a Large C-Terminus 85-Residue Segment

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks and the restart of stalled replication forks. A critical step in HR is the formation of Rad51 nucleofilaments, which perform homology search and strand invasion of a homologous DNA sequence required for repair synthesis. In the yeast Saccharomyces cerevisiae, Rad52 facilitates Rad51 nucleofilament formation by mediating Rad51 loading onto ssDNA and counteracting the dissociation of Rad51 filaments by the DNA translocase Srs2. The molecular basis of these two Rad52 functions remains unclear. Our integrative structural analyses of the Rad51-Rad52 interaction, combining NMR, SAXS, and modeling, reveal that an 85-residue segment of Rad52, conserved in fungi, folds upon binding to a broad surface of a Rad51 monomer. Notably, it includes an FxxA motif conserved in the BRC repeats of BRCA2 and at the Rad51-Rad51 interface. This binding mode was validated through an extensive set of mutations. Using in vivo assays and a functional fluorescent GFP-Rad51 fusion protein, we demonstrated that this entire segment is critical for Rad51 filament formation. These findings highlight how Rad52 functions as an assembly chaperone, preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and protecting Rad51 filaments from destabilization by Srs2.

molecular biology↗

bioRxiv2023

Cohesin complex oligomerization maintains end-tethering at DNA double-strand breaks

DNA double-strand breaks (DSB) must be repaired to ensure genome stability. Crucially, DSB ends must be kept together for timely repair. In Saccharomyces cerevisiae, two poorly understood pathways mediate DSB end-tethering. One employs the Mre11-Rad50-Xrs2 (MRX) complex to physically bridge DSB ends. Another requires the conversion of DSB ends into single-strand DNA (ssDNA) by Exo1, but the bridging proteins are unknown. We uncover that cohesin, its loader and Smc5/6 act with Exo1 to tether DSB ends. Remarkably, cohesin specifically impaired in oligomerization fails to tether DSB ends, revealing a new function for cohesin oligomerization. In addition to the known importance of sister chromatid cohesion, microscopy-based microfluidic experiments unveil a new role for cohesin in repair by ensuring DSB end-tethering. Altogether, our findings demonstrate that oligomerization of cohesin prevents DSB end separation and promotes DSB repair, revealing a novel mode of action and role for cohesin in safeguarding genome integrity.

molecular biology↗

bioRxiv2021

Sir3 Heterochromatin Protein Promotes NHEJ by Direct Inhibition of Sae2

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) complex assembles heterochromatin-like chromatin at subtelomeres. SIR-mediated repressive chromatin limits double strand break (DSB) resection protecting damaged chromosome ends during HR. As resection initiation marks the cross-road between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR- mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Sir3 physically interacts with Sae2 and this interaction impairs Sae2-dependent MRX functions. As a consequence, Sir3 limits Mre11-mediated resection, delays MRX removal from DSB ends and promotes NHEJ.

genetics↗