10/12
Our model:
🔹 Loop-forming cohesin sets a megabase-scale search radius within the TAD
🔹 Cohesive cohesin forms a local clamp that tethers DNA ends to the sister chromatid
Together, they focus RAD51 sampling and enable accurate repair!

9/12
ChIP-seq of specific cohesin subunits revealed distinct architectures:
🔹 Loop-forming cohesin (NIPBL-bound) spans broadly around the break
🔹 Cohesive cohesin (sororin-bound) concentrates at the break
Functionally, disturbing either loop extrusion or cohesion delays or blocks repair!

8/12
What happens if we disrupt sister cohesion?
Upon sororin depletion:
✅ Loops remain intact
❌ Sister contacts are lost
Without cohesion, the DSB cannot efficiently locate its sister chromatid 🚫

7/12
Sister-pore-C showed that DSBs locally increase looping and create strong trans-sister contact interactions, putting the DNA broken ends in contact with the sister chromatid.

6/12
Homology-directed repair uses the sister chromatid, but how does the broken end stay connected to its sister template? 🤔
We developed sister-pore-C, a high-resolution method combining BrdU labeling and nanopore reads to map cis-sister vs trans-sister contacts.

5/12
How is this local search space defined?
By perturbing loop extrusion, we found:
🔹 Reducing loops (NIPBL or RAD21 depletion) narrows RAD51 sampling
🔹 Hyper-extrusion (WAPL depletion) expands sampling too broadly
Cohesin loops regulate the search radius!

4/12
By mapping RAD51, we found that homology search occurs within the local TAD near the break and not across the whole genome 🎯 !
TAD boundaries act as barriers that confine sampling.

3/12
To study this, we induced DSBs at defined genomic sites in S/G2-synchronized human cells.
Most breaks were repaired by homologous recombination, allowing us to map the search process precisely.

We propose a dual function of cohesin in homology search:

🔹Loop-forming cohesin defines search space, limited by TADs
🔹Cohesive cohesin tethers the break to its sister, to favour productive interactions

This ensures efficient & accurate homology search!

Cohesion directs homology search towards sister chromatid

What happens if we disrupt sister cohesion? Upon sororin depletion (cohesion stabilizer):
✅ Loops remain intact
❌ Contacts between sister chromatids are lost

DSBs remodel sister chromatids in cis and trans

Sister-pore-C allows us to track how DNA architecture changes before and after DSBs. We found that DSBs locally increase loops and interactions between the broken ends and the intact sister chromatid.

A new tool: sister-pore-C

Homology-directed repair normally uses the sister chromatid as a template, but how does a DSB find its sister locus in 3D space? We developed sister-pore-C, a high-resolution method to map chromatin interactions within & between sister chromatids.

Cohesin-mediated loops control search range

When we disrupted cohesin regulators, search dynamics changed:
🔹 Reducing loops by NIPBL depletion narrowed homology search
🔹 Expanding loops by WAPL depletion made the search broader

Cohesin loops regulate homology search range!

RAD51 filaments sample local TADs

By mapping RAD51, a key repair protein, we found that homology search occurs within topologically associated domains near the breakrather than across the entire genome.
How does chromosome organization influence the search range?

Site-specific induction of homology-directed repair

To study how chromosome architecture influences homology search, we induced DSBs at specific genomic sites in S/G2-synchronized human cells. Most breaks were repaired by homologous recombination.