Multiple pathways regulate the repair of double-strand breaks (DSBs) to suppress potentially dangerous ectopic recombination. Both sequence and chromatin context are thought to influence pathway choice between non-homologous end-joining (NHEJ) and homology-driven recombination. To test the effect of repetitive sequences on break processing, we have inserted TG-rich repeats on one side of an inducible DSB at the budding yeast MAT locus on chromosome III. Five clustered Rap1 sites within a break-proximal TG repeat are sufficient to block Mre11-Rad50-Xrs2 recruitment, impair resection, and favor elongation by telomerase. The two sides of the break lose end-to-end tethering and show enhanced, uncoordinated movement. Only the TG-free side is resected and shifts to the nuclear periphery. In contrast to persistent DSBs without TG repeats that are repaired by imprecise NHEJ, nearly all survivors of repeat-proximal DSBs repair the break by a homology-driven, non-reciprocal translocation from ChrIII-R to ChrVII-L. This suppression of imprecise NHEJ at TG-repeat-flanked DSBs requires the Uls1 translocase activity. Marcomini et al. show that the presence of interstitial telomeric repeat sequences near a double-strand break alters the outcome of repair. A TG-flanked break loads MRX asymmetrically, supports resection only on one side, and allows uncoordinated movement of the break ends. The resected TG-free end invades homology on another chromosome driving a unidirectional translocation event.
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