TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination

Abstract

TRF2 (telomeric repeat binding factor 2) is an essential component of the telomeric cap, where it forms and stabilizes the T-loop junctions. TRF2 forms the T-loops by stimulating strand invasion of the 3 overhang into duplex DNA. TRF2 also has been shown to localize to nontelomeric DNA double-strand breaks , but its functional role in DNA repair has not been examined. Here, we present evidence that TRF2 is involved in homologous recombination (HR) repair of nontelomeric double-strand breaks. Depletion of TRF2 strongly inhibited HR and delayed the formation of Rad51 foci after ␥-irradiation, whereas overexpression of TRF2 stimulated HR. Depletion of TRF2 had no effect on nonhomologous end-joining, and overexpression of TRF2 inhibited nonhomologous end-joining. We propose, based on our results and on the ability of TRF2 to mediate strand invasion, that TRF2 plays an essential role in HR by facilitating the formation of early recombination intermediates.DNA repair ͉ telomeres T RF2 (telomeric repeat binding factor 2) was first identified as a major telomere binding protein that shields chromosome ends from degradation and from end-to-end fusions (1-3). Mammalian telomeres form large duplex loops in which the single-stranded 3Ј end is embedded within the double-stranded DNA (4). TRF2 generates T-loop structures and recently was shown to bind to DNA junctions (4, 5). The mechanism by which TRF2 facilitates folding of telomeric DNA into T-loops involves binding of TRF2 complexes to DNA and untwisting the neighboring DNA, thereby favoring strand invasion (6).In addition to evidence of TRF2's role in telomere maintenance, there are multiple pieces of evidence that implicate TRF2 in double-strand break (DSB) repair of nontelomeric DNA (7-9). TRF2 localizes to DSB sites at the early stages of cellular response to DSBs, appearing in the first few seconds after DSB induction and leaving as DSBs are being processed (7). TRF2 is phosphorylated by ATM in response to DNA damage (8). The phosphorylated form of TRF2 is not bound to telomeric DNA and is localized to DNA damage sites (8). In turn, overexpression of TRF2 inhibits autophosphorylation of ATM and induction of DNA damage response (9).DSBs are repaired by two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ). During HR, the missing information is copied into the DSB site from a homologous sequence, whereas NHEJ joins the broken ends without homology. Several proteins involved in DSB repair interact with TRF2, such as the MRE11/Rad50/NBS1 complex, Ku70, WRN, and BLM (10-12). Thus, TRF2 is phosphorylated in response to DNA damage, localizes to DNA breaks, and interacts with DSB repair proteins, but its functional role in DSB repair is unknown. Here, we examined the role of TRF2 in DSB repair by HR and NHEJ. ResultsReporter Cell Lines for Detection of NHEJ and HR. To study the role of TRF2 in NHEJ and HR, we used an in vivo assay that measures the repair of an induced chromosomal break. The reporter cassette for detection of NHEJ previously ...

Impaired DNA double-strand break repair contributes to the age-associated rise of genomic instability in humans

“…To further examine the fidelity of NHEJ in a more quantitative way, we sequenced the junctions of the rescued plasmids and compared the fidelity between the two groups. Although the process of NHEJ is often associated with deletions or insertions, 4,21,22,23 the chance of acquiring insertions in eyelids fibroblasts is extremely slim (0-15%) (Supplementary Table S2), so it is not feasible to analyze the change of insertion sizes with age in these cell lines. We therefore systematically compared the sizes of deletions at the junction areas.…”

Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence

“…HCA2 cells. Our previous analysis of integrated clones indicated that transfection with 0.1 μg of plasmid results in the integration of a single copy of the reporter per genome (18). Three separate transfections were performed, giving rise to three independent pools of integrants.…”

“…To examine the effect of replicative senescence on DNA DSB repair by HR, we integrated a HR reporter cassette (Fig. 1A) (6,10,18) into the genome of HCA2 cells, a primary foreskin fibroblast strain, at population doubling (PD)26. The challenge of studying HR in the context of replicative senescence is that the reporter construct must be integrated in primary (nonimmortalized cells).…”

“…We used a sensitive fluorescent reporter assay developed by our laboratory (18), which specifically A challenge of analyzing HR repair in the context of replicative senescence is that reporter constructs must be integrated in primary human cells, and by the time integrants are selected, cells exhaust their replicative potential. We overcame this problem by pooling integrating clones at low PD numbers.…”

Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere

“…These studies described first in this laboratory showed that telomeric DNA is highly prone to replication fork slippage, and thus, the binding of TRF2 to slipped structures may be key in stopping further fork slippage and preventing the generation of deleterious structures (19). Moreover, TRF2 prevents NHEJ at non-telomeric sites through its basic domain (22). * This work was supported by National Institutes of Health Grants GM31819 hRap1 was first identified by yeast two hybrid analysis with hTRF2 as a bait (10).…”