Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis. Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10–15% of human cancers. How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood. Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance. DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication. Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit. Break-induced telomere synthesis requires the RFC–PCNA–Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51. Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance.

Summary Telomere length maintenance is a requisite feature of cellular immortalization and a hallmark of human cancer. While most human cancers express telomerase activity, approximately 10-15% employ a recombination-dependent telomere maintenance pathway known as Alternative Lengthening of Telomeres (ALT) that is characterized by multi-telomere clusters and associated promyelocytic leukemia protein bodies. Here, we show that a DNA double-strand break (DSB) response at ALT telomeres triggers long-range movement and clustering between chromosome termini, resulting in homology-directed telomere synthesis. Damaged telomeres initiate increased random surveillance of nuclear space before displaying rapid directional movement and association with recipient telomeres over micron-range distances. This phenomenon required Rad51 and the Hop2-Mnd1 heterodimer, which are essential for homologous chromosome synapsis during meiosis. These findings implicate a specialized homology searching mechanism in ALT dependent telomere maintenance and provide a molecular basis underlying the preference for recombination between non- sister telomeres during ALT.

Activation of a telomere maintenance mechanism (TMM) is permissive for replicative immortality and a hallmark of human cancer. While most cancers rely on reactivation of telomerase, a significant fraction utilizes the recombination dependent alternative lengthening of telomeres (ALT) pathway. ALT is enriched in tumors of mesenchymal origin, including those arising from bone, soft tissue, and the nervous system, and usually portends a poor prognosis. Recent insights into the mechanisms of ALT are uncovering novel avenues to exploit vulnerabilities and may facilitate clinical development of ALT detection assays and personalized treatment decisions based on TMM status. Treatments targeting ALT may hold promise for a broadly applicable therapeutic modality specific to mesenchymal lineage tumors, something that has thus far remained elusive.