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DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG) n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replicatio