Telomeres stabilise DNA at the ends of chromosomes, preventing chromosome fusion and genetic instability. Telomeres differ from double strand breaks in that they activate neither DNA repair nor DNA damage checkpoint pathways. Paradoxically DNA repair and checkpoint genes play critical roles in telomere stability. Recent work has provided insights into the roles of DNA repair and DNA damage checkpoint pathways in the physiological maintenance of telomeres and in cellular responses when telomeres become uncapped. In budding yeast the Mre11p nuclease, along with other unidentified nucleases, plays critical roles in physiological telomere maintenance. However, when telomeres are uncapped, the 5′-to-3′ exonuclease, Exo1p, plays a critical role in generating single-stranded DNA and activating checkpoint pathways. Intriguingly Exo1p does not play an important role in normal telomere maintenance. Although checkpoint pathways are not normally activated by telomeres, at least four different types of telomere defect activate checkpoint pathways. Interestingly, each of these telomere defects depends on a different subset of checkpoint proteins to induce cell cycle arrest. A model for how a spectrum of telomeric states might interact with telomerase and checkpoint pathways is proposed.

Telomeric DNA ends are inert because they induce neither DNA repair nor DNA damage checkpoint responses. The efficiency with which telomeres are hidden from checkpoint and repair processes is illustrated by the fact that a haploid budding yeast cell that has 64 telomeres enters mitosis without delay or `repair' of the chromosome ends. In contrast, a yeast cell with a single unrepaired double strand break (DSB) does not (Lee et al., 1998; Sandell and Zakian, 1993). Therefore, special properties of telomeric DNA ends must explain why the ends of chromosomes are perceived differently from DNA ends elsewhere in the genome.

It is critical for genetic stability that telomeres and DSBs do not interconvert. If DSBs and telomeres were to switch properties, acentric fragments would be induced by DSBs that switched to telomeres and chromosome fusions would be induced by many of the telomeres that switched to DSBs. Since DSBs and telomeres rarely interconvert, it seemed reasonable to imagine that cells distinguish DSBs from telomeres by ensuring that different classes of protein bind to telomeric and DSB DNA ends. However, it is now clear that telomeres interact with numerous DNA repair and DNA damage checkpoint proteins. How DNA repair and checkpoint proteins interact at telomeres and yet induce neither DNA repair nor cell cycle arrest is a paradox that is not yet explained. Understanding this paradox will lead to a better understanding of the roles of DNA repair and checkpoint pathways not only in telomere stability but also in processing other types of DNA damage.

Here, I review recent insights into the roles of budding yeast DNA repair and checkpoint proteins in telomere physiology and pathology. Yeast telomeres are similar to those of many other organisms, including humans, and therefore lessons from budding yeast may be generally relevant. Other aspects of telomere biology are much better described in reviews on telomere-binding proteins (Cooper, 2000; McEachern et al., 2000; Rhodes et al., 2002), capping and replication (Blackburn, 2000; Blackburn, 2001; Cervantes and Lundblad, 2002; Chan and Blackburn, 2002; Dubrana et al., 2001; Evans and Lundblad, 2000; Shore, 2001), localisation (Hediger et al., 2002) and chromatin structure (Chan and Blackburn, 2002; Gasser, 2000). In addition, several recent reviews describing the roles of DNA damage checkpoint sensors, mediators and kinases in signaling cell cycle arrest have been published (Melo and Toczyski, 2002; Nyberg et al., 2002; Rouse and Jackson, 2002a).

To understand how telomeres protect chromosome ends it is important to know the DNA structures at telomeric ends and their interactions with repair and checkpoint pathways.

The inert nature of telomeric DNA must depend, at least in part, on specific DNA sequences found at telomeres. Two properties are common to all telomeres: repetitive DNA and short 3′ ssDNA tails.

Since telomeric repeats are found at all telomeres, they are presumably essential for telomeres to escape DNA repair or checkpoint responses. Across species, there is significant variation in the type of repetitive DNA sequence that forms the basis of functional telomeres (Louis, 2002; Mefford and Trask, 2002). Thus the presence of some type of repeat, rather than specific repeats, appears to allow telomeres to be inert. The considerable degree of variation in the type of repetitive DNA sequence that can form functional telomeres in yeast supports this view (Fig. 1). One view is that telomeric repeats allow the telomere to form a heterochromatic, silenced state, and thereby avoid repair and checkpoint pathways (Chan and Blackburn, 2002).

Fig. 1.

Six classes of functional telomere in budding yeast. Natural telomeres of budding yeast are illustrated in A and B (Pryde and Louis, 1997) and other types of functional telomere are illustrated in C to F. The data and colouring scheme are from the website of Ed Louis (http://www.le.ac.uk/ge/ejl12/research/telostruc/EndsSmall.html). (A) Y′ telomeres contain the three major repetitive sequences found at budding yeast telomeres: G-rich, Y′ and X repeats. G-rich and X repeats are found at all telomeres. The G-rich repeats are the product of telomerase activity and are approximately 300 bp in wild-type budding yeast strains. X repeats are based on a 473 bp core sequence that contains an ARS (autonomously replicating sequence) consensus sequence, the binding site of the origin recognition complex and a separate Abf1 (ARS binding factor 1) binding site (Pryde and Louis, 1997; Pryde and Louis, 1999). Y′ repeats are considerably larger than X repeats, with two predominant sizes of 5.2 and 6.7 kb (Lundblad and Blackburn, 1993). Y′ repeats also contain ARS consensus sequences and Abf1 binding sites and are therefore potential origins of replication (Pryde and Louis, 1997). In addition, Y′ repeats encode a functional helicase (Yamada et al., 1998). (B) X telomeres contain only G-rich and X repeats (C) In the absence of telomerase, or if telomere capping is defective, cells enter crisis and generate survivors. Type I survivors lose most of the G-rich repeats but amplify Y′ repeats by recombination-dependent mechanisms. (D,E) In the absence of telomerase, Type II survivors contain highly lengthened G-rich repeats that have been maintained by recombination-dependent mechanisms. (F) If a DSB is induced close to a G-rich telomere seed sequence a telomere can be formed de novo.

Fig. 1.

Six classes of functional telomere in budding yeast. Natural telomeres of budding yeast are illustrated in A and B (Pryde and Louis, 1997) and other types of functional telomere are illustrated in C to F. The data and colouring scheme are from the website of Ed Louis (http://www.le.ac.uk/ge/ejl12/research/telostruc/EndsSmall.html). (A) Y′ telomeres contain the three major repetitive sequences found at budding yeast telomeres: G-rich, Y′ and X repeats. G-rich and X repeats are found at all telomeres. The G-rich repeats are the product of telomerase activity and are approximately 300 bp in wild-type budding yeast strains. X repeats are based on a 473 bp core sequence that contains an ARS (autonomously replicating sequence) consensus sequence, the binding site of the origin recognition complex and a separate Abf1 (ARS binding factor 1) binding site (Pryde and Louis, 1997; Pryde and Louis, 1999). Y′ repeats are considerably larger than X repeats, with two predominant sizes of 5.2 and 6.7 kb (Lundblad and Blackburn, 1993). Y′ repeats also contain ARS consensus sequences and Abf1 binding sites and are therefore potential origins of replication (Pryde and Louis, 1997). In addition, Y′ repeats encode a functional helicase (Yamada et al., 1998). (B) X telomeres contain only G-rich and X repeats (C) In the absence of telomerase, or if telomere capping is defective, cells enter crisis and generate survivors. Type I survivors lose most of the G-rich repeats but amplify Y′ repeats by recombination-dependent mechanisms. (D,E) In the absence of telomerase, Type II survivors contain highly lengthened G-rich repeats that have been maintained by recombination-dependent mechanisms. (F) If a DSB is induced close to a G-rich telomere seed sequence a telomere can be formed de novo.

In most organisms the terminal telomeric repeats are generated by telomerase. Telomerase is a ribonucleoprotein with reverse transcriptase activity that circumvents the `end replication problem' by maintaining the presence of G-rich repeats at telomeres (Olovnikov, 1973; Watson, 1972). Telomerase extends the G-rich strand at the 3′ terminus of natural telomeres without a requirement for a complementary, template strand of DNA (Greider and Blackburn, 1985). Among species there is considerable variation in the precise sequence of the G-rich repeat added by telomerase (Wellinger and Sen, 1997). The C-rich strand is produced by standard, semi-conservative DNA replication.

Olovnikov and Watson pointed out that the ends of linear DNA molecules (telomeres) could not be completely replicated by the normal DNA replication machinery and that this `end replication problem' would lead to loss of telomeric DNA each cell cycle. Telomerase solves the end replication problem because it extends telomeres without the need for a template strand.

Telomerase can generate telomeres de novo, from DSBs, if G-rich telomeric seed sequences lie close to the site of the DSB (Diede and Gottschling, 1999; Kramer and Haber, 1993; Myung et al., 2001). DSB-derived telomeres contain G-rich repeats but lack sub-telomeric X or Y′ repeats that precede G-rich repeats at natural telomeres (see below and Fig. 1). DSB-induced telomeres act as fully functional chromosome caps but have silencing properties different from those of natural telomeres (Pryde and Louis, 1999).

A single sub-telomeric X repeat precedes G-rich repeats at all natural budding yeast telomeres (Pryde and Louis, 1997). Each X repeat is based on a 473 bp core sequence that contains an ARS (autonomously replicating sequence) consensus sequence, the binding site for the origin-recognition complex and a separate Abf1 (ARS binding factor 1) binding site (Pryde and Louis, 1997; Pryde and Louis, 1999). By these criteria the core X repeat is an origin of replication (Raychaudhuri et al., 1997). In addition, approximately half the budding yeast telomeres contain one to four copies of a Y′ repeat (Louis et al., 1994; Pryde and Louis, 1997) (Fig. 1A,B). Y′ repeats are considerably larger than X repeats and have two predominant sizes, 5.2 and 6.7 kb (Lundblad and Blackburn, 1993). Y′ repeats also contain ARS consensus sequences and Abf1-binding sites and are therefore potential origins of replication (Pryde and Louis, 1997). In addition Y′ repeats encode functional helicases (Yamada et al., 1998).

X and Y′ repeats are always orientated in the same direction at telomeres, presumably to ensure that recombination between sub-telomeric repeats does not generate dicentric chromosomes. The high degree of homology between Y′ repeats on different telomeres, and the variation in Y′ repeat number between different strains, indicates that there is a high frequency of recombination at sub-telomeric Y′ repeats (Louis et al., 1994). In contrast, the X repeats share less sequence similarity and are never present as more than one copy per subtelomere, which indicates that recombination is suppressed at X sequences.

Yeast and mammalian cells that do not express telomerase divide for a small number of cell divisions before entering crisis. At low frequency yeast cells in crisis generate `survivors' that can divide and maintain telomeres by recombination-dependent mechanisms. These alternative pathways of telomere maintenance may be analogous to the telomerase-independent ALT (alternative lengthening of telomeres) pathways that exist in mammalian cells (Henson et al., 2002). In budding yeast, Type I survivors amplify Y′ repeats (Fig. 1C) whereas Type II survivors amplify the G-rich repeats (Fig. 1D,E) (Le et al., 1999; Lundblad and Blackburn, 1993; Teng and Zakian, 1999). Type I survivors, containing amplified Y′ repeats, are particularly interesting because the telomeres of these cells effectively cap chromosome ends in the absence of extensive G-rich repeats at chromosome ends. This suggests that proteins that bind double-stranded G-rich repeats may not be essential for capping and that telomerase and other proteins binding the 3′ ssDNA tail at telomeres may be sufficient for capping. Alternatively, Type I survivors might cap telomeres by forming a heterochromatin type of structure (Chan and Blackburn, 2002).

After a number of divisions in the absence of telomerase, telomeres become critically short, and cells stop division and enter crisis.

Telomeres of all organisms examined, including budding yeast, Tetrahymena, human and Arabidopsis, terminate with a short 3′ overhang of the G-rich strand (Jacob et al., 2003; Makarov et al., 1997; McElligott and Wellinger, 1997; Riha et al., 2000; Wei and Price, 2003; Wellinger et al., 1996; Wellinger et al., 1993). This ssDNA tail probably exists to provide a substrate for telomerase, which requires a 3′ overhang on its substrate to function (Lingner and Cech, 1996; Wang and Blackburn, 1997). However, 3′ tails are also important for initiating recombination at DSBs. Since recombination-dependent mechanisms of telomere maintenance can be important, the 3′ tails at telomeres may also play a critical role in recombination-dependent telomere maintenance.

In mammalian and many other cell types the 3′ overhang appears to be folded back into a sub-telomeric location to create a `t-loop' (Wei and Price, 2003). To form t-loops the 3′ ssDNA G-rich repeat loops back and invades the dsDNA G-rich repeats (Griffith et al., 1999; Wei and Price, 2003). So far there is no evidence for t-loops in yeasts, which suggests that in yeasts the 3′ overhang may be exposed. In budding yeast the 3′ overhang is more pronounced in S phase (Dionne and Wellinger, 1996; Wellinger et al., 1996; Wellinger et al., 1993) and requires the passage of the replication fork (Dionne and Wellinger, 1998). In human and tetrahymena cells the ssDNA tail is detectable at all stages of the cell cycle (Jacob et al., 2003; McElligott and Wellinger, 1997). Recent experiments suggest that in rapidly dividing mammalian cells some telomeres instead have a 5′ C strand ssDNA overhang (Cimino-Reale et al., 2003).

The 3′ ssDNA overhang at telomeres is intriguing because mitotic and meiotic DSBs are resected to generate 3′ ssDNA overhangs as a prerequisite for genetic recombination (Sugawara and Haber, 1992; Sun et al., 1991). This raises the question: why are telomeres not undergoing continual recombination events? If telomeres were in a perpetual state of recombination then cell cycle progression might be inhibited either by checkpoint-dependent signaling or physically by inter-chromatid exchanges. Although there is clear evidence for elevated rates of recombination between Y′ repeats, recombination does not seem to be occurring continually because the cell cycle proceeds on schedule. Presumably some aspect of telomere capping is important for limiting resection and recombination at telomeres. Consistent with this idea yeast strains that are defective in telomere capping and/or replication show elevated levels of ssDNA and recombination at telomeres (see below; negative regulation of nucleases).

ssDNA at telomeres is also intriguing because ssDNA is thought to be an important component of the stimulus for checkpoint-dependent cell cycle arrest (Carr, 2003; Garvik et al., 1995; Maringele and Lydall, 2002; Vaze et al., 2002; Zou and Elledge, 2003). Analysis of cell cycle arrest in response to a single DSB suggests that 10 kb of ssDNA is necessary for cell cycle arrest (Vaze et al., 2002). In budding yeast it can be calculated that >150 bp of ssDNA per telomere would be required to generate 10 kb of ssDNA. Since each telomere contains approximately 300 bp of G-rich repeats, this would represent extremely high levels of ssDNA. Therefore it may simply be that there is normally insufficient ssDNA at telomeres to activate checkpoint-dependent cell cycle arrest.

Telomere capping ensures that telomeric DNA ends behave differently from DSB ends. A large number of telomere-binding proteins have been identified and these contribute to telomere capping and replication. Proteins that bind dsDNA at telomeres include Trf1, Trf2, Tin2, Tankyrase and hRap1 in mammalian cells (Rhodes et al., 2002), and Rap1, Sir2, Sir3, Sir4, Rif1 and Yku70/Yku80 in budding yeast, as well as components of telomerase (Est1 and Est2) and the DNA replication machinery (Cooper, 2000). In addition there are proteins that appear to be particularly involved in binding ssDNA at telomeres. Pot1 is a ssDNA-binding protein in mammalian and fission yeast cells, and Cdc13p binds ssDNA in budding yeast (Baumann and Cech, 2001; Garvik et al., 1995; Mitton-Fry et al., 2002; Nugent et al., 1996; Rhodes et al., 2002; Wei and Price, 2003).

Cells lacking telomere-binding proteins display several different phenotypes associated with improper capping and/or replication. These phenotypes include shortened telomeres, lengthened telomeres, telomere fusions, elevated levels of ssDNA, elevated levels of recombination, telomere loss and checkpoint activation. This range of phenotypes indicates that numerous different activities are normally coordinated to maintain and cap telomeres. For example, not only does telomerase need to be recruited to telomeres successfully but its activity needs to be inhibited to ensure that telomere length does not increase indefinitely. Similarly, lagging-strand DNA synthesis needs to be regulated coordinately with telomerase activity to ensure that the length of the 3′ single-stranded overhang does not become excessive (Fig. 2C). The large number of telomere-binding proteins, and the complexity of phenotypes associated with defects in these proteins, means that is difficult to understand precisely the roles of the various proteins in telomere capping and replication.

Fig. 2.

Telomere replication. (A) Telomeres in all organisms contain a short 3′ overhang on the G rich strand. (B) A replication fork moving towards the end of the chromosome. (C) The newly replicated, lagging C strand, will generate a natural 3′ overhang when the RNA primer is removed from the final Okazaki fragment, or if the lagging strand replication machinery cannot reach the end of the chromosome. In the absence of nuclease activity the unreplicated 3′ strand will be the same length as it was prior to replication. (D) The newly replicated leading G strand will be the same length as the parental 5′ C strand, and blunt ended if the replication fork reaches the end of the chromosome. Therefore the newly replicated 3′ G strand will be shorter than the parental 3′ strand and unable to act as a substrate for telomerase because it does not contain a 3′ overhang. If the leading strand replication fork does not reach the end of the chromosome a 5′ rather than 3′ overhang would be generated, but this would not be a suitable substrate for telomerase.

Fig. 2.

Telomere replication. (A) Telomeres in all organisms contain a short 3′ overhang on the G rich strand. (B) A replication fork moving towards the end of the chromosome. (C) The newly replicated, lagging C strand, will generate a natural 3′ overhang when the RNA primer is removed from the final Okazaki fragment, or if the lagging strand replication machinery cannot reach the end of the chromosome. In the absence of nuclease activity the unreplicated 3′ strand will be the same length as it was prior to replication. (D) The newly replicated leading G strand will be the same length as the parental 5′ C strand, and blunt ended if the replication fork reaches the end of the chromosome. Therefore the newly replicated 3′ G strand will be shorter than the parental 3′ strand and unable to act as a substrate for telomerase because it does not contain a 3′ overhang. If the leading strand replication fork does not reach the end of the chromosome a 5′ rather than 3′ overhang would be generated, but this would not be a suitable substrate for telomerase.

Nucleases are usually associated with DNA repair and replication processes but they are also critical for generating 3′ ssDNA overhangs at telomeres, particularly on leading strand telomeres (Fig. 2). Recent experiments in Tetrahymena indicate that unidentified nucleases degrade not only the 5′ strand but also the 3′ strand at telomeres (Jacob et al., 2003). Recruitment of nucleases to telomeres requires proper regulation because excessive nuclease activity might lead to telomere loss or high levels of ssDNA.

Telomere attrition occurs in mammalian and yeast cells that do not express telomerase. The end replication problem may explain the loss of telomeric DNA that occurs each cycle. However, it is equally possible that nucleases play a major role in degrading the ends of the chromosomes. Telomerase-deficient human cells lose approximately 150 bp (30-500 bp) of telomeric DNA per generation (Huffman et al., 2000). In contrast, telomerase-deficient yeast cells lose just 3-6 bp per generation (Lundblad and Blackburn, 1993). The difference in telomere loss rate in yeast and humans may reflect differing susceptibilities of human and yeast telomeres to nucleases. It is also known that different human cell types contain ssDNA overhangs of different lengths and lose telomeric DNA at different rates (Huffman et al., 2000). These cell-type-specific differences may be due to differing nuclease activities in different cell types rather than different abilities to replicate telomeres.

Several budding yeast genes that limit the extent of ssDNA at telomeres have been identified. These include genes encoding the DNA repair protein Yku70/Yku80 (Gravel et al., 1998; Maringele and Lydall, 2002; Polotnianka et al., 1998), the telomeric ssDNA-binding protein Cdc13 (Garvik et al., 1995; Polotnianka et al., 1998), DNA polymerase α (CDC17) (Adams Martin et al., 2000; Carson and Hartwell, 1985) and a FLAP endonuclease (RAD27) (Parenteau and Wellinger, 2002). In addition, other proteins that interact with Cdc13p, including Stn1p (Grandin et al., 1997) and Ten1p (Grandin et al., 2001) along with telomerase itself (Chan et al., 2001), contribute to limiting ssDNA at telomeres. Presumably many of these gene products play roles in capping and limiting nuclease activity at telomeres or in coordinating lagging-strand replication (Fig. 3C) (Diede and Gottschling, 1999; Parenteau and Wellinger, 2002).

Fig. 3.

A spectrum of telomeric states. A model showing three states at budding yeast telomeres. (A) A fully capped telomere that prevents checkpoint activation and repair pathways. It is capped by numerous telomere-binding proteins, indicated by T. (B) An uncapped telomere that has recruited the PIKK kinase Tel1p, the checkpoint protein Rad9p, and the MRX complex (encoded by MRE11, RAD50 and XRS2). This type of telomere is a weak inhibitor of cell division based on the fact that the TEL1-dependent response to unresected DSBs is weak (Usui et al., 2001) and that Tel1p overexpression causes transient arrest (Viscardi et al., 2003). Tel1p appears to be a potent activator of telomerases and contributes to telomere capping. (C) A resected, DSB-like telomere that has recruited the core members of the DNA damage checkpoint response, including MEC1, MEC3, RAD9, RAD17 and DDC1. This DSB-like telomere is a potent activator of cell cycle arrest but less efficient at recruiting telomerase.

Fig. 3.

A spectrum of telomeric states. A model showing three states at budding yeast telomeres. (A) A fully capped telomere that prevents checkpoint activation and repair pathways. It is capped by numerous telomere-binding proteins, indicated by T. (B) An uncapped telomere that has recruited the PIKK kinase Tel1p, the checkpoint protein Rad9p, and the MRX complex (encoded by MRE11, RAD50 and XRS2). This type of telomere is a weak inhibitor of cell division based on the fact that the TEL1-dependent response to unresected DSBs is weak (Usui et al., 2001) and that Tel1p overexpression causes transient arrest (Viscardi et al., 2003). Tel1p appears to be a potent activator of telomerases and contributes to telomere capping. (C) A resected, DSB-like telomere that has recruited the core members of the DNA damage checkpoint response, including MEC1, MEC3, RAD9, RAD17 and DDC1. This DSB-like telomere is a potent activator of cell cycle arrest but less efficient at recruiting telomerase.

Interestingly, checkpoint pathways also limit ssDNA production at telomeres. RAD9 was the first checkpoint gene to be so defined (Weinert and Hartwell, 1988) and is critical for cell cycle arrest in many strains that have telomere defects (Table 1). RAD9 inhibits ssDNA production in strains lacking the telomere-binding protein Cdc13p (Lydall and Weinert, 1995). The mechanism by which Rad9p inhibits nuclease activity at uncapped telomeres in cdc13-1 mutants is unclear. Rad9p is considered to be a `mediator' checkpoint protein, facilitating crosstalk between upstream checkpoint kinases, such as Mec1p, and downstream checkpoint kinases, such as Chk1p and Rad53p (Melo and Toczyski, 2002; Osborn et al., 2002). Our recent experiments suggest that Rad9p also inhibits ssDNA production at uncapped telomeres by mediating interactions between upstream and downstream checkpoint kinases (X. Jia, T. Weinert and D. Lydall, unpublished).

Table 1.

The role of checkpoint genes in responding to telomeric defects

S. cereviesiae gene Human/pombe orthologue Telomere damage
Function cdc13-1yku70Δtlc1Δ Tel1p op
MEC1  ATR/Rad3   PIK Kinase   Yes   Yes   Yes   No  
DDC2  ATRIP/Rad26   Kinase binding   Yes    Yes   No  
RAD53  CHK2/Cds1   Kinase   50%    Minor?   Yes  
DUN1   Kinase   50%   No    
CHK1  CHK1   Kinase   50%   Yes    Yes  
TEL1  ATM/Tel1   PIK Kinase     No   
RAD9  BRCA1 Rhp9 TOPBP1   Mediator   Yes   Yes   Minor?   Yes  
RAD24  RAD17   RFC like   Yes   No   Yes   
RAD17  RAD1   PCNA like   Yes   No    
MEC3  HUS1   PCNA like   Yes   No   Yes   
DDC1  RAD9   PCNA like   Yes   No    No  
S. cereviesiae gene Human/pombe orthologue Telomere damage
Function cdc13-1yku70Δtlc1Δ Tel1p op
MEC1  ATR/Rad3   PIK Kinase   Yes   Yes   Yes   No  
DDC2  ATRIP/Rad26   Kinase binding   Yes    Yes   No  
RAD53  CHK2/Cds1   Kinase   50%    Minor?   Yes  
DUN1   Kinase   50%   No    
CHK1  CHK1   Kinase   50%   Yes    Yes  
TEL1  ATM/Tel1   PIK Kinase     No   
RAD9  BRCA1 Rhp9 TOPBP1   Mediator   Yes   Yes   Minor?   Yes  
RAD24  RAD17   RFC like   Yes   No   Yes   
RAD17  RAD1   PCNA like   Yes   No    
MEC3  HUS1   PCNA like   Yes   No   Yes   
DDC1  RAD9   PCNA like   Yes   No    No  

The roles of different checkpoint genes in causing cell cycle arrest in cdc13-1, yku70Δ, tlc1Δ and TEL1 overexpressing strains are indicated. A blank indicates that the particular checkpoint gene has not been tested, 50% indicates that the checkpoint gene is only partially required for arrest (Clerici et al., 2001; Enomoto et al., 2002; Gardner et al., 1999; IJpma and Greider, 2003; Lydall and Weinert, 1995; Maringele and Lydall, 2002; Rouse and Jackson, 2002b; Sanchez et al., 1999; Viscardi et al., 2003). Checkpoint genes in bold are implicated in telomere maintenance or stability. mec1Δ tel1Δ double mutants and analogous rad3Δ tel1Δ double mutants of fission yeast are completely defective in telomere maintenance, erode telomeres and undergo telomere fusions (Craven et al., 2002; Matsuura et al., 1999; Naito et al., 1998). cdc13-1, yku70Δ, tlc1Δ and TEL1 over-expressing strains accumulate `DNA damage' at telomeres, but it is also possible that damage simultaneously induced elsewhere in the genome is an important stimulus for arrest. For tlc1Δ damage there is evidence that RAD9 and RAD53 play either no role (Enomoto et al., 2002) or a minor role (IJpma and Greider, 2003) in cell cycle arrest.

The nucleases responsible for generating the 3′ ssDNA tails at leading strand telomeres have yet to be unambiguously identified in budding yeast. However, there is evidence showing that MRX, EXO1 and the RAD24 group of checkpoint genes regulate or encode nucleases with differing activities at telomeres.

MRX

The MRX nuclease complex in yeast, comprising Mre11p, Rad50p and Xrs2p, is implicated in DNA repair, cell cycle arrest and telomere maintenance (D'Amours and Jackson, 2002). Null mutations in MRX genes result in short telomeres in most genetic backgrounds (Ritchie and Petes, 2000; Tsubouchi and Ogawa, 2000; Wilson et al., 1999), and in one background complete loss of telomeric DNA and senescence (Kironmai and Muniyappa, 1997). Although the MRX complex has numerous biochemical activities in vitro, including 3′-to-5′ nuclease activity, it is involved in the formation of a 3′ overhang at DSBs in vivo (D'Amours and Jackson, 2002; Haber, 1998).

mrx mutants are defective at generating telomeres de novo. In an elegant series of experiments, Diede and Gottschling showed that appropriately located DSBs are resected to generate 3′ ssDNA tails before telomerase converts them to functional capped telomeres (Diede and Gottschling, 2001). In this assay mrx mutants were defective in formation of 3′ ssDNA tails and generation of telomeres in vivo. However, four lines of evidence suggest that MRX-independent mechanisms to generate ssDNA also exist. (1) mrx mutants can generate telomeres at DSBs but with a delay (Diede and Gottschling, 2001). (2) The nuclease activity of the MRX complex does not seem to be required for telomere maintenance (Tsukamoto et al., 2001). (3) The ssDNA-binding protein Cdc13p binds telomeres in the absence of Mre11p (Tsukamoto et al., 2001). (4) In most genetic backgrounds mrx mutants do not become senescent and enter crisis. If MRX were the only nuclease required to generate 3′ ssDNA overhangs at leading strand telomeres (Fig. 2D) then mrx mutants should be unable to recruit telomerase and should enter crisis as do telomerase-deficient cells. Therefore, MRX-independent nucleases or mechanisms contribute to generating 3′ ssDNA overhangs at telomeres. A strong candidate for an alternative exonuclease at telomeres is Exo1p.

EXO1

Exo1p is a conserved 5′-to-3′ exonuclease with FLAP endouclease activity that appears to function redundantly with the MRX complex in resection of DSBs and DNA repair (Lee et al., 2002; Lewis et al., 2002; Moreau et al., 2001; Tran et al., 2002; Tsubouchi and Ogawa, 2000). Exo1p is also implicated in mismatch repair (Tishkoff et al., 1997) and meiotic recombination (Khazanehdari and Borts, 2000; Kirkpatrick et al., 2000). However, unlike mrx mutants, exo1Δ mutants show no telomere length defects (Moreau et al., 2001; Tsubouchi and Ogawa, 2000). Furthermore, mre11Δ single and mre11Δ exo1Δ double mutants have telomeres of similar length, which suggests that Exo1p does not function redundantly with MRX at telomeres.

Although Exo1p appears to play no essential role in telomere physiology, it plays a critical role in regulating ssDNA levels when telomere capping is defective. Specifically, an exo1Δ mutation suppresses the temperature-dependent growth defects and reduces ssDNA accumulation in capping-defective yku70Δ and cdc13-1 mutants cultured at non-permissive temperatures (Maringele and Lydall, 2002) (M. Zubko, S. Guillard and D. Lydall, unpublished). EXO1 is essential for generating all the ssDNA at telomeres of yku70Δ mutants at 37°C but other RAD24-dependent nuclease(s) appear to act in concert with Exo1p at telomeres of cdc13-1 mutants (Maringele and Lydall, 2002) (M. Zubko, S. Guillard and D. Lydall, unpublished).

The RAD24 group

RAD17, RAD24, MEC3 and DDC1 are termed the RAD24 group because deleting any or all of these genes results in similar checkpoint and DNA damage sensitivity phenotypes (Lydall and Weinert, 1995). In a variety of organisms, telomere defects are associated with defects in the RAD24 group of gene products. In C. elegans, mrt2 mutants lacking a RAD17 orthologue possess short telomeres and undergo end-to-end chromosome fusions (Ahmed and Hodgkin, 2000). In S. pombe, mutations in genes encoding the orthologues of the RAD24 group also result in generation of short telomeres (Dahlen et al., 1998; Matsuura et al., 1999; Nakamura et al., 2002). In budding yeast, mec3Δ mutants have long telomeres in one genetic background (Corda et al., 1999) but in another background rad17Δ, rad24Δ and ddc1Δ mutants have slightly shortened telomeres (Longhese et al., 2000).

Rad24p and the four small replication factor C subunits (Rfc2p-Rfc5p) appear to load Rad17p, Mec3p and Ddc1p, a heterotrimeric PCNA-like ring, at uncapped telomeres of cdc13-1 mutants (Griffith et al., 2002; Kondo et al., 2001; Majka and Burgers, 2003; Melo et al., 2001; Shiomi et al., 2002). RAD24 is important, similarly to EXO1, for generating ssDNA at telomeres of cdc13-1 mutants (Booth et al., 2001; Lydall and Weinert, 1995; Maringele and Lydall, 2002) (M. Zubko, S. Guillard and D. Lydall, unpublished). However, unlike EXO1, RAD24 is not important for generating ssDNA at telomeres of yku70Δ mutants (Maringele and Lydall, 2002). Therefore, an appealing model to explain the role the RAD24 group in generating ssDNA at telomeres of cdc13-1 mutants is that the Rad17p-Mec3p-Ddc1p PCNA-type complex tethers some type of nuclease(s) onto DNA. However, Rad17p, Mec3p and Ddc1p do not appear to tether Exo1p to DNA because RAD24 and EXO1 encode or control nucleases with different properties (Maringele and Lydall, 2002) (M. Zubko, S. Guillard and D. Lydall, unpublished).

As described above, checkpoint proteins play critical roles in responding to uncapped telomeres. Blackburn and others have proposed that capped telomeres prevent telomerase, DNA repair and checkpoint pathways from being activated, whereas uncapped telomeres activate telomerase, repair and checkpoint pathways (Blackburn, 2000; Blackburn, 2001; Chan and Blackburn, 2002). According to these models uncapped telomeres are short lived because telomerase rapidly restores the telomere length required for capping. However, the question remains as to how a cell distinguishes between telomeric and DSB ends such that telomeres induce telomerase-dependent rather than repair-dependent pathways to heal the end.

Building on the concept of capped and uncapped telomeres, I suggest that telomeres may vary between the extremes of capped, telomere-like states, and uncapped, DSB-like states. Fig. 3 illustrates this model and is based on the finding that at least four seemingly different DNA damage checkpoint pathways can be activated by uncapped telomeres. According to this model, a fully capped telomere is essentially inert within the cell (Fig. 3A), whereas a fully uncapped telomere behaves like a DSB and is a potent inducer of cell cycle arrest and DNA repair events (Fig. 3C). Between these two extremes, a spectrum of alternative states might exist and just one is shown in Fig. 3B. Telomere capping proteins favour the formation of capped telomeres (Fig. 3A), whereas nucleases favour the formation of DSB-like telomeres (Fig. 3C).

In budding yeast four different types of telomere defect activate checkpoint-dependent cell cycle arrest. Although the roles of all checkpoint genes at each type of damage have not been tested, it is clear that each type of damage relies on different subsets of DNA-damage checkpoint genes to signal arrest (Table 1). Intriguingly, spindle checkpoint genes have also been implicated in arrest of cells with telomere damage (Maringele and Lydall, 2002; Miller and Cooper, 2003). The role of spindle checkpoints in responding to telomere damage is poorly understood.

cdc13-1 mutants cultured at 36°C arrest cell division very rapidly and rely on all classes of checkpoint gene for efficient cell cycle arrest. This is completely dependent on MEC1, DDC2, RAD9 and the RAD24 group but only partially on RAD53, DUN1 and CHK1. The Rad53p and Dun1p protein kinases appear to function in a pathway parallel to that involving Chk1p (and Pds1p) because double mutants are completely arrest-defective (Gardner et al., 1999; Sanchez et al., 1999).

yku70Δ mutants cultured at 37°C arrest more slowly than cdc13-1 mutants and their arrest depends on only a subset of checkpoint genes. It is particularly notable that there is no role for DUN1 [RAD53; see discussion in Maringele and Lydall (Maringele and Lydall, 2002)] or the members of the RAD24 group in arrest of yku70Δ mutants at 37°C (Maringele and Lydall, 2002).

Telomerase-deficient yeast, in crisis, accumulate in G2/M phase of the cell cycle. Two recent papers showed that disruption of checkpoint genes reduces the G2/M delay observed in such cells (Enomoto et al., 2002; IJpma and Greider, 2003). The two papers show that the delay depends on MEC1, DDC2 and the RAD24 group of genes but less so on TEL1, RAD9 and RAD53. Interestingly, Rad53p is phosphorylated in a RAD9-dependent manner in senescing cells, although neither RAD9 nor RAD53 plays a major role in cell cycle arrest (IJpma and Greider, 2003; Enomoto et al., 2002).

Finally, Tel1p overexpression in cells that have short telomeres induces transient checkpoint-dependent cell cycle arrest (Clerici et al., 2001; Viscardi et al., 2003). This type of damage is unique in that it is the only type of telomere-specific damage that can induce arrest independently of MEC1 and DDC2. Arrest of cells overexpressing TEL1 is transient and correlates with the length of time it takes for telomeres to stabilise at a new length. Tel1p overexpression may exaggerate a weak checkpoint that occurs each time telomeres are elongated.

In summary, each of four different types of telomere defect relies on a different subset of checkpoint genes. One explanation for these data is that each type of damage recruits and activates a different constellation of checkpoint proteins at the telomere. I suggest that in yeast different telomeric stimuli represent states that exist between the extremes of a fully capped telomere and a DSB-like telomere. Damage induced by the cdc13-1 mutation is perhaps most `DSB-like' because it relies on most of the genes required to induce arrest at DSBs, whereas TEL1 overexpression is most `telomere-like' since arrest is transient and independent of MEC1. It may also be relevant that different checkpoint genes respond to blunt or resected DSBs. MEC1, RAD17, RAD24 and MEC3 signal cell cycle arrest in meiotic cells that contain resected DSBs, but RAD9 and TEL1 do not (Lydall et al., 1996; Usui et al., 2001). However, all six genes, and the MRX genes, are essential to signal arrest in response to unresected (blunt) DSBs (Usui et al., 2001). Aspects of this model may also be relevant in mouse and human cells, where different checkpoint pathways respond to similar telomere defects (Smogorzewska and de Lange, 2002).

Telomeres do not normally activate DNA repair and DNA damage checkpoint responses. It came as a surprise to discover that DNA repair and DNA damage checkpoint genes play important roles at telomeres. Although we are still far from understanding the precise roles of repair and checkpoint proteins at telomeres, some important clues are emerging. It is now clear that many different types of telomere defect exist and each type requires a different subset of checkpoint genes to induce arrest. It may be that a spectrum of states, each activating different checkpoint pathways, explains the roles of checkpoint proteins in telomere physiology and pathology. If so, then understanding these states will have implications not only for how cells respond to defective telomeres but also for how cells respond to damaged DNA elsewhere in the genome.

I thank Richard Blankley, Julie Cooper, Xindan Jia, Pia Longhese, Ed Louis, Laura Maringele, Misha Zubko and anonymous referees for input and comments on the manuscript. I apologise to those whose work was not cited owing to space constraints. D.L. is supported by the Wellcome Trust.

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