Cruciform-forming AT/TA repeats are acted upon by structure-selective endonucleases and Rad51 prior to repositioning to the nuclear periphery for repair.

Structure-forming DNA repeats can pose a barrier to DNA replication and repair, creating chromosomal fragile sites. An AT/TA DNA repeat, derived from the Flex1 region of human common fragile site FRA16D, can form hairpin and cruciform structures, which interfere with DNA replication. When inserted into the Saccharomyces cerevisiae genome, the Flex1(AT)34 repeat stimulates chromosome deletions in a manner dependent on the Mus81-Mms4 nuclease and the SLX4 nuclease scaffold. It was previously found that hairpin-forming CAG/CTG repeats move to the nuclear periphery to maintain genomic stability. Here, we show that a structure-forming AT/TA repeat also relocalizes to the nuclear periphery in late S/G2 phase in a replication- and length-dependent manner. In contrast to the CAG repeat, this shift in nuclear positioning is dependent on polySUMOylation and the activity of the Mus81-Mms4 nuclease. Processing by the Mre11 nuclease and Rad51-dependent strand exchange occurs prior to repositioning. Replication analysis indicates that the replisome likely bypasses the AT/TA repeat, leaving behind a DNA structure that initiates relocation to the nuclear periphery. We conclude that AT/TA repeats form post-replicative DNA structures that are targeted for nuclease cleavage and require hairpin processing and repositioning to the nuclear periphery for homologous recombination-dependent repair.