Formation of cancerous translocations requires the illegitimate joining of chromosomes containing double-strand breaks (DSBs). It is unknown how broken chromosome ends find their translocation partners within the cell nucleus. Here, we have visualized and quantitatively analysed the dynamics of single DSBs in living mammalian cells. We demonstrate that broken ends are positionally stable and unable to roam the cell nucleus. Immobilization of broken chromosome ends requires the DNAend binding protein Ku80, but is independent of DNA repair factors, H2AX, the MRN complex and the cohesion complex. DSBs preferentially undergo translocations with neighbouring chromosomes and loss of local positional constraint correlates with elevated genomic instability. These results support a contact-first model in which chromosome translocations predominantly form among spatially proximal DSBs.DSBs occur frequently in the genome through the action of DNA-damaging agents or during genome replication 1,2 . DSBs are hazardous to the cell because failure to properly repair them may lead to tumorigenic trans-locations 3 . How broken ends of different chromosomes meet in the cell nucleus to eventually form a translocation is poorly understood 4 . Two hypotheses have been put forth: the 'contact-first' model proposes that interactions between breaks on distinct chromosomes can only take place when the breaks are created in chromatid fibres that colocalize at the time of DNA damage 5 . In contrast, the 'breakage-first' hypothesis postulates that breaks formed at distant locations are able to scan the nuclear space for potential partners and come together to produce translocations 6 . The two models make divergent predictions as to the dynamic behaviour of broken chromosome ends. In the breakage-first model, single DSBs are required to undergo large-scale motions within the cell nucleus and must be able to roam the nuclear space in search of appropriate interaction partners. In the contact-first model, only limited local positional motion of DSBs is expected. The available experimental data is contradictory: in mammalian cells, induction of extensive chromosome damage using ultrasoft X-rays 7 , laser microirradiation 8 or γ-irradiation 8 indicates that damaged DNA is largely stationary. In contrast, α-particle irradiation leads to large-scale motion and clustering of 6Correspondence should be addressed to T.M. (e-mail: mistelit@mail.nih.gov). AUTHOR CONTRIBUTIONS E.S. and T.M. designed the study, E.S., J.F.D. and K.S. performed the experiments, J.M., A.N. and T.R. provided reagents and advice, and E.S. and T.M. wrote the manuscript. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.Note: Supplementary Information is available on the Nature Cell Biology website. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2008 July 3. Published in final edited form as:Nat Cell Biol. 2007 June ; 9(6): 675-682. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manus...

DNA repair and maintenance of genome stability are crucial to cellular and organismal function, and defects in these processes have been implicated in cancer and ageing. Detailed molecular, biochemical and genetic analyses have outlined the molecular framework involved in cellular DNA-repair pathways, but recent cell-biological approaches have revealed important roles for the spatial and temporal organization of the DNA-repair machinery during the recognition of DNA lesions and the assembly of repair complexes. It has also become clear that local higher-order chromatin structure, chromatin dynamics and non-random global genome organization are key factors in genome maintenance. These cell-biological features of DNA repair illustrate an emerging role for nuclear architecture in multiple aspects of genome maintenance.

Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position.