New Research Reveals Novel Role of DNA Loops in Repairing Genetic Damage

The homologous recombination (HR) pathway repairs DNA damage, such as double-strand breaks (DSBs), by copying sequence information from a homologous donor DNA template (typically the sister chromatid) to the damaged site. During HR in mammals, the RAD51 (radiation-sensitive protein 51) recombinase forms a nucleoprotein filament around the resected single-stranded DNA (ssDNA) generated during DSB end processing at the DSB site, known as the presynaptic filament. Subsequently, the presynaptic filament scans the genome for a suitable homologous DNA donor through a process called homology search. However, how this homology search occurs in the context of the three-dimensional (3D) genome remains largely unexplored.

To investigate DSB repair, researchers utilized an optogenetic multi-target CRISPR-Cas9 system capable of generating DSBs in a high-throughput and temporally controlled manner. Following multi-target CRISPR-induced damage in human embryonic kidney (HEK) 293T cells, they performed time-resolved Hi-C and chromatin immunoprecipitation sequencing (ChIP-seq) analyses to capture changes in genome structure and chromatin interactions of key factors during the repair process. Additionally, they conducted recombination assays using an HR-GFP reporter in mouse embryonic stem cells to measure HR efficiency for Cas9-induced DSBs targeting donor sites located at different distances.

Figure 1. Chromatin loops accelerate the homology search. (Marin-Gonzalez A, et al., 2025)

The researchers discovered that Cas9-induced DSBs induce the formation of chromatin loops anchored at the break sites in a cohesin-dependent manner. However, contrary to expectations, these break-anchored chromatin loops do not form immediately after DSB generation but rather form relatively late (>1 hour) during the repair process, coinciding temporally with DNA end resection and presynaptic filament formation. ChIP-seq data further revealed that, in addition to binding to ssDNA around the approximately 5 kb long presynaptic filament, RAD51 also binds to double-stranded DNA (dsDNA), covering extensive chromatin regions extending hundreds of kilobases flanking the DSB site. Additional experiments indicated that this extensive chromatin region reflects RAD51-chromatin interactions occurring during the homology search.

This extensive RAD51 chromatin region coincides temporally with the formation of break-anchored chromatin loops, is confined by TAD boundaries, and is regulated by the cohesin unloading factor WAPL. Thus, break-anchored chromatin loops participate in the homology search. Indeed, depletion of NIPBL, the cohesin subunit responsible for loop extrusion, leads to reduced HR efficiency, and this effect is more pronounced when the donor is located intrachromosomally at a greater distance (hundreds of kilobases) from the DSB. These results suggest that cohesin, responsible for loop extrusion, plays a role in homology search, and its importance in this search increases with the increasing genomic distance between the DSB and the intrachromosomal homologous donor within a TAD.

In summary, cohesin, by extruding break-anchored chromatin loops, likely facilitates the homology search during HR, thereby transforming the intrachromosomal scanning of DSBs from a 3D problem into an ordered 1D process. These studies reveal the unexpected role of chromatin loops organized by cohesin as mediators of HR repair and capture the genomic search occurring during mammalian HR. These findings pave the way for further exploration of the interplay between 3D genome structure and DSB repair processes, as well as the mechanisms of homology search in the cellular context.

Reference

Marin-Gonzalez A, et al. Cohesin drives chromatin scanning during the RAD51-mediated homology search. Science, 2025, 390(6777): eadw1928.