Summary Mammalian telomeres are associated with shelterin, the telomere specific protein complex that solves the end-protection problem. The telomeric shelterin binding sites, TTAGGG repeats, are maintained by telomerase, which solves the end-replication problem. We report that the TTAGGG repeat arrays of human and mouse telomeres challenge the DNA replication machinery, giving rise to replication-dependent defects that resemble those of the aphidicolin-induced common fragile sites. Conditional gene deletion experiments showed that efficient duplication of telomeric DNA requires the shelterin component TRF1. In the absence of TRF1, telomeres activate the ATR kinase in S phase and show a fragile site phenotype in metaphase. SMARD showed that TRF1 promotes efficient replication of TTAGGG repeats and prevents fork stalling. Two helicases that can remove G4 DNA structures, BLM and RTEL1, were required to repress the fragile telomere phenotype. These results identify a second telomere replication problem that is solved by the shelterin component TRF1.

Although broken chromosomes can induce apoptosis, natural chromosome ends (telomeres) do not trigger this response. It is shown that this suppression of apoptosis involves the telomeric-repeat binding factor 2 (TRF2). Inhibition of TRF2 resulted in apoptosis in a subset of mammalian cell types. The response was mediated by p53 and the ATM (ataxia telangiectasia mutated) kinase, consistent with activation of a DNA damage checkpoint. Apoptosis was not due to rupture of dicentric chromosomes formed by end-to-end fusion, indicating that telomeres lacking TRF2 directly signal apoptosis, possibly because they resemble damaged DNA. Thus, in some cells, telomere shortening may signal cell death rather than senescence.

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Cells from Werner syndrome patients are characterized by slow growth rates, premature senescence, accelerated telomere shortening rates, and genome instability. The syndrome is caused by the loss of the RecQ helicase WRN, but the underlying molecular mechanism is unclear. Here we report that cells lacking WRN exhibit deletion of telomeres from single sister chromatids. Only telomeres replicated by lagging strand synthesis were affected, and prevention of loss of individual telomeres was dependent on the helicase activity of WRN. Telomere loss could be counteracted by telomerase activity. We propose that WRN is necessary for efficient replication of G-rich telomeric DNA, preventing telomere dysfunction and consequent genomic instability.

Primary human cells in culture invariably stop dividing and enter a state of growth arrest called replicative senescence. This transition is induced by programmed telomere shortening, but the underlying mechanisms are unclear. Here, we report that overexpression of TRF2, a telomeric DNA binding protein, increased the rate of telomere shortening in primary cells without accelerating senescence. TRF2 reduced the senescence setpoint, defined as telomere length at senescence, from 7 to 4 kilobases. TRF2 protected critically short telomeres from fusion and repressed chromosome-end fusions in presenescent cultures, which explains the ability of TRF2 to delay senescence. Thus, replicative senescence is induced by a change in the protected status of shortened telomeres rather than by a complete loss of telomeric DNA.