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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.
Tremendous progress has been made in identifying the events responsible for recognizing and repairing DNA double-strand breaks (DSBs)1. A complex aspect of this response is homology-directed DNA repair (HDR), which can involve numerous possibilities to capture homologous regions of the genome to use for templated DNA synthesis and repair. The detailed order of molecular events that ensues after the initial sensing of DSBs to allow the execution of homology-directed synthesis remains enigmatic. Specifically, how the DNA damage response coordinates productive interactions between DNA replication complexes to perform break-induced DNA synthesis has not been extensively demonstrated in mammalian cells. ALT is a clinically relevant example of a DNA repair pathway that requires homology-directed synthesis to maintain telomeres in ~10-15% of human cancers2,3. Additionally, such synthesis could represent an attractive therapeutic target against cancers, especially if it proves to be different from canonical S-phase replication.
Telomere breaks stimulate long-tract synthesis
To study homology-directed synthesis at ALT telomeres, we developed a bromodeoxyuridine (BrdU) pulldown approach to isolate and quantify nascent telomeres synthesized following telomere-targeted DSBs generated by the fusion of the Shelterin component TRFlto the FokI endonuclease (Fig. 1a). Using stable ALT-positive U2OS cell lines expressing TRFl-FokI under tetracycline-control, a 2-h damage induction with wild-type TRFl-FokI, but not the FokI(D450A) nuclease-null mutant, resulted in a ~ 10-fold increase...