Telomeres are essential for genome stability, because they prevent chromosome ends from being ligated together by the repair pathways that act on DNA double-strand breaks1,2
. The telomeric DNA is shielded from double-strand break repair activities by specialized telomere proteins that bind along the duplex region of the telomere and to the overhang on the 3′ G-rich strand. In vertebrate cells the telomeric duplex is packaged by a complex of six proteins (Trf1, Trf2, Rap1, Tin2, Tpp1 and Pot1) termed shelterin1
. Two of the proteins, Pot1 and Tpp1, also bind to the G-strand overhang. Pot1 is a specialized G-strand binding protein, whereas Tpp1 interacts with both telomerase and other components of the shelterin complex3,4
Although telomere proteins shield the chromosome end from unwanted repair activities, a wide range of DNA damage response proteins are also found at telomeres (for example, ATM, ATR, MRN and Ku)1,2,5
. The presence of these proteins is not merely a hallmark of a dysfunctional telomere. Instead, damage response proteins are recruited to functional telomeres during replication, where they cooperate with telomere proteins to ensure correct processing of the G-overhang and appropriate telomere length regulation6-10
. Notably, the recruitment of ATM and ATR in late S/G2 does not activate a full DNA damage response11,12
, indicating that downstream signaling must be quenched during telomere replication. However, if telomeres are critically short or otherwise defective, ATM-ATR signaling is not quenched and a full DNA damage response ensues5,13,14
. Although the telomeric defects leading to ATM signaling have been explored in some detail, little is known about the stimuli leading to ATR activation. Our results now point to a role for Pot1 in quenching ATR-Chk1 signaling.
In Schizosaccharomyces pombe
, Pot1 depletion leads to rapid loss of telomeric and subtelomeric DNA and widespread cell death15
. This observation suggested that Pot1 proteins function by protecting the G-strand overhang from degradation, thus preventing telomere loss and chromosome fusions. However, gene knockouts in mice and chicken cells indicate that this is not the case in all eukaryotes, because vertebrate cells lacking Pot1 retain telomeric G-overhangs and show only a slight increase in chromosome fusions16-18
. We have studied Pot1 function using a conditional chicken DT40 cell line in which one POT1
allele is disrupted and the other is replaced by a cDNA encoding an estrogen receptor-Pot1 fusion protein (Er-Pot1)17
. The Er-Pot1 cells grow normally in tamoxifen, but drug removal results in loss of Pot1 from telomeres, a telomeric DNA damage response and cell cycle arrest in late S/G2. Pot1 loss also leads to an increase in G-overhang length and, as described below, to rapid telomere growth. The absence of G-overhang degradation indicates that other proteins must substitute for Pot1 in overhang protection. However, they are unable to prevent the telomere from being detected as DNA damage. Our findings indicate that a major function of Pot1 is to prevent the G-overhang from activating a DNA damage response. A second function is to regulate telomere length.
Although ATM is involved in damage signaling at critically short or Trf2-depleted telomeres5,13,14
, several observations suggested that the checkpoint activated after Pot1 loss might instead be mediated by ATR. First, a telomere with its 3′ G-strand overhang provides the classic structure (a 5′ double-stranded/single-stranded DNA (ds/ssDNA) junction) for ATR activation19
. Second, Rpa (replication protein A), a key protein in ATR recruitment, is known to bind to telomeres during S phase12
and hence is likely to protect the overhangs in late S/G2 in cells that lack functional Pot1. We have used the Er-Pot1 cell line to explore the role of ATM and ATR in the checkpoint response to Pot1 removal. We show that the checkpoint is ATM independent but requires Chk1 signaling, a hallmark of ATR activation. We also show that Pot1 removal leads to unusually rapid telomere growth but that the rate of growth is decreased if Chk1 signaling is inhibited and the Er-Pot1 cells can cycle. Our results indicate that cell cycle progression is an important component of telomere length regulation.