Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay in human cells (Indel-Seq) to monitor rearrangements at the TRIM37 acceptor locus which reports indels stemming from experimentally-induced and spontaneous genome instability. Templated insertions derive from sequences genome-wide and are enriched within 100 kb of donor regions flanking a DSB. Insertions require contact between donor and acceptor loci as well as DNA-PK catalytic activity. Notably, these templated insertions originate from actively transcribed loci, underscoring transcription as a critical source of spontaneous genome instability. Transcription-coupled insertions involve a DNA/RNA hybrid intermediate and are stimulated by DNA end-processing. Using engineered Cas9 breaks, we establish that ssDNA overhangs at the acceptor site greatly stimulate insertions. Indel-Seq revels that insertions are generated via at least three distinct pathways. Our studies indicate that insertions result from movement and subsequent contact between acceptor and donor loci followed invasion or annealing, then by non-homologous end-joining at the acceptor site.

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.