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 cell-free 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.