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    • Telomerase action at the telomere is highly regulated it

    2023-09-18

    Telomerase action at the telomere is highly regulated; it preferentially elongates the shortest telomeres, and recruitment of the enzyme complex to the telomere occurs in mid-S phase of the cell cycle (Bianchi and Shore, 2007, Britt-Compton et al., 2009, Teixeira et al., 2004, Hemann et al., 2001, Tomlinson et al., 2006, Jády et al., 2006). In both Amprolium HCl and fission yeasts, the preference of telomerase to extend the shortest telomeres requires the activity of Tel1, the yeast homolog of human ATM (Sabourin et al., 2007, Hector et al., 2007, Arnerić and Lingner, 2007). ATM and ATR are kinases within the phosphatidylinositol-3 kinase-related kinase (PIKK) family, which regulates cellular responses to DNA damage, mRNA decay, and nutrient-dependent signaling (Lovejoy and Cortez, 2009). Activation of these DNA damage pathways is dampened at telomeres; in mammalian cells, TRF2 represses activation of ATM while POT1 represses ATR (Karlseder et al., 2004, Celli and de Lange, 2005, Denchi and de Lange, 2007, Guo et al., 2007, Okamoto et al., 2013). Nevertheless, there is a large amount of evidence that their yeast homologs play a positive role in facilitating telomere extension by telomerase (Moser et al., 2009, Moser et al., 2011, Yamazaki et al., 2012, Churikov et al., 2013). It is not known whether the role of the ATM and ATR pathways in recruiting telomerase is conserved in mammals. Although ATM deficiency or ATR mutations can induce telomere shortening or instability in human and mouse cells (Metcalfe et al., 1996, Smilenov et al., 1997, Wong et al., 2003, Wu et al., 2007, Pennarun et al., 2010), these kinases were reported to be dispensable for elongation of the shortest telomeres in mouse models (Feldser et al., 2006, McNees et al., 2010). Also, immortalized cell lines from human patients with ATM mutations are able to maintain their telomeres with telomerase, albeit at short lengths (Sprung et al., 1997). Nonetheless, there is evidence that TRF1-mediated telomere-length regulation in human cells involves ATM. Inhibition of human ATM resulted in increased TRF1 at the telomere, and phosphorylation of TRF1 on serine 367, an ATM/ATR target site, reduced the interaction of TRF1 with telomeres and abrogated its ability to inhibit telomere lengthening (McKerlie et al., 2012, Wu et al., 2007).
    Results
    Discussion In this study, we have demonstrated that ATM and ATR are both necessary for full telomerase recruitment to telomeres in human cell lines. This conclusion is supported by an independent study using complementary approaches in which ATM was demonstrated to be necessary for telomerase-mediated telomere addition in both human and mouse cells (Lee et al., 2015). This provides an explanation, at least in part, for the long-standing observation of short telomeres in the ATM-deficient cells of ataxia telangiectasia (AT) patients (Metcalfe et al., 1996, Smilenov et al., 1997) and the telomere shortening observed upon inhibition of ATM in telomerase-positive immortal human cell lines (Wu et al., 2007). Consistently, while AT patient cells can become immortalized by activation of telomerase, most of these cell lines harbor very short telomeres (Sprung et al., 1997). Our data show that TRF1 is involved in the signaling pathway restricting telomerase access to the telomere to S phase; loss of TRF1 results in an increase in telomerase at the telomere in both G1 and G2/M phases. Evidence from this and previous studies suggests that phosphorylation of TRF1 at serine 367 results in partial TRF1 dissociation from telomeres and its degradation; removal of this phosphate is necessary for correct cell-cycle control of telomerase presence at the telomere. Constitutive expression of a phosphomimetic of S367 TRF1 also leads to inappropriate retention of telomerase at the telomere outside S phase, leading to a telomere-length increase (Wu et al., 2007, McKerlie et al., 2012; this study; Figure 3). The mechanism for this regulatory function of TRF1 may include its known role in facilitating telomere replication; TRF1 dissociation from telomeres induces replication fork stalling that activates ATR (Sfeir et al., 2009), and we provide evidence that replication fork stalling leads to an ATR-dependent increase in telomerase recruitment (Figure 4). The control of telomere replication and telomerase presence at the telomere by human TRF1 (Figure 7A) appears analogous to the situation in Schizosaccharomyces pombe, in which deletion of the double-stranded telomeric-binding protein Taz1 results in stalled telomeric replication forks (Miller et al., 2006) and leads to deregulation of the cell-cycle control of telomerase at the telomere (Dehé et al., 2012, Chang et al., 2013). It is possible that in the absence of TRF1, aberrant products of stalled replication forks may persist into the subsequent G2/M and G1 phases, forming substrates for telomerase, as has been postulated in the case of Taz1 in S. pombe (Dehé et al., 2012). It has been proposed that it is the tendency of the replication machinery to stall in repetitive DNA that forms the signal for telomerase recruitment specifically in S phase (Rog and Cooper, 2008, Verdun and Karlseder, 2006, Wu et al., 2007, Stern and Bryan, 2008, Dehé et al., 2012, Chang et al., 2013). In this report, we provide direct evidence for this concept.