TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

Abstract Inappropriate homology-directed repair (HDR) of telomeres results in catastrophic telomere loss and aberrant chromosome fusions, leading to genome instability. We have previously shown that the TRF2–RAP1 heterodimer protects telomeres from engaging in aberrant telomere HDR. Cells lacking the basic domain of TRF2 and functional RAP1 display HDR-mediated telomere clustering, resulting in the formation of ultrabright telomeres (UTs) and massive chromosome fusions. Using purified proteins, we uncover three distinct molecular pathways that the TRF2–RAP1 heterodimer utilizes to protect telomeres from engaging in aberrant HDR. We show mechanistically that TRF2–RAP1 inhibits RAD51-initiated telomeric D-loop formation. Both the TRF2 basic domain and RAP1-binding to TRF2 are required to block RAD51-mediated homology search. TRF2 recruits the BLM helicase to telomeres through its TRFH domain to promote BLM-mediated unwinding of telomere D-loops. In addition, TRF2–RAP1 inhibits BLM–DNA2-mediated 5′ telomere end resection, preventing the generation of 3′ single-stranded telomere overhangs necessary for RAD51-dependent HDR. Importantly, cells expressing BLM mutants unable to interact with TRF2 accumulate telomere D-loops and UTs. Our findings uncover distinct molecular mechanisms coordinated by TRF2–RAP1 to protect telomeres from engaging in aberrant HDR.