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 favor productive interactions This ensures efficient & accurate homology search! 10/12

DSBs recruit two cohesin pools to distinct domains 🔹Loop-forming cohesin accumulates across ~1 Mb, controlling homology sampling range. 🔹Cohesive cohesin accumulates at DSBs, tethering the broken DNA ends to the intact sister chromatid. 9/12

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 8/12

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. 7/12

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. 6/12

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! 5/12

RAD51 filaments sample local TADs By mapping RAD51, a key repair protein, we found that homology search occurs within topologically associated domains near the break rather than across the entire genome. How does chromosome organization influence the search range? 4/12

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. 3/12

Just had our annual lab christmas dinner, featuring curry and Schrottwichteln (a not-quite-secret-santa, rules here: geschenke.de/blogs/wichteln/…). Wishing you a festive end to 2024!

A great big congratulations to Sofia, who successfully defended her PhD thesis @IMBA_Vienna yesterday. Here’s one happy graduate complete with traditional lab-made defense hat 🎩 Well done Sofia! @SofiaKolesni29 @viennabiocenter @TrainingVbc

… also at the symposium is new PhD student @neos_cruz, who presented his Master’s thesis in today’s 3-minute thesis competition - nicely done, Neos!

Massive congratulations to @pauldanielbatty, who has succesfully defended his PhD thesis @IMBA_Vienna @viennabiocenter, titled 'The mechanics of sister chromatid resolution'. Well done Paul!!

Together these results provide evidence that cohesin not only holds sister chromatids together, but also actively promotes their separation by its loop extrusion activity. Furthermore, removal of cohesion is not itself a prerequisite for high levels of sister resolution 8/9

To quantify genomic intervals over which sister chromatids resolve from each other, we performed sister-chromatid-sensitive Hi-C. WAPL depletion resolved sister chromatids over more than 10Mb, whereas depletion of NIPBL strongly reduced sister resolution vs wildtype 7/9

Sister DNAs in WAPL-depleted cells resolve around a single cohesin axis, which splits upon depletion of the cohesion cofactor Sororin. Thus, although cohesion prevents the separation of cohesin axes, it imposes only small constraints on sister chromatid resolution 6/9

Depletion of WAPL after chromosome replication promotes further resolution of sister chromatids into two parallel threads in G2 cells, resembling the morphology of wildtype chromosomes in mitotic prophase 5/9

This allows us to visualise substantial resolution of sisters in G2. Resolution is reduced by degradation of the cohesin loop-extrusion cofactor NIPBL, but not by degradation of condensins, indicating cohesin-mediated sister chromatid resolution already prior to mitotic entry 4/9