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DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cellfree extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.
DNA double-strand breaks (DSBs) induce chromatin movement. In budding yeast, which repair DSBs primarily by homology-directed repair (HDR), induction of a single chromosomal break triggers increased local mobility: the DSB mean-square displacement is substantially higher than that of an undamaged region1,2. Moreover, multiple DSBs form clusters after traversing long distances3. DSB clustering may facilitate homology search, increase repair efficiency or shield breaks from misrepair4,5. These movements are intricately related to HDR. Factors that are critical for initiation of resection and downstream recombination are essential for DSB mobility in yeast1,2. In mammalian cells, DSBs are often described as more stable, suggesting that nonhomologous end joining (NHEJ), the predominant repair pathway, limits movement6-8. However, in human HeLa cells, RAD51-positive DSBs induced by alpha particles form clusters4. Similarly, damaged telomeres in human U2OS cells that are maintained by recombination merge in a RAD51-dependent manner9. Moreover, damaged active genes cluster in preparation for HDR5. Movement of deprotected mouse telomeres requires the LINC (linker of nucleoskeleton and cytoskeleton) complex, which transmits cytoskeletal forces from the cytoplasm to the nucleus10. The molecular basis of DSB movement and its role in DNA repair remain unclear.
The machinery that drives actin polymerization in the cytoplasm is also found in the nucleus11. Specifically, the ARP2/3 complex and its activator WASP, a Wiskott-Aldrich syndrome (WAS) protein family member, are located...