Telomeres are protein-DNA complexes at the ends of linear chromosomes that are important for genome stability. Telomeric DNA in humans, as in many eukaryotic organisms, consists of tandem repeats (Blackburn and Gall 1978; Moyzis et al. 1988; Meyne et al. 1989). The repeats at human telomeres are composed of TTAGGG sequences and stretch for several kilobase pairs. Another feature of telomeric DNA in many eukaryotes is a G-rich 3' single strand overhang, which in humans is estimated to be approximately 50-300 bases long (Makarov et al. 1997; Wright et al. 1997; Huffman et al. 2000). Telomeric DNA isolated from humans and several other organisms can form a lasso-type structure called a t-loop in which the 3' single-strand end is presumed to invade the double stranded telomeric DNA repeat tract (Griffith et al. 1999). Telomeric DNA is bound by multiple protein factors that play important roles in regulating telomere length and in protecting the chromosome end from recombination, non-homologous end-joining, DNA damage signaling, and unregulated nucleolytic attack (reviewed in de Lange 2005).
DNA attrition can occur at telomeres, which can impact cell viability. Attrition can occur owing to the "end-replication problem", a consequence of the mechanism of lagging-strand synthesis (Watson 1972; Olovnikov 1973). Besides incomplete replication, nucleolytic processing also likely contributes to telomere attrition (Huffman et al. 2000). If telomeres become critically shortened, replicative senescence can result (Harley et al. 1990). Thus, in order to undergo multiple divisions, cells need a mechanism to replenish the sequence at their chromosome ends.
The primary means for maintaining the sequence at chromosome ends in many eukaryotic organisms, including humans, is based on telomerase (Greider and Blackburn, 1985; Morin 1989). Telomerase is a ribonucleoprotein complex minimally composed of a conserved protein subunit containing a reverse transcriptase domain (telomerase reverse transcriptase, TERT) (Lingner et al. 1997; Nakamura et al. 1997) and a template-containing RNA (telomerase RNA component, TERC, TR, TER) (Greider and Blackburn, 1987; Feng et al 1995). Telomerase uses the RNA template to direct addition of multiple tandem repeats to the 3' G-rich single strand overhang. Besides extension by telomerase, maintenance of telomeric DNA involves additional activities, including C-strand synthesis, which fills in the opposing strand, and nucleolytic processing, which likely contributes to the generation of the 3' overhang.
View original pathway at Reactome.
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Reactive oxygen species (ROS), whose concentration increases in senescent cells due to oncogenic RAS-induced mitochondrial dysfunction (Moiseeva et al. 2009) or due to environmental stress, cause DNA damage in the form of double strand breaks (DSBs) (Yu and Anderson 1997). In addition, persistent cell division fueled by oncogenic signaling leads to replicative exhaustion, manifested in critically short telomeres (Harley et al. 1990, Hastie et al. 1990). Shortened telomeres are no longer able to bind the protective shelterin complex (Smogorzewska et al. 2000, de Lange 2005) and are recognized as damaged DNA.
The evolutionarily conserved MRN complex, consisting of MRE11A (MRE11), RAD50 and NBN (NBS1) subunits, binds DSBs (Lee and Paull 2005) and shortened telomeres that are no longer protected by shelterin (Wu et al. 2007). Once bound to the DNA, the MRN complex recruits and activates ATM kinase (Lee and Paull 2005, Wu et al. 2007), leading to phosphorylation of ATM targets, including TP53 (p53) (Banin et al. 1998, Canman et al. 1998, Khanna et al. 1998). TP53, phosphorylated on serine S15 by ATM, binds the CDKN1A (also known as p21, CIP1 or WAF1) promoter and induces CDKN1A transcription (El-Deiry et al. 1993, Karlseder et al. 1999). CDKN1A inhibits the activity of CDK2, leading to G1/S cell cycle arrest (Harper et al. 1993, El-Deiry et al. 1993).
SMURF2 is upregulated in response to telomere attrition in human fibroblasts and induces senecscent phenotype through RB1 and TP53, independently of its role in TGF-beta-1 signaling (Zhang and Cohen 2004). The exact mechanism of SMURF2 involvement is senescence has not been elucidated.
This is a generic nucleosome created for the telomerase module. It contains Histones H2A, H2B, and H3 as candidate sets where all of the variants of each histone protein are entered as candidates (as opposed to members). The list for each is not exhaustive, but rather is a list of histones known to Reactome at the time of the creation of the nucleosome complex. Histone H4 is only documented once in Uniprot, so for now it is an EWAS.
The template of hTERC directs the sequential addition of nucleotides to the 3' telomeric DNA end. Following addition of a nucleotide, the template and catalytic site must move relative to one another within the telomerase RNP to place the appropriate template residue in the active site. As base-pairing and nucleotide addition occur at one end of the template, base pair melting occurs at the other (Collins and Greider 1993; Wang and Blackburn, 1997; Hammond and Cech 1998; Benjamin et al. 2000; Forstemann and Lingner 2005). This un-pairing is thought to reduce the energy used for mediating the subsequent translocation step. Nucleotide addition can occur up until the template boundary which in hTERC is defined by a helix called P1b (Chen and Greider 2003).
Studies in yeast and human cells indicate that recruitment of telomerase to a telomere may be influenced by multiple variables, including regulatory protein factors, hTERT domains, telomere length, and the cell cycle stage. First, in yeast, the telomerase associated factor Est1 and the single-strand DNA binding protein Cdc13 play roles in telomerase recruitment (Pennock et al. 2001; Bianchi et al. 2004). Analogous proteins exist in human cells (Est1A, Est1B, Est1C, and POT1, respectively); however, how or whether these proteins are directly involved in telomerase recruitment remains to be elucidated. Second, N-terminal residues of hTERT within the DAT (dissociate the activities of telomerase) domain may have a role in binding single stranded telomeric DNA as the "anchor site" (Lee et al. 1993; Moriarty et al. 2005). Third, a cis-acting mechanism in yeast and humans that regulates telomere length maintenance may modulate telomerase access to the telomere (reviewed in Blackburn 2001; Smogorzewska and de Lange, 2004). Long telomeres, which have more associated protein factors, are in a state that is acted on by telomerase less frequently than that of short telomeres, which have fewer associated factors. Whether short telomeres actively recruit telomerase remains to be determined. Last, the recruitment of telomerase to telomeres shows cell-cycle regulation (Taggart et al. 2002; Smith et al. 2003; Fisher et al. 2004; Jady et al. 2006; Tomlinson et al. 2006). Presence of the telomeric protection complex shelterin at telomeres is necessary for the recruitment of telomerase. ACD (TPP1), the subunit of the shelterin complex, directly interacts, through its TEL patch region, with telomerase and is required for telomerase function in vivo (Abreu et al. 2010, Nandakumar et al. 2012, Sexton et al. 2014). The interaction involves the TEN domain of TERT (Schmidt et al. 2014). The helicase RTEL1 is recruited to telomeres in S phase via direct interaction with the shelterin complex subunit TREF2. RTEL1 is needed for T-loop unwinding and resolution of telomeric G-quadruplex (G4) DNA structures, necessary steps for efficient telomere replication (Vannier et al. 2012, Sarek et al. 2015). Germline mutations in RTEL1 cause a severe form of dyskeratosis congenita, a telomere disorder syndrome, called Hoyeraal Hreidarsson syndrome (Ballew, Yeager et al. 2013; Walne et al. 2013; Ballew, Joseph et al. 2013, Le Guen et al. 2013, Deng et al. 2013). Loading of RTEL1 to telomere ends is negatively regulated outside of S phase by CDK2:CCNA-mediated phosphorylation of the shelterin complex subunit TERF2 at serine residue S365. At the S phase entry, TERF2 is dephosphorylated by the PP6 phosphatase, thus allowing timely RTEL1 loading (Sarek et al. 2019).
In vitro studies of telomerase complexes derived from multiple organisms indicate that at least two types of interactions are important for telomerase RNP catalytic site alignment at the 3' G-rich single-strand telomere end. In one interaction, an alignment region in hTERC base-pairs with the 3' G-rich single-strand telomeric DNA end to form an RNA-DNA hybrid, which positions the template adjacent to the 3' end of the telomere. In a second interaction, a portion of hTERT is proposed to interact with the DNA 5' of the telomerase RNA/DNA primer hybrid (Harrington and Greider 1991; Morin 1991; Moriarty et al. 2005), which is important for the catalytic rate (Lee and Blackburn, 1993) and presumably allows telomerase to maintain contact with the chromosome during the translocation step. How the anchor site binding and template hybridization are coordinated is not known.
In vitro, telomerase can disassociate from the primer following addition of each nucleotide or during the translocation step. The regulation of telomerase disassociation from the telomere in vivo is not well-characterized (de Lange 2002, Zhu et al. 2003, Ye et al. 2004, Smorgozewska and de Lange 2004, Tomita 2018). One factor that may be involved is a helicase termed hPIF1, which can unanneal the telomerase RNA/telomeric DNA hybrid (Boule et al. 2005; Zhang et al. 2006). PIF1 can directly interact with telomerase (Mateyak and Zakian 2006) and it acts to inhibit telomerase activity and telomere lengthening (Zhang et al. 2006, Paeschke et al. 2013). In addition to regulation of telomere lengthening, PIF1 is also involved in resolution of G-quadruplex (G4) structures in single-stranded nucleic acid intermediates that form during DNA replication and gene expression (Sanders 2010, Paeschke et al. 2013).
hTERC is transcribed as a precursor and is processed at its 3' end to yield a 451 nucleotide RNA (Zaug et al. 1996). The accumulation of hTERC that has undergone this processing event requires a conserved region of sequence termed the box H/ACA motif (Mitchell et al. 1999a). This motif is bound by a complex containing DKC1 (dyskerin), and mutations in dyskerin affect the processing and accumulation of hTERC (Mitchell et al. 1999b; Mitchell and Collins 2000; Fu and Collins 2003). Studies of purified, catalytically active telomerase indicate that the minimal structure that has telomerase activity in vitro is a complex of one molecule of hTERC RNA, one molecule of hTERT and two molecules of DKC1 (dyskerin) (Cohen et al. 2007). A cryo-electron microscopy (EM) structure of human substrate-bound telomerase holoenzyme revealed that, in addition to one molecule of hTERC RNA, one molecule of hTERT and two molecules of DKC1, the holoenzyme also contains one molecule of WRAP53 (TCAB1, also known as telomere Cajal body protein 1) and two molecules of each NOP10, NHP2 (NOLA2) and GAR1 (Nguyen et al. 2018). WRAP53 is needed for the activity and localization of the telomerase holoenzyme to Cajal bodies (Venteicher et al. 2009). Homozygosity for NHP2 mutations is associated with telomerase failure (dyskeratosis congenita) in humans (Vuillamy et al. 2008). Several additional proteins may associate with the holoenzyme, promoting its assembly and modulating its activity. RUVBL1 (pontin) and RUVBL2 (reptin) are found associated with human telomerase RNPs purified from HeLa cells, and activities of these proteins are required for telomerase RNP assembly in vivo (Venteicher et al. 2008). Pontin and reptin may modulate the interaction between SHQ1 and DKC1 (Machado-Pinilla et al. 2012), but as their exact roles in the assembly and function of telomerase RNP remain unclear, they are annotated simply as positive regulators of telomerase RNP formation.
The core components hTERC and hTERT undergo trafficking in the cell that may be important for telomerase function. hTERC has been found localized in multiple nuclear structures, including Cajal bodies, nucleoli, and at telomeres (Mitchell et al. 1999a; Jady et al. 2004; Zhu et al. 2004; Jady et al. 2006; Tomlinson et al. 2006). hTERT is also reported localize in Cajal bodies, nucleoli, and to associate with telomeres (Etheridge et al. 2002; Wong et al. 2002; Yang et al. 2002; Zhu et al. 2004; Tomlinson et al. 2006). Some of the factors that regulate trafficking of these two core components of telomerase have been identified, such as nucleolin (Khurts et al. 2004), SMN (Bachand et al. 2002), and 14-3-3 (Seimiya et al. 2000). Cytological studies of HeLa cells suggest that the localization of the telomerase core components can change through the cell-cycle (Jady et al. 2006; Tomlinson et al. 2006). Despite these studies, it is not clear in which compartment hTERT and hTERC assemble to form functional telomerase RNP.
The assembly of telomerase involves the chaperone proteins p23 and Hsp90, which stably associate with telomerase in vitro (Holt et al. 1999; Forsythe et al. 2001; Keppler et al. 2006). A number of other proteins interact with the telomerase RNP, but it is not clear if they play a role in telomerase assembly. Interestingly, assembled human telomerase RNP can multimerize, though the function of multimerization remains unclear (Beattie et al. 2001; Wenz et al. 2001; Arai et al. 2002).
The elongation reaction proceeds as follows: The template of hTERC directs the sequential addition of nucleotides to the 3' telomeric DNA end. Following addition of a nucleotide, the template and catalytic site must move relative to one another within the telomerase RNP to place the appropriate template residue in the active site. As base-pairing and nucleotide addition occur at one end of the template, base pair melting occurs at the other (Collins and Greider 1993; Wang and Blackburn, 1997; Hammond and Cech 1998; Benjamin et al. 2000; Forstemann and Lingner 2005). This un-pairing is thought to reduce the energy used for mediating the subsequent translocation step. Nucleotide addition can occur up until the template boundary which in hTERC is defined by a helix called P1b (Chen and Greider 2003).
The human telomerase RNP can catalyze multiple rounds of repeat addition on the same telomeric substrate in vitro. Before initiating synthesis of another repeat, telomerase undergoes a translocation step to reposition itself on the telomere. Base pairs in the DNA/RNA hybrid are unannealed, the RNA template is repositioned relative to the active site, and the template base-pairs at the 3' end of the newly synthesized DNA. The anchor site interaction with DNA 5' of the RNA-DNA duplex is thought to maintain the interaction of telomerase with DNA during the translocation step.
The complementary C-strand at telomeres is synthesized by the DNA polymerase alpha:primase complex (Nakamura et al. 2005) using conventional RNA priming (Wang et al. 1984). Interaction of the DNA polymerase alpha complex with the G-strand-bound CST complex is needed for successful priming of the C-strand (Feng et al. 2018).
The complementary C-strand at telomeres is synthesized by the polymerase alpha:primase complex using conventional RNA priming (Nakamura et al. 2005, Dai et al. 2010). This process is regulated by the CST complex (Dai et al. 2010, Feng et al. 2017, Feng et al. 2018) and CDK1 (Dai et al. 2010, Dai et al. 2012).
When the polymerase delta:PCNA complex reaches a downstream Okazaki fragment, strand displacement synthesis occurs. The primer containing 5'-terminus of the downstream Okazaki fragment is folded into a single-stranded flap (Podust et al. 1995, Bae et al. 2001, Maga et al. 2001). The helicase activity of either WRN (Werner syndrome protein) or BLM (Bloom syndrome helicase) is needed for DNA polymerase delta progression and strand displacement synthesis across G-rich telomeric repeats during lagging strand (C-strand) synthesis (Li et al. 2017).
The binding of the primer recognition complex involves the loading of the proliferating cell nuclear antigen (PCNA). Replication Factor C (RFC) transiently opens the PCNA toroid in an ATP-dependent reaction, and then allows PCNA to re-close around the double helix adjacent to the primer terminus. This leads to the formation of the "sliding clamp" (Tsurimoto et al. 1990, Mossi and Hubscher 1998). In a human telomere replication model, RFC-mediated PCNA loading increases the processivity of telomeric C-strand synthesis, but does not eliminate polymerase delta stalling on the G-rich template (Lormand et al. 2013). Interaction of RTEL1 with PCNA is needed for telomere replication and maintenance of telomere integrity (Vannier et al. 2013).
The DNA2 endonuclease removes the initiator RNA along with several downstream deoxyribonucleotides. The cleavage of the single-stranded RNA substrate results in the disassembly of RPA and DNA2. The current data for the role of the DNA2 endonuclease has been derived from studies of yeast (Budd et al. 2000, Bae et al. 2001) and Xenopus DNA2 (Liu et al. 2000, Liao et al. 2008). DNA2-mediated cleavage of G-quadruplexes (G4), DNA structures commonly formed by polyguanine-rich telomeric DNA sequences, is necessary for completion of telomeric DNA synthesis (Masuda-Sasa et al. 2008, Lin et al. 2013).
After RFC initiates the assembly of the primer recognition complex, the complex of pol delta and PCNA is responsible for incorporating the additional nucleotides prior to the position of the next downstream initiator RNA primer. On the lagging strand, short discontinuous segments of DNA, called Okazaki fragments, are synthesized on RNA primers. The average length of the Okazaki fragments is 100 nucleotides. Polymerase switching is a key event that allows the processive synthesis of DNA by the pol delta and PCNA complex (Lee and Hurwitz 1990, Tsurimoto and Stillman 1991, Nethanel et al. 1992, Brown and Campbell 1993, Waga et al.1994, Bambara et al. 1997). PCNA increases the processivity of the DNA polymerase delta during telomeric C-strand synthesis in a human telomere replication model, but it does not eliminate the DNA polymerase delta stalling on the G-rich template (Lormand et al. 2013).
The first step in the removal of the flap intermediate is the binding of Replication Protein A (RPA) to the long flap structure. RPA is a eukaryotic single-stranded DNA binding protein (Bae et al. 2001). Binding of RPA to the single strand DNA during telomeric strand displacement synthesis is necessary for the recruitment of DNA2. DNA2 is a helicase/endonuclease that resolves G quadruplexes (G4), which are DNA structures that commonly form in polyguanine-rich telomeric DNA sequences (Masuda-Sasa et al. 2008). DNA2 also removes the initiator RNA primers of Okazaki fragments (Bae et al. 2001).
The remaining flap, which is too short to support RPA binding, is then processed by FEN1. There is evidence that binding of RPA to the displaced end of the RNA-containing Okazaki fragment prevents FEN1 from accessing the substrate. FEN1 is a structure-specific endonuclease that cleaves near the base of the flap at a position one nucleotide into the annealed region. Biochemical studies have shown that the preferred substrate for FEN1 consists of a one-nucleotide 3'-tail on the upstream primer in addition to the 5'-flap of the downstream primer (Harrington and Lieber 1994, Harrington and Lieber 1995, Murante et al. 1996, Lieber 1997, Kaiser et al. 1999, Xu et al. 2000, Kao et al. 2002). The interaction of FEN1 with WRN, a RECQ family DNA helicase, is needed for successful flap cleavage during telomeric strand displacement synthesis (Saharia et al. 2010, Li et al. 2017).
It is assumed that, as shown for generic DNA replication (Podust et al. 1998), the RFC complex dissociates from PCNA following sliding clamp formation at the telomere, and the DNA toroid alone tethers pol delta to the DNA.
The loading of proliferating cell nuclear antigen (PCNA) leads to recruitment of pol delta, the process of polymerase switching. Human PCNA is a homotrimer of 36 kDa subunits that form a toroidal structure. The loading of PCNA by RFC is a key event in the transition from the priming mode to the extension mode of DNA synthesis. The processive complex is composed of the pol delta holoenzyme and PCNA (Lee and Hurwitz 1990, Podust et al. 1998). While PCNA increases the processivity of the DNA polymerase delta during telomeric C-strand synthesis in a human telomere replication model, it does not eliminate the DNA polymerase delta stalling on the G-rich template (Lormand et al. 2013).
After RPA binds the long flap, it recruits the DNA2 helicase/endonuclease which removes the initiator RNA primers of Okazaki fragments (Bae et al. 2001). DNA2 is also needed to resolve G quadruplexes (G4), DNA structures commonly formed by polyguanine-rich telomeric DNA sequences (Masuda-Sasa et al. 2008, Lin et al. 2013).
Once the RNA-DNA primer is synthesized, replication factor C (RFC) initiates a reaction called "polymerase switching"; pol delta, the processive enzyme, replaces pol alpha, the priming enzyme. RFC binds to the 3'-end of the RNA-DNA primer on the Primosome, to displace the pol alpha primase complex. The binding of RFC triggers the binding of the primer recognition complex (Tsurimoto and Stillman 1991, Maga et al. 2000, Mossi et al. 2000). RFC is recruited to telomeres via interaction with 5'-phosphate ends of a telomere repeat sequence (Uchiumi et al. 1996, Uchiumi et al. 1999). In budding yeast, the alternative evolutionarily conserved RFC complex in which the RFC1 subunit is substituted with the CTF18 complex (composed of CHTF18, CHTF8 and DSCC1) plays a critical role in telomere maintenance (Hiraga et al. 2006, Gao et al. 2014). The CTF18-RFC complex is also implicated in telomere maintenance in fission yeast (Khair et al. 2010). It was shown that the human CTF18-RFC complex has a redundant function with the RFC pentamer in PCNA loading and DNA replication (Bermudez et al. 2003), but its role in human telomere maintenance has not been studied. Mouse CFT18 complex is necessary for proper development of germ cells (Berkowitz et al. 2012).
Removal of the flap by FEN1 leads to the generation of a nick between the 3'-end of the upstream Okazaki fragment and the 5'-end of the downstream Okazaki fragment. DNA ligase I (LIG1) then seals the nicks between adjacent processed Okazaki fragments to generate intact double-stranded DNA (Turchi and Bambara 1993, Bambara et al. 1997, Waga and Stillman 1998, Levin et al. 2000). LIG1 is necessary for ligation of Okazaki fragments at the lagging telomere DNA strand. LIG1 deficiency results in telomere instability, manifested through telomere sister fusions, which is a consequence of DNA breaks in the lagging strand (C-strand) (Le Chalony et al. 2012).
In addition to telomerase-mediated elongation and C-strand synthesis, other DNA processing steps are likely involved in telomere maintenance. In humans, nucleolytic activity is proposed to be involved in generating the G-rich 3' single strand overhang. In addition, differences in the structure of the overhang at telomeres that have undergone leading vs. lagging strand replication suggest that DNA processing may be different at these telomeres (Chai et al. 2006).
Electron microscopy studies of purified human telomeric DNA have provided evidence for telomeric loops, or t-loops (Griffith et al. 1999). t-loops are proposed to result from invasion of the 3' G-rich single strand overhang into the double stranded portion of the telomeric TTAGGG repeat tract. The strand displaced by invasion forms a structure called a D loop. The function of the t-loop is presumed to be the protection of the 3' telomeric end. In vitro, the double strand telomeric DNA binding protein TRF2 can increase the frequency of t-loop formation. The prevalence of the t-loops in vivo is not known.
Many proteins associate with telomeric DNA. One complex that binds telomeres is called shelterin. Shelterin is a six-protein complex composed of TRF1 and TRF2, which can bind double-stranded telomeric DNA, POT1, which can bind single-stranded telomeric DNA, and three other factors, RAP1, TIN2, and TPP1 (reviewed in de Lange 2006 "Telomeres"). Human telomeric DNA is also bound by nucleosomes (Makarov et al. 1993; Nikitina and Woodcock 2004). A number of other proteins, including some that play roles in the DNA damage response, can be found at telomeres (Zhu et al. 2000; Verdun et al. 2005).
Studies in yeast and humans indicate that the association of many proteins with telomeres is regulated through the cell cycle (Smith et al. 1993; Zhu et al. 2000; Taggart et al. 2002; Fisher et al. 2004; Takata et al. 2004; Takata et al. 2005; Verdun et al. 2005). For instance, TRF1, MRE11, POT1, ATM, and NBS1 display cell cycle regulated chromatin immunoprecipitation of telomeric DNA (Zhu et al. 2000; Verdun et al. 2005), and cytologically observable hTERT and hTERC localize to a subset of telomeres only in S-phase (Jady et al. 2006; Tomlinson et al. 2006). These data indicate that telomeres are dynamically remodeled through the cell cycle.
At some point in the extension process a sufficient number of regulatory factors that repress telomere extension become bound to the extending telomere. These factors include the TRF1 complexes, TRF2 complexes, telomerase, other factors, and the telomere itself. As repeats are added to the G-rich strand, and once lagging strand synthesis completes the duplex, new binding sites become available for these repressive factors. Once a balance is reached between telomere extension and the telomere repression factors, extension ceases. In this state extension machinery disassociates, leaving the telomere to be folded into a stable conformation.
This module details a single transit through the telomere extension process, detailing the addition of two repeats, and the corresponding synthesis of a section of lagging strand. An actual round of in vivo telomere extension would require thousands of telomere repeat additions, and it is the repressive effect of the factors bound to these repeats that turns off telomere extension (Moldovan et al. 2007).
In addition to telomerase-mediated elongation and C-strand synthesis, other DNA processing steps are likely involved in telomere maintenance. In humans, nucleolytic activity is proposed to be involved in generating the G-rich 3' single strand overhang. In addition, differences in the structure of the overhang at telomeres that have undergone leading vs. lagging strand replication suggest that DNA processing may be different at these telomeres (Chai et al. 2006).
Many proteins associate with telomeric DNA. One complex that binds telomeres is called shelterin. Shelterin is a six-protein complex composed of TRF1 and TRF2, which can bind double-stranded telomeric DNA, POT1, which can bind single-stranded telomeric DNA, and three other factors, RAP1, TIN2, and TPP1 (reviewed in de Lange 2006 "Telomeres"). Human telomeric DNA is also bound by nucleosomes (Makarov et al. 1993; Nikitina and Woodcock 2004). A number of other proteins, including some that play roles in the DNA damage response, can be found at telomeres (Zhu et al. 2000; Verdun et al. 2005).
Studies in yeast and humans indicate that the association of many proteins with telomeres is regulated through the cell cycle (Zhu et al. 2000; Taggart et al. 2002; Fisher et al. 2004; Takata et al. 2004; Takata et al. 2005; Verdun et al. 2005). For instance, TRF1, MRE11, POT1, ATM, and NBS1 display cell cycle regulated chromatin immunoprecipitation of telomeric DNA (Zhu et al. 2000; Verdun et al. 2005), and cytologically observable hTERT and hTERC localize to a subset of telomeres only in S-phase (Jady et al. 2006; Tomlinson et al. 2006). These data indicate that telomeres are dynamically remodeled through the cell cycle.
ATRX (Alpha-thalassemia mental retardation syndrome X-linked) binds to transcriptional co-activator DAXX (Death domain-associated protein 6) to form an ATP-dependent chromatin remodeling complex with triple-helix displacement activity (Xue et al. 2003).
Binding of the polymerase alpha complex and the CST (Ctc1-Stn1-Ten1) complex to the telomeric DNA ends inhibits further extension of the G-strand by telomerase. The loading of the DNA polymerase alpha to telomeres does not depend on the CST complex, but the CST complex is needed for cessation of telomerase activity and for synthesis of RNA primers by the primase component of the DNA polymerase alpha (Feng et al. 2018. Gu et al. 2018).
CTC1, STN1 and TEN1, orthologs of S. cerrevisiae proteins Cdc13, Stn1 and Ten1, respectively, form the CST complex. This evolutionarily conserved complex plays a role in telomere maintenance (Miyake et al. 2009).
The CTF18 complex, composed of RFC1 homolog CHTF18 (CTF18), CHTF8 (CTF8) and DSCC1 (DCC1) binds to RFC2, RFC3, RFC4 and RFC5 to form the evolutionarily conserved heteroheptameric CTF18-RFC complex (CTF18-RFC(7s)), in which the RFC1 subunit of the RFC complex is replaced with the CTF18 complex (Bermudez et al. 2003, Merkle et al. 2003). CHTF18 is able to form a heteropentameric CTF18-RFC complex (CTF18-RFC(5s)) with RFC2, RFC3, RFC4 and RFC5 in the absence of CHTF8 and DSCC1 (Bermudez et al. 2003, Shiomi et al. 2004).
CHTF18 (CTF18), a homolog of the RFC complex subunit RFC1, binds to CHTF8 (CTF8) and DSCC1 (DCC1) to form the evolutionarily conserved CTF18 complex (Merkle et al. 2003, Bermudez et al. 2003). Formation of a heterodimer between DSCC1 and CHTF8 may precede formation of a heterotrimer (Bermudez et al. 2003).
The complex of ATRX (Alpha-thalassemia mental retardation syndrome X-linked) and DAXX (Death domain-associated protein 6) binds to subtelomeric chromosomal regions and plays a role in the recruitment of cohesin to subtelomeric regions and in the regulation of transcription of the noncoding telomeric repeat-containing RNA (TERRA) (Eid et al. 2015).
ATRX (Alpha-thalassemia mental retardation syndrome X-linked) and its binding partner DAXX (Death domain-associated protein 6) are required for deposition of histone H3.3, encoded by either the H3F3A gene or the H3F3B gene, to telomeres, independently of the H3.3 chaperone HIRA, in both human and mouse embryonic stem cells. Highly evolutionarily conserved N-terminus of DAXX interacts directly with the H3.3 core (Goldberg et al. 2010, Lewis et al. 2010).
Transcription of the telomeric noncoding RNA TERRA (the telomere repeat-containing RNA) is inhibited by ATRX (Flynn et al. 2015). Tumors with ATRX and DAXX mutations associated with the alternative lengthening of telomeres (ALT) show increased TERRA levels (Barthel et al. 2017).
SHQ1 is an evolutionarily conserved protein involved in assembly of H/ACA ribonucleoparticles, including telomerase RNPs. SHQ1 binds to DKC1 (dyskerin) and, by sequestering DKC1, it regulates the step-wise functional assembly of the telomerase holoenzyme (Grozdanov, Roy et al. 2009). A subset of DKC1 (dyskerin) mutations that cause dyskeratosis congenita, a rare bone marrow failure syndrome, modulate the affinity of DKC1 for SHQ1, thus preventing the assembly of telomerase RNPs (Grozdanov, Fernandez-Fuentes et al. 2009). Rarely, mutations in SHQ1 that impair binding to DKC1 cause a dyskeratosis congenita-like disease phenotype (Bizarro and Meier 2017).
Outside of the S phase of the cell cycle, the shelterin complex subunit TERF2 (TRF2) is phosphorylated on serine residue S365. This phosphorylation is performed by the complex of CDK2 and cyclin A (CCNA). Phosphorylation of TERF2 at S365 prevents association of RTEL1 with telomeres, thus protecting t-loops from promiscuous unwinding and inappropriate activation of ATM (Sarek et al. 2019).
The regulatory subunit PPP6R3 of the protein phosphatase complex PP6 facilitates PP6-mediated dephosphorylation of the shelterin subunit TERF2 at serine residue S365. PP6-mediated dephosphorylation of TERF2 occurs at the S phase entry and enables timely loading of RTEL1 to telomere ends (Sarek et al. 2019).
RFC
Heteropentamer:RNA
primer-DNA
primer:G-strand
extended telomere
end duplex:PCNA
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Stress Induced
SenescenceThe evolutionarily conserved MRN complex, consisting of MRE11A (MRE11), RAD50 and NBN (NBS1) subunits, binds DSBs (Lee and Paull 2005) and shortened telomeres that are no longer protected by shelterin (Wu et al. 2007). Once bound to the DNA, the MRN complex recruits and activates ATM kinase (Lee and Paull 2005, Wu et al. 2007), leading to phosphorylation of ATM targets, including TP53 (p53) (Banin et al. 1998, Canman et al. 1998, Khanna et al. 1998). TP53, phosphorylated on serine S15 by ATM, binds the CDKN1A (also known as p21, CIP1 or WAF1) promoter and induces CDKN1A transcription (El-Deiry et al. 1993, Karlseder et al. 1999). CDKN1A inhibits the activity of CDK2, leading to G1/S cell cycle arrest (Harper et al. 1993, El-Deiry et al. 1993).
SMURF2 is upregulated in response to telomere attrition in human fibroblasts and induces senecscent phenotype through RB1 and TP53, independently of its role in TGF-beta-1 signaling (Zhang and Cohen 2004). The exact mechanism of SMURF2 involvement is senescence has not been elucidated.
Processed Telomere End and Associated DNA Binding and Packaging Protein Complex Folded Into Higher Order
StructureProcessed Telomere End and Associated DNA Binding and Packaging Protein
ComplexProcessed Telomere
Endloaded on telomere:Okazaki
fragment complexloaded on telomere:Okazaki fragment:Flap:RPA
heterotrimer:DNA2loaded on telomere:Okazaki fragment:Flap:RPA
heterotrimerloaded on telomere:Okazaki
fragment:Flaploaded on telomere:Okazaki fragments:Remaining
Flaploaded on telomere:ligated C-strand Okazaki
fragmentsloaded on telomere:nicked DNA from adjacent
Okazaki fragmentsHeteropentamer:RNA primer-DNA primer:G-strand extended telomere end duplex:PCNA
homotrimerHeteropentamer:RNA primer-DNA primer:G-strand extended telomere
endprimer:DNA primer:G-strand extended
telomere:POLA:primaseholoenzyme complex
(generic)primer:G-strand extended
telomere:PCNAextended telomere
end:POLA:primaseHoloenzyme Base-paired to the Telomeric Chromosome End with an Additional single Stranded
Telomere repeatRNP:G-strand telomeric
chromosome endRNP:Telomeric Chromosome End with an Additional single Stranded
Telomere repeatand base-paired to the Telomeric
Chromosome Endchromosome end:Shelterin
(p-S365-TERF2)chromosome
end:Shelterinchromosome end with two additional single strand
repeatsAnnotated Interactions
Processed Telomere End and Associated DNA Binding and Packaging Protein Complex Folded Into Higher Order
StructureProcessed Telomere End and Associated DNA Binding and Packaging Protein Complex Folded Into Higher Order
StructureProcessed Telomere End and Associated DNA Binding and Packaging Protein
ComplexProcessed Telomere End and Associated DNA Binding and Packaging Protein
ComplexProcessed Telomere
EndProcessed Telomere
Endloaded on telomere:Okazaki
fragment complexloaded on telomere:Okazaki
fragment complexloaded on telomere:Okazaki fragment:Flap:RPA
heterotrimer:DNA2loaded on telomere:Okazaki fragment:Flap:RPA
heterotrimer:DNA2loaded on telomere:Okazaki fragment:Flap:RPA
heterotrimer:DNA2loaded on telomere:Okazaki fragment:Flap:RPA
heterotrimerloaded on telomere:Okazaki fragment:Flap:RPA
heterotrimerloaded on telomere:Okazaki
fragment:Flaploaded on telomere:Okazaki
fragment:Flaploaded on telomere:Okazaki fragments:Remaining
Flaploaded on telomere:Okazaki fragments:Remaining
Flaploaded on telomere:ligated C-strand Okazaki
fragmentsloaded on telomere:ligated C-strand Okazaki
fragmentsloaded on telomere:nicked DNA from adjacent
Okazaki fragmentsloaded on telomere:nicked DNA from adjacent
Okazaki fragmentsThe helicase RTEL1 is recruited to telomeres in S phase via direct interaction with the shelterin complex subunit TREF2. RTEL1 is needed for T-loop unwinding and resolution of telomeric G-quadruplex (G4) DNA structures, necessary steps for efficient telomere replication (Vannier et al. 2012, Sarek et al. 2015). Germline mutations in RTEL1 cause a severe form of dyskeratosis congenita, a telomere disorder syndrome, called Hoyeraal Hreidarsson syndrome (Ballew, Yeager et al. 2013; Walne et al. 2013; Ballew, Joseph et al. 2013, Le Guen et al. 2013, Deng et al. 2013). Loading of RTEL1 to telomere ends is negatively regulated outside of S phase by CDK2:CCNA-mediated phosphorylation of the shelterin complex subunit TERF2 at serine residue S365. At the S phase entry, TERF2 is dephosphorylated by the PP6 phosphatase, thus allowing timely RTEL1 loading (Sarek et al. 2019).
In addition to regulation of telomere lengthening, PIF1 is also involved in resolution of G-quadruplex (G4) structures in single-stranded nucleic acid intermediates that form during DNA replication and gene expression (Sanders 2010, Paeschke et al. 2013).
The core components hTERC and hTERT undergo trafficking in the cell that may be important for telomerase function. hTERC has been found localized in multiple nuclear structures, including Cajal bodies, nucleoli, and at telomeres (Mitchell et al. 1999a; Jady et al. 2004; Zhu et al. 2004; Jady et al. 2006; Tomlinson et al. 2006). hTERT is also reported localize in Cajal bodies, nucleoli, and to associate with telomeres (Etheridge et al. 2002; Wong et al. 2002; Yang et al. 2002; Zhu et al. 2004; Tomlinson et al. 2006). Some of the factors that regulate trafficking of these two core components of telomerase have been identified, such as nucleolin (Khurts et al. 2004), SMN (Bachand et al. 2002), and 14-3-3 (Seimiya et al. 2000). Cytological studies of HeLa cells suggest that the localization of the telomerase core components can change through the cell-cycle (Jady et al. 2006; Tomlinson et al. 2006). Despite these studies, it is not clear in which compartment hTERT and hTERC assemble to form functional telomerase RNP.
The assembly of telomerase involves the chaperone proteins p23 and Hsp90, which stably associate with telomerase in vitro (Holt et al. 1999; Forsythe et al. 2001; Keppler et al. 2006). A number of other proteins interact with the telomerase RNP, but it is not clear if they play a role in telomerase assembly. Interestingly, assembled human telomerase RNP can multimerize, though the function of multimerization remains unclear (Beattie et al. 2001; Wenz et al. 2001; Arai et al. 2002).
Interaction of RTEL1 with PCNA is needed for telomere replication and maintenance of telomere integrity (Vannier et al. 2013).
Electron microscopy studies of purified human telomeric DNA have provided evidence for telomeric loops, or t-loops (Griffith et al. 1999). t-loops are proposed to result from invasion of the 3' G-rich single strand overhang into the double stranded portion of the telomeric TTAGGG repeat tract. The strand displaced by invasion forms a structure called a D loop. The function of the t-loop is presumed to be the protection of the 3' telomeric end. In vitro, the double strand telomeric DNA binding protein TRF2 can increase the frequency of t-loop formation. The prevalence of the t-loops in vivo is not known.
Many proteins associate with telomeric DNA. One complex that binds telomeres is called shelterin. Shelterin is a six-protein complex composed of TRF1 and TRF2, which can bind double-stranded telomeric DNA, POT1, which can bind single-stranded telomeric DNA, and three other factors, RAP1, TIN2, and TPP1 (reviewed in de Lange 2006 "Telomeres"). Human telomeric DNA is also bound by nucleosomes (Makarov et al. 1993; Nikitina and Woodcock 2004). A number of other proteins, including some that play roles in the DNA damage response, can be found at telomeres (Zhu et al. 2000; Verdun et al. 2005).
Studies in yeast and humans indicate that the association of many proteins with telomeres is regulated through the cell cycle (Smith et al. 1993; Zhu et al. 2000; Taggart et al. 2002; Fisher et al. 2004; Takata et al. 2004; Takata et al. 2005; Verdun et al. 2005). For instance, TRF1, MRE11, POT1, ATM, and NBS1 display cell cycle regulated chromatin immunoprecipitation of telomeric DNA (Zhu et al. 2000; Verdun et al. 2005), and cytologically observable hTERT and hTERC localize to a subset of telomeres only in S-phase (Jady et al. 2006; Tomlinson et al. 2006). These data indicate that telomeres are dynamically remodeled through the cell cycle.
This module details a single transit through the telomere extension process, detailing the addition of two repeats, and the corresponding synthesis of a section of lagging strand. An actual round of in vivo telomere extension would require thousands of telomere repeat additions, and it is the repressive effect of the factors bound to these repeats that turns off telomere extension (Moldovan et al. 2007).
Many proteins associate with telomeric DNA. One complex that binds telomeres is called shelterin. Shelterin is a six-protein complex composed of TRF1 and TRF2, which can bind double-stranded telomeric DNA, POT1, which can bind single-stranded telomeric DNA, and three other factors, RAP1, TIN2, and TPP1 (reviewed in de Lange 2006 "Telomeres"). Human telomeric DNA is also bound by nucleosomes (Makarov et al. 1993; Nikitina and Woodcock 2004). A number of other proteins, including some that play roles in the DNA damage response, can be found at telomeres (Zhu et al. 2000; Verdun et al. 2005).
Studies in yeast and humans indicate that the association of many proteins with telomeres is regulated through the cell cycle (Zhu et al. 2000; Taggart et al. 2002; Fisher et al. 2004; Takata et al. 2004; Takata et al. 2005; Verdun et al. 2005). For instance, TRF1, MRE11, POT1, ATM, and NBS1 display cell cycle regulated chromatin immunoprecipitation of telomeric DNA (Zhu et al. 2000; Verdun et al. 2005), and cytologically observable hTERT and hTERC localize to a subset of telomeres only in S-phase (Jady et al. 2006; Tomlinson et al. 2006). These data indicate that telomeres are dynamically remodeled through the cell cycle.
Heteropentamer:RNA primer-DNA primer:G-strand extended telomere end duplex:PCNA
homotrimerHeteropentamer:RNA primer-DNA primer:G-strand extended telomere end duplex:PCNA
homotrimerHeteropentamer:RNA primer-DNA primer:G-strand extended telomere
endHeteropentamer:RNA primer-DNA primer:G-strand extended telomere
endHeteropentamer:RNA primer-DNA primer:G-strand extended telomere
endprimer:DNA primer:G-strand extended
telomere:POLA:primaseprimer:DNA primer:G-strand extended
telomere:POLA:primaseholoenzyme complex
(generic)primer:G-strand extended
telomere:PCNAprimer:G-strand extended
telomere:PCNAextended telomere
end:POLA:primaseextended telomere
end:POLA:primaseextended telomere
end:POLA:primaseHoloenzyme Base-paired to the Telomeric Chromosome End with an Additional single Stranded
Telomere repeatHoloenzyme Base-paired to the Telomeric Chromosome End with an Additional single Stranded
Telomere repeatHoloenzyme Base-paired to the Telomeric Chromosome End with an Additional single Stranded
Telomere repeatRNP:G-strand telomeric
chromosome endRNP:G-strand telomeric
chromosome endRNP:Telomeric Chromosome End with an Additional single Stranded
Telomere repeatRNP:Telomeric Chromosome End with an Additional single Stranded
Telomere repeatand base-paired to the Telomeric
Chromosome Endand base-paired to the Telomeric
Chromosome Endand base-paired to the Telomeric
Chromosome Endchromosome end:Shelterin
(p-S365-TERF2)chromosome end:Shelterin
(p-S365-TERF2)chromosome
end:Shelterinchromosome
end:Shelterinchromosome
end:Shelterinchromosome end with two additional single strand
repeatschromosome end with two additional single strand
repeats