Biology subjects

Savocco, J.

Publications and source records attributed to Savocco, J..

3 recordsLinked to original sources

bioRxiv2024

Condensin loop extrusion properties, roadblocks, and role in homology search in S. cerevisiae

The in vivo mechanism, cis-acting roadblocks, and biological functions of loop extrusion by eukaryotic SMC complexes are incompletely defined. Here, we identify condensin-dependent Hi-C contact stripes at the Recombination Enhancer (RE) and the rDNA in S. cerevisiae. The RE is an autonomous condensin loading site only active in MATa cells from which oriented, unidirectional loop extrusion proceeds with an estimated processivity [~]150-250 kb and a density [~]0.04-0.18 that varies across the cell cycle. Centromeres, replication forks and highly-transcribed RNA PolII-dependent genes are roadblocks for condensin. Cohesin is not an obstacle for condensin while Top2 promotes its loop extrusion activity. A DNA double-strand break at MAT blocks loop extrusion, resulting in the establishment of a [~]170 kb-long RE-MAT loop. The RE and the DSB are required and sufficient to form this site-specific loop, which promotes RE-proximal homology identification in the early stages of recombinational DNA break repair. We propose that the juxtaposition of the broken MATa site and its target HML donor is the relevant structure by which condensin promotes MATa-to- switching.

molecular biology↗

bioRxiv2023

Mechanism of homology search expansion during recombinational DNA break repair

Homology search catalyzed by a RecA/Rad51 nucleoprotein filament (NPF) is a central step of DNA double-strand break (DSB) repair by homologous recombination. How it operates in cells remains elusive. Here we developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short NPFs is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection by Exo1 drives this transition from local to genome-wide search by providing ssDNA substrates for assembly of extensive NPFs. DSB end-tethering promotes coordinated homology search by NPFs formed on the two DSB ends. Finally, an autonomous genetic element on chromosome III engages the NPF and stimulates homology search in its vicinity. This work reveals the mechanism of the progressive and uneven expansion of homology search orchestrated by chromatin organizers, long-range resection, end-tethering, specialized genetic elements, and that exploits the stiff NPF structure conferred by Rad51 oligomerization. Highlights- Cohesin-mediated chromatin loops constrain homology search in cis for NPF regions close to the resection front - Stiffening of ssDNA by Rad51 enables genome-wide homology search by DSB-proximal sites - Exo1-mediated long-range resection promotes genome-wide homology search - DSB end-tethering promotes coordinated homology search by NPFs formed on both DSB ends - The recombination enhancer focuses homology search in its vicinity

genetics↗

bioRxiv2020

Cohesin regulates homology search during recombinational DNA repair

Homologous recombination (HR) is a ubiquitous DNA double-strand break (DSB) repair mechanism that promotes cell survival. It entails a potentially genome-wide homology search step, carried out along a conserved RecA/Rad51-ssDNA nucleoprotein filament (NPF) assembled on each DSB ends1-3. This search is subdued to NPF-dsDNA collision probability, dictated in part by chromatin conformation2,4. In contrast to the extensive knowledge about chromatin composition and mobility changes elicited by the DNA damage checkpoint (DDC)5-7, whether, how, and to which extent a DSB impacts spatial chromatin organization, and whether this organization in turns influences the homology search process, remains ill-defined8,9. Here we characterize two layers of spatial chromatin reorganization following DSB formation in S. cerevisiae. While cohesin folds chromosomes into cohesive arrays of 10-20 kb long chromatin loops as cells arrest in G2/M10,11, the DSB-flanking regions locally interact in a resection- and 9-1-1 clamp-dependent manner, independently of cohesin and HR proteins. This local structure blocks cohesin progression, constraining the extending NPF at loop base. Functionally this organization promotes side-specific cis DSB-dsDNA interactions that scales with loop expansion span, and provides a kinetic advantage for identification of intra- over inter-chromosomal homologies. We propose that cohesins regulate homology search by promoting cis dsDNA over-sampling, both upon loop expansion-coupled unidimensional dsDNA scanning, NPF trapping, and chromosome individualization, largely independent of their role in sister chromatid cohesion.

genetics↗