Cohesin-mediated replication fork tethering enables fork slowing and reversal

Technical Summary: Cohesin-mediated replication fork tethering enables fork slowing and reversal

Problem Statement
DNA replication forks face frequent challenges from genotoxic stress, such as cancer chemotherapeutic treatments, leading to the accumulation of single-stranded DNA (ssDNA) and slowed DNA synthesis. While the plasticity of replication forks is known to ensure genome stability, the mechanisms governing how the three-dimensionally organized genome influences fork behavior under stress remain unclear. Specifically, the role of the cohesin complex—which organizes the genome via loop extrusion and associates with replication origins—in the immediate response to replication stress (RS) and the regulation of fork plasticity (slowing and reversal) was previously unknown.

Methodology
The study employed a multidisciplinary approach combining proteomics, high-resolution chromatin conformation capture, and functional genetics:

• Proteomics: "Isolation of proteins on nascent DNA" (iPOND) coupled with mass spectrometry (MS) was used to characterize the proteome of replication forks under different Hydroxyurea (HU) treatments (2 mM for 2 hours to stall forks; 100 µM for 1 hour to slow forks).
• Proximity Ligation Assays (SIRF): Used to visualize and quantify the interaction between cohesin subunits and nascent DNA (EdU-labeled) in situ.
• Genetic Perturbations: The authors utilized auxin-inducible degron (AID) systems to rapidly deplete specific cohesin factors (NIPBL, SMC1, RAD21, CTCF, Sororin, WAPL) and employed separation-of-function mutants of SMC1 (SMC1^3D^ and SMC1^4E^) to distinguish between loop extrusion and sister chromatid cohesion functions.
• Repli-C: A novel Micro-C-based technique developed to capture chromatin contacts specifically at nascent DNA (EdU-labeled) by ligating DNA contacts after pull-down, allowing for high-resolution mapping of replication-associated genomic interactions.
• Functional Assays: DNA fiber assays (including S1 nuclease sensitivity), electron microscopy for fork reversal visualization, and fork degradation assays in BRCA2-deficient backgrounds were used to assess fork progression, reversal, and stability.

Key Results

1. Cohesin Accumulation at Stalled Forks: iPOND-MS and SIRF assays revealed that cohesin subunits (SMC1, RAD21, STAG2) and the loader NIPBL accumulate at stalled replication forks upon HU treatment. This accumulation is dependent on NIPBL and the loop extrusion activity of cohesin, rather than on sister chromatid cohesion establishment (mediated by Sororin) or TAD insulation (mediated by CTCF).
2. Reorganization of Replication Contacts: Using Repli-C, the authors observed "fountain-like" structures at replication origins representing coupled sister forks. Upon HU treatment, cohesin is required to stabilize intra-replicon contacts near origins and, crucially, to establish physical contacts between forks of adjacent replicons (inter-replicon tethering). These inter-replicon contacts are dependent on cohesin loop extrusion and are significantly enhanced by replication stress.
3. Loop Extrusion Drives Fork Slowing and Reversal: Depletion of NIPBL or expression of the loop-extrusion-deficient SMC1^4E^ mutant resulted in unrestrained fork progression during stress. Conversely, wild-type cohesin and the SMC1^3D^ mutant (which retains loop extrusion but lacks cohesion) restored fork slowing.
4. Mechanism of Action via PRIMPOL Inhibition: The study demonstrates that cohesin-mediated tethering prevents the action of the primase PRIMPOL. In the absence of functional loop extrusion (NIPBL depletion or SMC1^4E^), PRIMPOL levels increase at stalled forks, leading to discontinuous DNA synthesis and preventing fork reversal. Depletion of PRIMPOL in these cohesin-deficient contexts restored the ability of forks to undergo reversal and degradation in BRCA2-deficient cells.
5. Cellular Fitness: Cells lacking NIPBL or expressing SMC1^4E^ showed increased sensitivity to prolonged low-dose genotoxic treatments, comparable to cells with defective fork reversal machinery.

Significance and Claims
The paper claims to redefine the functions of loop-extruding cohesin, positioning it not merely as a structural organizer of the genome but as an active mediator of the replication stress response. The authors propose that cohesin-mediated loop extrusion promotes the tethering of replication forks from adjacent origins. This tethering restricts fork progression and prevents the uncoupling of helicase and polymerase activities, thereby limiting the exposure of ssDNA that would otherwise trigger PRIMPOL-mediated re-priming. By suppressing PRIMPOL, cohesin facilitates the biochemical rearrangements necessary for fork reversal, a critical mechanism for genome stability.

The findings suggest that the response to replication stress is integrated into the organized genome architecture via cohesin loop extrusion. Furthermore, the authors highlight the potential clinical relevance of these findings, noting that frequent cohesin mutations found in tumors may impact the efficacy of cancer chemotherapies that induce replication stress by compromising this tethering and fork plasticity mechanism.