Cells were transfected with plasmid DNA using jetPEI, according to the manufacturer's instructions. For silencing experiments, 5 x 10^4 cells were seeded 24 h before transfection and transfected with 20 nmol of siRNA using INTERFERin according to the manufacturer's instructions.

DNA double-strand breaks (DSB) are very harmful lesions that can generate genome rearrangements. In this study, we used intrachromosomal reporters to compare both the efficiency and accuracy of end-joining occurring with close (34 bp apart) vs. distant DSBs (3200 bp apart) in human fibroblasts. We showed that a few kb between two intrachromosomal I-SceI-induced DSBs are sufficient to foster deletions and capture/insertions at the junction scar. Captured sequences are mostly coupled to deletions and can be partial duplications of the reporter (i.e., sequences adjacent to the DSB) or insertions of ectopic chromosomal sequences (ECS). Interestingly, silencing 53BP1 stimulates capture/insertions with distant but not with close double-strand ends (DSEs), although deletions were stimulated in both case. This shows that 53BP1 protects both close and distant DSEs from degradation and that the association of unprotection with distance between DSEs favors ECS capture. Reciprocally, silencing CtIP lessens ECS capture both in control and 53BP1-depleted cells. We propose that close ends are immediately/rapidly tethered and ligated, whereas distant ends first require synapsis of the distant DSEs prior to ligation. This "spatio-temporal" gap gives time and space for CtIP to initiate DNA resection, suggesting an involvement of single-stranded DNA tails for ECS capture. We therefore speculate that the resulting single-stranded DNA copies ECS through microhomology-mediated template switching.