Abstract The telomere long noncoding RNA TERRA forms R-loops in vitro at telomeres in a RAD51AP1-dependent manner. In classic DNA double-strand break repair, RAD51AP1 promotes R-loop formation and enables RAD51 to form D-loops by promoting the invasion of local RNA transcripts into donor DNA to form DNA–RNA (DR)-loops. We have previously shown that cells lacking the basic domain of TRF2 and functional RAP1 accumulate telomere D-loops, resulting in homology-directed repair (HDR)-mediated telomere-telomere clustering and formation of ultrabright telomeres (UTs). TRF2B also cooperates with RAP1 to repress telomere R-loop formation in UTs. TERRA has been shown to promote telomere HDR, associating telomeres during R-loop formation. However, the mechanism behind TERRA-mediated telomere HDR and how the TRF2–RAP1 complex regulates telomere R-loops remain unclear. Using reconstituted biochemical systems, we found that RAD51AP1 and TERRA-dependent R-loops promote RAD51-mediated telomere D-loop formation in a TERRA length- and sequence-dependent manner. Specifically, RAD51–ssDNA filaments capture telomere R-loops preferentially over dsDNA. We also discovered that BLM’s interaction with the TRF2–RAP1 complex is required to promote BLM helicase-mediated unwinding of telomere R-loops. Importantly, BLM-deficient cells and cells reconstituted with BLM mutants unable to interact with TRF2 accumulate telomere R-loops in UTs. Our findings highlight a novel mechanism revealing that the TRF2–RAP1–BLM complex removes R-loops at telomeres to inhibit the generation of telomere D-loops, thus repressing telomere HDR and UT formation.
Liang et al. (Thu,) studied this question.