TRF binds to doublestranded telomeric DNA and has been implicated in the formation of the tloop structure.Human telomeres undergo acute deprotection when the telomeric protein TRF is removed and also become dysfunctional after excessive shortening in the absence of telomerase. Telomeres represent a unique opportunity to study these issues because they provide molecularly marked sites that can activate the canonical DNA damage response.To study the DNA damage response at telomeres, we targeted the mouse TRF gene on chromosome. Intercrosses of TRF mice failed to generate TRF offspring. Embryonic lethality occurred early in development before embryonic day. TRF and TIN are both required to stabilize TRF at telomeres, suggesting that the embryonic lethal phenotype of TRF, TIN and TRF deficiency will be substantially similar.Because TRF deficiency resulted in a rapid growth defect and genomewide telomere fusions in cultured cells, the events leading to early death of the embryos were not studied in detail.The cellular consequences of TRF deficiency were determined using mouse embryo fibroblasts retrovirus resulted in rapid deletion of the TRF gene, and concomitant loss of TRF protein was detectable at hpostinfection. By contrast, TRF was still detectable in immunoblots and by immunofluorescence and was present at telomeres, providing a marker for telomeres in cells lacking TRF.This arrest was abrogated by p deficiency, consistent with previous findings.In general, the telomeric signals were retained and were of equal intensity on sister telomeres.Partial loss of telomeric DNA from all chromosome ends was observed sporadically and its significance is not clear.The telomere fusion phenotype was rescued when TRFF p cells were complemented with a mouse TRF cDNA, demonstrating that it is caused by TRF deficiency.Although p cells yielded metaphase chromosomes after depletion of TRF, the cultures eventually perished, presumably due to the inability of the cells to segregate chromosomes in mitosis.Metaphase spreads with telomeres detected by FISH.The induction of telomere fusion upon inhibition of TRF function and their dependence on DNA ligase IV is consistent with previous experiments in which TRF was partially deactivated with a dominantnegative allele.The generation of a homogeneous population of cells, in which nearly all telomeres persisted in a free state without the protection of TRF, provided the opportunity to correlate the structure of the chromosome ends with the cellular response.The status of the telomeric overhangs was assessed using an ingel hybridization method that allows quantification of the singlestranded TTAGGG repeats.In this assay, a labelled CCCTAA probe is annealed to native DNA and the overhang signal is normalized to the total TTAGGG repeat signal obtained after ingel denaturation of the DNA.Degradation of the telomeric overhang upon TRF inhibition is known to require the nucleotide excision repair endonuclease ERCCXPF.ERCCXPF has not yet been implicated in global NHEJ but most NHEJ takes place in the context of short overhangs, and directed analysis of joining events at ends with long overhangs has not been performed.Telomeric DNA was denatured in gel and hybridized with an endlabelled probe.The asterisks indicate presumed chromosome internal sites with homology to the probe.The bracket on the left shows the position of most mouse telomeric fragments.