1. CST Meets Shelterin to Keep Telomeres in Check
Marie-Josèphe Giraud-Panis, M. Teresa Teixeira, Vincent Géli, Eric Gilson 
Molecular Cell 
Volume 39, Issue 5, Pages (September 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1 Knowledge before the Discovery of the CST Conservation
Before the discovery of the evolutionary conservation of the CST complex, the shelterin mode of capping found in most eukaryotes was opposed to the CST protective functions described in budding yeasts. The relative position of the different complexes on the duplex and single-stranded portions of the telomeric DNA is arbitrary. The gap due to the filling reaction might be transitory, because internal single-stranded stretches at telomeres have not been reported.
(A) Saccharomyces cerevisiae telomeres are bound by Rap1-Rif1-Rif2 complex in their double-stranded region and by Cdc13 in their single-stranded region. Cdc13 may interact with Stn1 and Ten1 to form the CST complex. However, the current model establishes that Cdc13-dependent activation of telomerase does not involve Ten1 and Stn1, because these compete with Est1-telomerase complex (blue: telomerase catalytic subunit and Est1 telomerase subunit; dark line: telomerase template RNA) for Cdc13 binding. Telomerase action results in the formation of a long G tail (blue line) that is potentially covered with CST complexes. These, in turn, would stimulate the C-strand synthesis through their interaction with Polα-primase (Pol, red). Subsequent newly formed double-stranded telomeric repeats (with red line indicating the newly synthesized C strand) are then covered by additional Rap1-Rif1-Rif2 complexes that downregulate telomerase.
(B) In many other eukaryotes, including humans, the shelterin complex, composed of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, covers the telomeric chromatin, both the double-stranded part and the single-stranded part. The complex TPP1-POT1 was shown to stimulate telomerase, participating in the increase of the G tail. It is supposed that this G tail is converted into double-stranded telomeric chromatin through the priming by Polα-primase (red as in A), but until recently, no telomeric candidate was convincingly found responsible for coordinating this activity. As in S. cerevisiae, the resulting structure downregulates telomerase from adding additional repeats.
(C) Cdc13 was shown to be potentially phosphorylated by Tel1 (S225, S249, S255), Mec1 (S225, S249, S255, S306), and Cdk1 (T308, S336) kinases (Li et al., 2009; Tseng et al., 2006; Tseng et al., 2009; Zhang and Durocher, 2010). In vivo functions of these phosphorylations were reported for S249, S255, S306, and T308. Indeed, phosphorylation of S249 and S255 by Tel1 or Mec1was suggested to be required for telomerase activation (Tseng et al., 2006). Mec1 was shown to have a preponderant role during the establishment of senescence in telomerase-negative cells, but its target or targets in these conditions is or are not known (Abdallah et al., 2009; Khadaroo et al., 2009). Very recently, it was shown that phosphorylation of Cdc13-S306 by Mec1 suppresses Cdc13 accumulation at double-stranded breaks (DSBs) and thereby inhibits telomere healing (Zhang and Durocher, 2010). A C-terminal truncation from position 695 is found in cdc13-5 that leads to overelongated G tails (Chandra et al., 2001). Another allele of CDC13 affecting telomere protection maps within Stn1-binding domain (cdc13-1) (Nugent et al., 1996). Red asterisks mark the single positions mutated in CDC13 alleles that show loss of Pol1 interaction (K50Q, C124R, L129S, S228P, L392P, I523V) (Qi and Zakian, 2000).
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3. Figure 2 A CST-Counting Model for the Regulation of G-Tail Length
(A) Model of leading telomere processing. Telomere replication may generate a 3′ overhang and a blunt end in the strands synthesized by the lagging or the leading strand synthesis machinery, respectively. MRX is recruited to the leading strand telomere (Faure et al., 2010), initiating the processing of this telomere that leads to 3′ to 5′ resection. Sae2, Sgs1, Dna 2, and Exo1 helicase or nuclease activities are involved in this process in an order of events very similar to the processing of DSBs (Bonetti et al., 2009; Mimitou and Symington, 2008). The telomere nucleolytic processing is limited by Rap1 and Ku (Bonetti et al., 2010) and by CST (Frank et al., 2006; Vodenicharov and Wellinger, 2006).
(B) Asymmetric versus symmetric mode of telomere replication model. Semiconservative DNA replication machinery is asymmetric, with distinct machineries replicating the continuous and discontinuous strands. At telomeres, further 5′ to 3′ resection at leading strand telomere, and eventual telomerase-dependent telomere elongation, generates a long G tail. This latter may bind CST, which, in turn, activates the Polα-primase. Subsequently, in this model, the long, single-stranded DNA formed at leading telomeres is filled by discontinuous synthesis of the C strand.
(C) CST may block the access to Exo1 and/or other nuclease and helicase activities.
(D) CST might activate Polα-primase-dependent C-strand synthesis, compensating overhang production activities.
(E) As the G tail increases, it may be bound by an increasing number of CST complexes that, in turn, prevent its further extension and/or actively compensate by C-strand resynthesis. This dynamic equilibrium determined by the CST complex would provide a finely tuned control of the G-tail length.
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4. Figure 3 Structural Similarities between RPA and CST
(A) Schematic view of human RPA subunits (RPA70, RPA32, and RPA14) and CST proteins (ScCdc13, Stn1, and Ten1). Light orange and light purple domains correspond to putative OB folds and wHTH domains, respectively.
(B) Detail of the X-ray structure of RPA70 AB OB folds; only the B domain is shown (PDB 1FGU) (Bochkareva et al., 2001).
(C) Detail of the solution structure of Cdc13 OB fold bound to ssDNA; only the protein is shown (PDB 1S40) (Mitton-Fry et al., 2004).
(D) X-ray structure of the heterodimer RPA32D-RPA14 (PDB 1QUQ) (Bochkarev et al., 1999). Conserved secondary structures between RPA32C and Stn1 proteins are shown in cyan and red for conservation between RPA14 and Ten1 proteins. These regions were drawn according to Sun et al. (2009).
(E) X-ray structure of the heterodimer between the N-terminal domain of SpStn1 and SpTen1 (PDB 3KF6) (Sun et al., 2009).
(F) X-ray structure of the heterodimer between the N-terminal domain of CtStn1 and CtTen1 (PDB 3KF8) (Sun et al., 2009).
(G) Detail of the solution structure of the complex between the C-terminal wHTH domain of human RPA32 (RPA32D) and UNG2; only RPA32D is shown (PDB 1DPU) (Mer et al., 2000).
(H) X-ray structure of the C-terminal wHTH domains of ScStn1 (PDB 3K10) (Gelinas et al., 2009). These figures were built from Protein Data Bank data using the PyMOL software.
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5. Figure 4 A Shelterin-CST Alliance to Keep the G Tail in Check
The double-stranded region of telomeres is covered with shelterin complexes that inhibit the activation of ATM. The inhibition of ATR may be achieved by both limiting the length of the G tail through a CST-dependent stimulation of C-strand synthesis and covering it with single-stranded telomeric proteins such as POT1 and CST itself.
Molecular Cell  , DOI: ( /j.molcel )
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