Biology of Telomeres, Lessons From Budding Yeast

R EV I EW AR T I C L E

Biology of telomeres: lessons from budding yeast

Martin Kupiec

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Correspondence: Martin Kupiec,

Department of Molecular Microbiology and

Biotechnology, Tel Aviv University, Ramat

Aviv 69978, Israel. Tel.: +972 3 640 9031;

fax: +972 3 640 9407;

e-mail: [email protected]

Received 17 July 2013; revised 12 November

2013; accepted 3 December 2013. Final

version published online 8 January 2014.

DOI: 10.1111/1574-6976.12054

Editor: Jure Piskur

Keywords

genome stability; aging; cancer; DNA

replication; DNA damage response.

Abstract

Telomeres are nucleoprotein structures that cap the ends of the linear eukary-

otic chromosomes and thereby protect their stability and integrity. Telomeres

play central roles in maintaining the genome’s integrity, distinguishing between

the natural chromosomal ends and unwanted double-stranded breaks. In addi-

tion, telomeres are replicated by a special reverse transcriptase called telomer-

ase, in a complex mechanism that is coordinated with the genome’s

replication. Telomeres also play an important role in tethering the chromo-

somes to the nuclear envelope, thus helping in positioning the chromosomes

within the nucleus. The special chromatin configuration of telomeres affects

the expression of nearby genes; nonetheless, telomeres are transcribed, creating

noncoding RNA molecules that hybridize to the chromosomal ends and seem

to play regulatory roles. The yeast Saccharomyces cerevisiae, with its sophisti-

cated genetics and molecular biology, has provided many fundamental concepts

in telomere biology, which were later found to be conserved in all organisms.

Here, we present an overview of all the aspects of telomere biology investigated

in yeast, which continues to provide new insights into this complex and

important subject, which has significant medical implications, especially in the

fields of aging and cancer.

Introduction

The genome of most eukaryotic organisms is divided into

linear chromosomes. Each chromosomal end is protected

by a special nucleoprotein structure called telomere. Telo-

meres play central roles in maintaining the stability of the

genome: they differentiate the natural chromosomal ends,

which should not be repaired, from double-stranded

DNA breaks (DSBs), which occur often by accident in the

cells and need to be repaired urgently to prevent loss of

genomic information (Dewar & Lydall, 2012). Protection

of the chromosomal ends is conferred by the special fold-

ing of telomeres, as well as by specific telomeric proteins.

In addition, telomeres provide a solution to the end-

replication problem: the regular DNA replication machin-

ery is unable to fully replicate the chromosomal ends

(Olovnikov, 1971; Watson, 1972); as a consequence,

information is lost with each cell division, eventually

resulting in senescence and cell death (Hayflick, 1979;

Lundblad & Szostak, 1989; Harley et al., 1990).

Highly proliferative cells, such as mammalian embry-

onic cells and unicellular organisms, solve this problem

by expressing the specialized reverse transcriptase telo-

merase (Greider & Blackburn, 1987; de Lange, 2009),

which is able to extend the telomeres by copying telo-

meric sequences from an internal RNA template. Indeed,

it is enough to express active telomerase to overcome cel-

lular senescence in somatic cells (Bodnar et al., 1998).

Cancer cells also require functional telomeres: in about

80% of tumors, the telomerase gene is expressed

(DeMasters et al., 1997); in the rest, an alternative mech-

anism, based on homologous recombination (HR), allows

telomere length extension (ALT; reviewed in Conomos

et al., 2013). Moreover, experiments have shown that

replenishing telomeres is one of the few essential and

earliest steps that a normal mammalian fibroblast must

take to become cancerous (Hahn et al., 1999). Mutations

that affect telomere function result in human diseases,

such as dyskeratosis Congenita, idiopathic pulmonary

fibrosis, and others (Calado & Young, 2009; Armanios,

2012; Gramatges & Bertuch, 2013). Thus, our under-

standing of the biology of telomeres has significant medi-

cal implications and is especially relevant to the fields of

aging and cancer.

FEMS Microbiol Rev 38 (2014) 144–171ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

ROBI

OLO

GY

REV

IEW

S

Although some differences exist between the organiza-

tion of telomeres in yeast and mammals, many basic rules

are universal. In 2009, Elizabeth Blackburn, Carol

Greider, and Jack Szostak received the Nobel Prize in

Medicine for their work on telomeres and telomerase.

Much of this work was carried out in model organisms,

including the yeast Saccharomyces cerevisiae.

Several excellent reviews on various aspects of telomere

biology have been published in the last years (Palm & de

Lange, 2008; Lydall, 2009; Shore & Bianchi, 2009; Artandi

& DePinho, 2010; Giraud-Panis et al., 2010; Dewar &

Lydall, 2012; Smekalova et al., 2012; Stewart et al.,

2012a, b; Wellinger & Zakian, 2012; Churikov et al.,

2013; Conomos et al., 2013; Gramatges & Bertuch, 2013;

Lu et al., 2013; Nandakumar & Cech, 2013; Teixeira,

2013). Here, we will concentrate on what we have learnt

from the yeast S. cerevisiae, an organism that, by virtue of

its fast growth, excellent biochemistry, and superb genet-

ics, has become extensively used for the molecular genetic

dissection of many universal cellular processes.

Throughout this review, we will refer to four intercon-

nected aspects of telomere biology: (1) telomere ‘capping’,

which prevents the recognition of the natural ends as if

they were DSBs that require repair; (2) telomere replica-

tion, which is necessary to solve the end-replication prob-

lem and must be coordinated with regular DNA

polymerases; (3) telomere localization at the periphery of

the nucleus and its role in silencing of genes located nearby;

finally, (4) telomere length regulation: a typical ‘wild-type’

length is achieved by a complex homeostasis, which is

exquisitely regulated. As expected, all these aspects of

telomere biology are interconnected, interrelated, and

interdependent, and it is not always possible to separate

one from another.

Structure and sequence of telomeres

The telomeres of most organisms are composed of simple

tandem repeats, and, although telomeres vary in length

(from c. 350 bp in yeast to several kb in mammals), their

general structure and functions are conserved (Fig. 1).

The yeast telomeric sequence is not regular and can be

described as T(G1–3) (Shampay et al., 1984; McEachern &

Blackburn, 1994). These sequences are copied by the cata-

lytic subunit of telomerase from the telomerase RNA

template (TLC1 in yeast), whose template sequence is

CACACACCCACACCAC (Lin et al., 2004). Thus, the

RNA template is partially used, and only very short

stretches are copied in each round of telomerase activity

from different regions of the template (Forstemann &

Lingner, 2001). Although the heterogeneity of the telo-

meric sequence in yeast makes it difficult to know the

exact sequence present at any chromosomal end, the same

heterogeneity has been exploited to monitor recently

added telomeric DNA: Individual clones differ in the

sequences added to the pre-existing telomere; this has

been used to ask what telomeres are chosen for elonga-

tion and how this mechanism is controlled (Teixeira

et al., 2004; Arneric & Lingner, 2007; Chang et al., 2007).

The yeast telomeres, as their mammalian counterparts,

are not blunt, but exhibit a 3′ extension of the G-rich

strand (also called a ‘G-tail’ or ‘G-overhang’). This tail

varies in length during the cell cycle, remaining very short

for most of the cycle (about 12 nt; Larrivee et al., 2004),

CSTRif2Rif1 Rap1

Sir2Sir3Sir4

Cdc13Stn1Ten1

Ku70ku80

Telomerase

RPASIR KU

X 0–4 x Y’

TG repeats

TG repeats

5’3’

Rfa1Rfa2Rfa3 Telomerase

Est1Est2Est3TLC1

(a)

(b)

(c)

Fig. 1. Structure of the yeast telomere.

(a) Schematic representation of a yeast

telomere, showing the X and Y′ sequences

and the internal and terminal TG overhangs.

(b) ‘Fold-back’ structure of the yeast telomere,

with representative proteins. Rap1 binds the

telomeric repeats; and Rif1, Rif2, and the SIR

proteins bind to Rap1. The Ku heterodimer

binds to telomeric dsDNA, and the CST

complex binds the terminal ssDNA end.

(c) Telomerase is recruited to telomeres

present in an ‘extensible’ configuration.

FEMS Microbiol Rev 38 (2014) 144–171 ª 2013 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Yeast telomeres 145

but elongating at late S by a mechanism that involves

both elongation of the G-strand by telomerase and degra-

dation of the C-strand and is highly coordinated with

genomic DNA replication (Dionne & Wellinger, 1998;

Frank et al., 2006).

In addition to the telomeric sequences, yeast chromo-

somal ends, as most other eukaryotes, have subtelomeric

repeats. Two repeat families occur exclusively at subtelo-

meric regions: the X and Y′ elements. X units are present

in almost all telomeres, although they may slightly vary in

size and in sequence. In about half of the telomeres, and

distally to the X units (i.e. toward the chromosome’s

end), Y′ elements are present, in 1–4 tandem repeats, bor-

dered by the telomeric repeats on the distal end (Fig. 1a).

TG repeats are sometimes found between the X and the

Y′ elements, as well as between Y′ and Y′ (when more

than one Y′ repeat is present in tandem; Walmsley et al.,

1984). These are potential sources of genomic instability,

as they can recombine with telomeric sequences (Akseno-

va et al., 2013; Gazy & Kupiec, 2013). Potential origins of

replication (ARSs) are present within these elements

(Chan & Tye, 1983). Interestingly, subtelomeric repeats

appear to be extremely variable from strain to strain: for

example, the identity of the Y′-less chromosomal arms

differs among related strains (Horowitz et al., 1984;

Zakian et al., 1986); this is correlated with a high

frequency of recombination among subtelomeric regions

(Louis & Haber, 1990).

Linear chromosomes lacking telomeric TG repeats have

extremely low stability and tend to be lost (Szostak &

Blackburn, 1982; Shampay et al., 1984; Lundblad &

Szostak, 1989). However, normal chromosomal arms nat-

urally exist lacking Y′ elements, and chromosomes engi-

neered to lack both X and Y′ elements are very stable

(Sandell & Zakian, 1993). Despite this apparent dispens-

ability, subtelomeric regions, in particular Y′, are used to

maintain stable chromosomes in cells defective for telo-

merase activity (Lundblad & Blackburn, 1993; Maxwell

et al., 2004).

Telomeric proteins

A number of proteins bind the subtelomeric and telomer-

ic DNA and serve several roles in end protection, DNA

replication, and chromatin establishment and mainte-

nance. Most proteins participate in more than one aspect

of telomere biology (Fig. 1b and c).

Rap1

Rap1 is an abundant essential protein (c. 4000 molecules

per haploid cell; Buchman et al., 1988) that binds

double-stranded telomeric DNA via its two tandem myb

domains (Konig et al., 1996). In addition, Rap1 works as

a general cellular transcriptional activator that binds to

upstream promoter regions at a large number of genes

and interacts with various coactivator proteins (Tornow

et al., 1993; Lieb et al., 2001; Zhao et al., 2006).

It has been calculated that about 20 Rap1 molecules

bind each individual telomere in wt cells (Wright &

Zakian, 1995). Rap1 binds telomeres with high affinity

(Conrad et al., 1990; Lustig et al., 1990) although in a

noncooperative manner (Gilson et al., 1993; Williams

et al., 2010) and plays a central role in determining telo-

mere length: indeed, it has been proposed that a ‘count-

ing mechanism’ is able to monitor (and respond to) the

number of Rap1 molecules (and its partners Rif1 and

Rif2, see below) bound to each individual telomere

(Krauskopf & Blackburn, 1996, 1998; Marcand et al.,

1997; Levy & Blackburn, 2004; Poschke et al., 2012).

Rap1 is an essential protein; however, N-terminal dele-

tions that ablate its BRCT domain (which usually inter-

acts with phosphorylated proteins) are not lethal (Moretti

et al., 1994; Graham et al., 1999). The telomeric functions

of the protein are concentrated in its C-terminus, which

interacts with the Rif1/Rif2 proteins, as well as with the

gene silencing Sir3/Sir4 complex (Kyrion et al., 1992;

Wotton & Shore, 1997; Graham et al., 1999). Rap1 plays

several interrelated roles at the telomere: it prevents

telomere–telomere fusions (Pardo & Marcand, 2005;

Marcand et al., 2008), determines its localization to the

nuclear periphery (Gotta & Gasser, 1996; Laroche et al.,

1998), affects silencing (Hardy et al., 1992a, b; Kyrion

et al., 1993; Palladino et al., 1993), and protects the chro-

mosomal ends (Negrini et al., 2007; Vodenicharov et al.,

2010). Moreover, its dual role as a telomere component

and a general transcription regulator allows Rap1 to serve

as an effector of stress-specific expression programs. Rap1

gets relocalized from the telomeres to additional genomic

sites upon DNA damage (Tomar et al., 2008), glucose

starvation (Buck & Lieb, 2006), and interestingly, senes-

cence initiated by telomere shortening (Platt et al., 2013).

Among hundreds of genes affected by these relocatization

events, it is possible to find the core histone genes, which

are repressed by Rap1 upon senescence (Platt et al.,

2013).

Rif1 and Rif2

Using the yeast two-hybrid method, two factors were iso-

lated, Rif1 and Rif2, that bind the C-terminal region of

Rap1 (Hardy et al., 1992a, b; Wotton & Shore, 1997).

Cells defective for each of these factors exhibit long telo-

meres, indicating that the function of these proteins is to

negatively regulate the elongation of telomeres (Hardy

et al., 1992a, b; Wotton & Shore, 1997). Rif1 and Rif2


146 M. Kupiec

binding confers to Rap1-bound telomeric DNA, a higher

order structure by interconnecting different Rap1 units.

This structure is functionally important, although its

function remains enigmatic (Shi et al., 2013). Despite

these common structural function, Rif1 and Rif2 seem to

play several roles independently of each other. As

explained above, rif1 and rif2 mutants show elongated

telomeres. The double mutant, however, exhibits much

longer and unregulated telomeres, indicating that the two

proteins participate in alternative regulatory mechanisms

(Wotton & Shore, 1997; Romano et al., 2013).

Although telomeric DNA resembles one half of a bro-

ken chromosome, one of the main functions of telomeres

is to ‘cap’ this end to prevent its recognition by the DNA

repair machinery of the cell. Rif1 and Rif2 play an

important role in this process. They both play nonover-

lapping roles in masking a DSB flanked by a very short

array of telomeric repeats (Ribeyre & Shore, 2012). Sur-

prisingly, however, Rap1 and Rif2, but not Rif1, inhibit

the access of nucleases and the nonhomologous end-

joining (NHEJ) machinery (Marcand et al., 2008; Bonetti

et al., 2010a, b; Cornacchia et al., 2012); Rif2 also pre-

vents the association of the Tel1/MRX complex (the yeast

version of ATM/MRN) with telomeres (Hirano et al.,

2009; Chapman et al., 2013) and plays a role in recruit-

ing the histone deacetylase Rpd3L (Poschke et al., 2012).

In contrast, Rif1, but not Rif2, is essential for cell viabil-

ity when the CST activity fails (CST is a protein complex

with a role in telomere capping, see below; Addinall

et al., 2011; Anbalagan et al., 2011; Di Virgilio et al.,

2013). Rif1 seems to play an independent role in trans-

ducing environmental signals to the telomere-maintaining

machinery (Harari et al., 2013; Romano et al., 2013), a

role not shared with Rif2. Surprisingly, Rif1 seems also to

carry out checkpoint-regulating functions at the telo-

meres independently of Rap1 (Feldheim et al., 2011;

Harari et al., 2011; Xue et al., 2011; Escribano-Diaz et al.,

2013; Zimmermann et al., 2013). This finding suggests

that Rif1 may be able to bind DNA sequences by itself.

Consistently, recent work has uncovered functions carried

out by Rif1 that are independent of telomeres, but are

related to DNA transactions. Both fission yeast and mam-

malian Rif1 control the replication-timing program,

determining which regions should replicate at any given

time (Hardy et al., 1992a, b; Cornacchia et al., 2012;

Hayano et al., 2012; Yamazaki et al., 2012). The mamma-

lian ortholog of Rif1 has also recently been found to play

a central role in determining whether a DSB will be pro-

cessed by the HR or the NHEJ pathways (Chapman

et al., 2013; Di Virgilio et al., 2013; Escribano-Diaz et al.,

2013; Zimmermann et al., 2013). The exact mechanism

of these Rif1-regulated events is the current subject of

much investigation.

Yku70 and Yku80

The conserved Ku complex, composed of two proteins of

c. 70 and 85 kDa (Yku70 and Yku80 in yeast), plays

central roles in NHEJ in all eukaryotes studied to date

(Gilson et al., 1993; Palladino et al., 1993; Hirano &

Sugimoto, 2007; Vodenicharov & Wellinger, 2007; Bonetti

et al., 2010a, b). Yeast cells, however, lack the DNA-PK

activity associated with Ku in mammals (Collis et al.,

2005). As NHEJ must be avoided at telomeres, it is sur-

prising that Ku is also a natural component of telomeres.

However, Ku plays an essential role in telomere mainte-

nance (Porter et al., 1996; reviewed in Bertuch &

Lundblad, 2003; Dewar & Lydall, 2012). In yeast, the Ku

complex seems to be recruited to the telomeres in a num-

ber of ways: (1) via the interactions between Yku80 and

Sir4 [Sir4 is a member of the heterochromatin-specific

complex silent information regulator (SIR), see below]

(Martin et al., 1999; Bonetti et al., 2010a, b). (2) In a

Sir4-independent fashion, to subtelomeric X sequences

(Boulton & Jackson, 1996a, b). (3) The Ku complex is

associated with telomerase RNA (TLC1) and participates

in the import of TLC1 to the nucleus (Rathmell & Chu,

1994) and possibly in the recruitment of telomerase

(Taccioli et al., 1994; Gravel et al., 1998; Roy et al.,

2004). (4) The Ku proteins play a role in anchoring the

telomeres to the perinuclear space (Laroche et al., 1998)

by a still mysterious mechanism that involves the small

protein modifier SUMO (Marvin et al., 2009a, b).

(5) Finally, Ku activity has been shown to prevent exonu-

cleolytic activity at broken chromosomes and at telomeres

(Bonetti et al., 2010a, b; Mimitou & Symington, 2010).

Thus, Ku affects almost all aspects of telomere biology.

Interestingly, specific mutations have been found, which

separate the roles that Ku plays in NHEJ and in telomere

biology (Ribes-Zamora et al., 2007; Lopez et al., 2011).

The CST complex

A third conserved complex is composed of the Cdc13,

Stn1, and Ten1 proteins. This complex is structurally sim-

ilar to replication protein A (RPA), which binds ssDNA

during cellular DNA replication and DNA repair (Nugent

et al., 1996; Grandin et al., 1997, 2000, 2001a, b; Qi &

Zakian, 2000; Pennock et al., 2001; Petreaca et al., 2006;

Churikov et al., 2013). Indeed, domains can be swapped

between the two complexes, without losing functionality

(Gao et al., 2007; Gelinas et al., 2009).

The CST binds single-stranded telomeric repeats

through oligosaccharide/oligonucleotide/oligopeptide bind-

ing (OB) folds, a common motif in ssDNA and RNA

binding proteins (Pennock et al., 2001; Sun et al., 2009,

2011). It has been proposed that the CST outcompetes


Yeast telomeres 147

and replaces RPA at telomeres; however, RPA can also be

detected at telomeres and is probably functional during

DNA replication (Schramke et al., 2004; Luciano et al.,

2012; Grandin & Charbonneau, 2013). Thus, both com-

plexes are able to bind the telomeric repeats, and the

division of work between them may be intricately linked

to the mechanism of replication at telomeres.

Conversely, despite high affinity of Cdc13 for single-

stranded TG repeats (Lin & Zakian, 1996; Nugent et al.,

1996), Cdc13 can in principle be recruited to broken

chromosomes (DSBs) to promote telomere addition at

nontelomeric ssDNA sequences (Mandell et al., 2011).

This process is tightly monitored by phosphorylation and

de-phosphorylation of Cdc13 at position S306 by the

checkpoint kinases and phosphatases (Zhang & Durocher,

2010). The two proteins associated with Cdc13, Stn1, and

Ten1, were isolated as genetic and physical interactors of

Cdc13 (Grandin et al., 1997, 2001a, b). Mutations that

inactivate Cdc13, such as the temperature-sensitive

cdc13-1 allele, result in telomere uncapping and cell death

(Grandin et al., 2001a, b; Maringele & Lydall, 2004a, b).

Complete lack of Cdc13 activity leads to telomeric DNA

resection, generating ssDNA that stimulates a checkpoint-

mediated cell cycle arrest (Garvik et al., 1995; Maringele

& Lydall, 2004a, b; Vodenicharov & Wellinger, 2006). At

the permissive temperature, many cdc13 strains (such as

those carrying the cdc13-1 allele), as well as stn1 and ten1

mutants, elongate their telomeres (Garvik et al., 1995; Lin

& Zakian, 1996; Grandin et al., 1997, 2000, 2001a, b;

Evans & Lundblad, 1999; Meier et al., 2001; Petreaca

et al., 2006), indicating that their normal activity prevents

telomere elongation. However, the interactions between

the three proteins are not completely understood. Stn1

and Ten1 appear to regulate the activity of Cdc13

(Churikov et al., 2013) possibly by modulating the inter-

actions with subunits of polymerase alpha (see below).

On the other hand, mutations in STN1 (Grandin et al.,

1997) or overexpression of both Stn1 and Ten1 (Petreaca

et al., 2006; Sun et al., 2009) suppresses the lethality of

cdc13-1 mutants.

The SIR complex

In many organisms, genes located close to telomeres

undergo silencing (also called telomere position effect or

TPE). This phenomenon is due to the heterochromatic

nature of subtelomeric regions, which represses promoter

activity independently of the specific promoter sequence.

The area silenced varies among strains and chromosomal

ends, but can be as long as 10–15 kb from the telomere

ends (Pryde & Louis, 1999).

The SIR complex consists of three proteins, Sir2, Sir3,

and Sir4. The SIR complex interacts with histones to

form the silencing machinery in S. cerevisiae (Rusche

et al., 2003; Liou et al., 2005; Cubizolles et al., 2006). The

complex is recruited to telomeres by interactions with

Rap1. Interestingly, although Rap1 binds to the TG

repeats at the telomere ends, bound Rap1 can also be

found by chromatin immunoprecipitation (ChIP) at a

distance from the telomere end, several kb away (Strahl-

Bolsinger et al., 1997; Poschke et al., 2012). The SIR com-

plex is present in this subtelomeric region too, and it has

been suggested that the yeast chromosome folds back,

allowing contact between terminal and subtelomeric

regions (Fourel et al., 1999, 2001; Ferrari et al., 2004;

Poschke et al., 2012), in a way that may protect the DNA

ends, akin to the mammalian T-loop (Griffith et al.,

1999) (Fig. 1b). Transcription factors that bind to the X

regions participate in the nucleation of SIR-dependent

repression, as well as the Ku complex (Enomoto et al.,

1994; Laroche et al., 1998; Fourel et al., 1999, 2001;

Mishra & Shore, 1999; Ferrari et al., 2004; Radman-Livaja

et al., 2011).

From these nucleation sites, the SIR complex spreads

along the chromatin fiber (Hecht et al., 1996; Strahl-

Bolsinger et al., 1997). This spreading is dependent on the

deacetylation activity of Sir2 (Tanny et al., 1999; Imai

et al., 2000; Smith et al., 2000), which, interestingly, is

stimulated by its interactions with Sir4 (Ghidelli et al.,

2001; Tanny et al., 2004; Hsu et al., 2013) and generates

high-affinity nucleosomal binding sites for Sir3. The

Sir3–Sir4 dimer constitutes the structural backbone of

silent chromatin (reviewed in Moazed et al., 2004). The

SIR complex plays a still enigmatic role in tethering telo-

meres to the nuclear envelope (see below). Among other

interactions, Sir4 interacts with the Mps3 nuclear envelope

protein (Bupp et al., 2007), and this interaction may con-

tribute to organizing chromosomes within the nucleus.

Telomerase

The genetic screens carried out by Lundblad and Szostak

(1989) that identified ‘ever shorter telomere’ (est)

mutants defective in components of the telomerase holo-

enzyme (est1, est2, est3) also found a fourth complemen-

tation group that was allelic to CDC13 (Lendvay et al.,

1996). However, contrary to the defective capping pheno-

type of the cdc13-1 allele, which leads to G2/M cell cycle

arrest and massive telomeric DNA resection, the est4

allele of CDC13 (re-named cdc13-2; Nugent et al., 1996)

showed a senescent behavior, characteristic of cells unable

to support telomerase activity. This suggested that Cdc13

could function in both telomere capping and replication.

Est1, Est2, Est3, and Tlc1 form the yeast telomerase holo-

enzyme (Hughes et al., 2000). A fusion between Cdc13

and Est1 was shown to lead to telomere elongation, even


148 M. Kupiec

in the presence of cdc13-2 mutations or Est1 alleles

unable to interact with telomerase (Evans & Lundblad,

1999). Moreover, if Cdc13 was fused directly to the Est2

catalytic subunit, the essential Est1 subunit became dis-

pensable, demonstrating that the Cdc13-Est1 interaction

has as its goal the recruitment of telomerase.

Telomere capping

One of the main functions of the telomere is to prevent

the cell from repairing its natural chromosomal ends as if

they were DSBs. This function, called telomere capping, is

extremely important: DSBs are among the most serious

types of DNA damage a cell can undergo, and efficient

response mechanisms have evolved to cope with the pres-

ence of even a single DSB. Below, I summarize the cellu-

lar response to a broken chromosome and then compare

it to that observed when the telomeric capping function

is missing. For a more in-depth comparison, see Dewar

and Lydall (2012).

The DNA damage response

A single DSB [created by external insults (radiation,

chemical treatment) or internal cellular metabolism (reac-

tive oxygen species, errors during DNA replication)] elic-

its a robust DNA damage response (DDR, sometimes

referred to as the DNA damage checkpoint), which

includes cell cycle arrest and attempts to repair the break

(Sandell & Zakian, 1993; Aylon & Kupiec, 2003).

Depending on the cell cycle phase (Ira et al., 2004; Aylon

et al., 2004; Aylon & Kupiec, 2005), the broken arms are

then either ligated in a sequence-independent manner (by

the nonhomologous end-joining, or NHEJ, mechanism)

or processed by nucleases, to generate ssDNA that can

then engage in HR with similar sequences at other geno-

mic locations (Aylon et al., 2003).

The decision of whether to repair the break by NHEJ

or by HR falls early in the process: once the ends start to

be resected, they are committed to repair by HR (Aylon

et al., 2003). The first step in the resection process

depends on the MRX complex (Mre11–Xrs2–Rad50) and

the nuclease Sae2, which together generate a short

(50–100 nucleotide) overhang of 3′ ssDNA (Mimitou &

Symington, 2008). This first step is followed by a more

extensive resection carried out by a combination of the

exonuclease Exo1, the helicase Sgs1, and the helicase/

nuclease Dna2 (Tsubouchi & Ogawa, 2000; Gravel et al.,

2008; Mimitou & Symington, 2008; Zhu et al., 2008;

Bonetti et al., 2009). The specific functions of these

enzymes and their interactions are still being elucidated.

A triple mutant devoid of Sae2, Exo1, and Sgs1 or the

double mutants defective for Sae2 and Sgs1 or Sgs1 and

Exo1 show no resection, whereas a sae2D exo1D double

mutant still shows resection. This suggested a model in

which MRX/Sae2 acts first in combination with Sgs1 and

Dna2, and then the Sgs1/Dna2 pair allows Exo1 to extend

the resection. Apparently, Exo1 cannot initiate resection

by itself; double mutants sae2D sgs1D are inviable,

whereas sae2D exo1D cells are viable (Tsubouchi & Oga-

wa, 2000; Gravel et al., 2008; Mimitou & Symington,

2008; Zhu et al., 2008; Bonetti et al., 2009). The activity

of Exo1 is inhibited by the Ku complex (Yku70/Yku80):

in the absence of Ku, sae2D sgs1D cells become viable

(Bonetti et al., 2010a, b; Mimitou & Symington, 2010).

Also consistent with this model, the resection reaction

can be carried out in vitro by combining MRX with Sgs1

and Dna2 (Cejka et al., 2010; Niu et al., 2010) or MRX/

Sae2 and Exo1 (Nicolette et al., 2010; Nimonkar et al.,

2011). Chromatin configuration also plays a still enig-

matic role in controlling resection: the Sgs1–Dna2-depen-dent machinery requires a nucleosome-free gap adjacent

to the DSB for efficient resection, and Exo1 activity is

blocked by regular nucleosomes and may require the

incorporation of the H2A.Z for its activity (Adkins et al.,

2013).

As a result of the resection activity, ssDNA is created,

which gets rapidly covered by the ssDNA binding protein

RPA. This leads to the activation of the DDR kinases

(Zou & Elledge, 2003) through two main branches: Mec1,

the yeast ortholog of ATR, and its partner Ddc2 (ATRIP)

are recruited to the ssDNA-RPA (Paciotti et al., 2000;

Rouse & Jackson, 2000; Kondo et al., 2001; Zou & Ell-

edge, 2003). In parallel, the 9-1-1 complex (composed in

yeast by the Rad17, Ddc1, and Mec3 proteins) is also

loaded onto RPA-coated ssDNA by the Rad24 (Rad17 in

humans) clamp loader (Kondo et al., 2001; Melo et al.,

2001; Majka & Burgers, 2003). Mec1-phosphorylated his-

tone H2A at the site of DNA damage attracts the Rad9

adaptor protein (Downs et al., 2004; Naiki et al., 2004;

Toh et al., 2006; Hammet et al., 2007; Usui et al., 2009),

which also interacts with methylated H3K79 (Wysocki

et al., 2005; Lazzaro et al., 2008). In addition, Dpb11

serves as a bridging partner connecting the Mec1/Ddc2,

the 9-1-1, and the Rad9 branches (Mordes et al., 2008;

Pfander & Diffley, 2011). Rad9 undergoes phosphoryla-

tion by the single yeast cyclin-dependent kinase (CDK1),

generating a substrate for Dpb11 binding. By simulta-

neously binding Mec1 and phosphorylated Rad9, Dpb11

enforces the checkpoint signal transduction and restricts

it to the proper cell cycle phase (Pfander & Diffley, 2011).

Once all these players are in place at the DNA damage

site, Mec1 phosphorylates and activates the downstream

effector kinases Rad53 (Chk2) and Chk1 (Sun et al.,

1998; Sanchez et al., 1999; Blankley & Lydall, 2004; Usui

et al., 2009). The activity of these kinases prevents cell


Yeast telomeres 149

cycle progression: Rad53 phosphorylates the anaphase-

promoting complex (Hu et al., 2001; Agarwal et al.,

2003), and Chk1 phosphorylates the securin Pds1 (San-

chez et al., 1999), blocking progress through anaphase. In

a parallel branch, Rad53 activates the Dun1 kinase, thus

upregulating DNA damage-responsive genes and resulting

in an increase in the cellular dNTP levels (Zhou &

Elledge, 1993; Gardner et al., 1999; Zhao & Rothstein,

2002; Andreson et al., 2010).

Importantly, a feedback mechanism seems to operate,

in which Mec1, Rad9, and Rad53 play roles in inhibiting

resection (Lydall & Weinert, 1995; Jia et al., 2004; Lazzaro

et al., 2008; Morin et al., 2008; Segurado & Diffley, 2008)

thus preventing excessive chromosomal DNA degrada-

tion.

In human cells, two major, evolutionarily related

checkpoint kinases control the response to DNA damage:

ATR and ATM. Whereas ATR responds to problems

during DNA replication by activating Chk1, the ATM

is elicited by DSBs and activates Chk2 (Cimprich &

Cortez, 2008; Shiloh & Ziv, 2013). In S. cerevisiae and

Schizosaccharomyces pombe, in contrast, the ATR ortholog

(Mec1 and Rad3) activates both Chk1 and Chk2/Rad53,

whereas Tel1, the ATM ortholog, plays a role mainly at

telomeres, or when the Mec1 pathway is dysfunctional

(Sabourin & Zakian, 2008). Tel1 is recruited to DSBs by

the Mre11/Rad50/Xrs2 (MRX) complex, where it usually

plays a secondary role, helping to activate the Mec1

checkpoint pathway (Mantiero et al., 2007). However, a

large number of DSBs is able to elicit a Tel1-dependent,

Mec1-independent response (Mantiero et al., 2007). The

activity of Sae2, which initiates resection, also stimulates

the Mre11 nuclease, which helps to liberate MRX from

the DSB, committing the cells to the Mec1-dependent

response (Langerak et al., 2011; Limbo et al., 2011). In

the absence of resection or Mec1, Tel1 is recruited via

interactions with Xrs2 (Usui et al., 2001; Nakada et al.,

2003). Tel1 is then able to elicit the checkpoint response

similarly to Mec1 (Usui et al., 2001; Mantiero et al.,

2007; Limbo et al., 2011).

As we have seen, the broken DNA ends are resected in

cells with an active CDK1 to generate RPA-covered

ssDNA. With the help of several mediator proteins, the

RPA is displaced and replaced by the Rad51 protein, the

eukaryotic ortholog of bacterial RecA. This Rad51 nucleo-

filament is the main intermediate in the HR process: it

allows strand exchange and pairing between molecules

sharing sequences (reviewed in Krejci et al., 2012).

Sequence homology can be usually found in sister chro-

matids, homologs, or just similar sequences ectopically

located. A genome-wide search for homology allows the

Rad51 filament to find its recombination partner (Barzel

& Kupiec, 2008; Agmon et al., 2013). Repair by HR

results in the joining of the broken ends, sometimes

incorporating information transferred from the ‘donor

sequence’ to the originally broken molecule (called ‘gene

conversion’). If the two DNA molecules share a long

stretch of sequence identity, gene conversion can be asso-

ciated with a crossing over event that exchanges informa-

tion reciprocally between partners (Inbar et al., 2000).

Telomere uncapping

As we have seen, telomeres naturally carry a ssDNA end

covered by the RPA-like CST complex (Lin & Zakian,

1996; Grandin et al., 1997, 2001a, b; Gao et al., 2007).

The cdc13-1 allele is temperature sensitive: at the restric-

tive temperature, telomeres become uncapped (Garvik

et al., 1995), resulting in their recognition by the cell as a

DSB. A robust DDR ensues, which includes extensive

ssDNA resection and cell cycle arrest (Garvik et al., 1995;

Jia et al., 2004). A similar response is observed with other

temperature-sensitive alleles of the CST partners (Gao

et al., 2007) or even with a strain deleted for the CDC13

gene (Vodenicharov & Wellinger, 2006; see below). The

genetic control of resection differs from the one observed

at nontelomeric DSBs: the MRX complex, which usually

participates in the initiation of resection, inhibits resec-

tion at telomeres, and mutations in the MRX genes exhi-

bit increased ssDNA levels (Foster et al., 2006). An

elegant labeling experiment demonstrated that MRX

binds specifically to leading-strand telomeres, where it

could generate ssDNA for the CST to bind (Faure et al.,

2010).

When telomeres become uncapped, or in the absence

of active Tel1 and Mec1 pathways, the cells also repair

some of the exposed telomere DNA by NHEJ, creating

telomere–telomere fusions (Mieczkowski et al., 2003;

Pardo & Marcand, 2005; Marcand et al., 2008), which

contribute to the formation of gross chromosomal rear-

rangements (Myung et al., 2001). The genetic control of

fusion formation is complex, involving several alternative

pathways (Mieczkowski et al., 2003; Pardo & Marcand,

2005; Marcand et al., 2008).

Telomerase is also found at the leading-strand telo-

meres; it could play a protective role preventing resection

(Vega et al., 2007). In contrast to the relatively small

effect of mutations in EXO1 on the resection of DSBs

(Mimitou & Symington, 2008), Exo1 is the main nuclease

at uncapped telomeres, and exo1D mutants show reduced

resection levels (Maringele & Lydall, 2002; Zubko et al.,

2004). This effect is probably due to the requirement by

Exo1 for overhangs to initiate its activity: these naturally

occur at telomeres. Sgs1 is also active in the resection of

uncapped cdc13-1 telomeres (Ngo & Lydall, 2010),

and Dna2 is likely to be involved too, as its human


150 M. Kupiec

counterpart participates in telomere processing (Lin et al.,

2013). Surprisingly, however, extensive resection is

observed upon Cdc13 inactivation in the absence of both

Exo1 and Sgs1, suggesting the existence of an alternative

nuclease (Ngo & Lydall, 2010). The Pif1 helicase plays a

role in controlling access of this nuclease, as its inactiva-

tion abolishes all resection in an exo1D mutant (Dewar &

Lydall, 2010). Another important player at Cdc13-inacti-

vated telomeres is the 9-1-1 complex and its loader,

Rad24, which contribute to regulate resection. A similar

role has been observed in nontelomeric DSBs (Aylon &

Kupiec, 2003).

The Ku complex negatively regulates resection at both

DSBs (Mimitou & Symington, 2010) and at telomeres

(Maringele & Lydall, 2002; Bonetti et al., 2010a, b), where

it also plays a capping role. Surprisingly, however, at telo-

meres lacking Ku, Chk1, rather than Rad53, is in charge

of the cell cycle arrest (Teo & Jackson, 2001; Maringele &

Lydall, 2002). In addition, in uncapped telomeres that

lack Ku70, the 9-1-1 complex seems to play no role in

checkpoint activation, which is entirely dependent on

Exo1 (Booth et al., 2001; Maringele & Lydall, 2002;

Zubko et al., 2004).

Thorough experiments from the Wellinger laboratory

have shown (Vodenicharov & Wellinger, 2006, 2007;

Vodenicharov et al., 2010) that the Cdc13 complex is

only required for capping during late S and G2/M phases,

but not in G1. Several explanations are possible for this

cell cycle dependency: it may be related to the CDK1

activity, which is required for regulating the resection

machinery (Aylon et al., 2004; Ira et al., 2004; Aylon &

Kupiec, 2005). Alternatively, this cell cycle dependency

may be related to the timing of DNA replication: physical

interactions have been observed between CST members

and lagging-strand replication components (Nugent et al.,

1996; Qi & Zakian, 2000; Grossi et al., 2004). Interest-

ingly, mutations in Ku components promote a change in

the pattern of end-processing, allowing MRX-dependent

resection in G1 (Clerici et al., 2008; Bonetti et al.,

2010a, b; Vodenicharov et al., 2010) even in cells having

an intact CST.

Similarly to the CST and Ku complexes, the Rap1-Rif1-

Rif2 complex plays a role in telomere capping. Rap1 inac-

tivation leads to Exo1-driven resection that, surprisingly,

leads to cell cycle arrest at G1 instead of the Mec1-depen-

dent G2 arrest (Vodenicharov et al., 2010). Elimination

of the C-terminal region of Rap1 or mutations in Rif2

leads to an MRX-dependent, but Exo-independent accu-

mulation of telomeric ssDNA (Bonetti et al., 2010a, b).

These results highlight a separation of function in con-

trolling resection, with Ku inhibiting Exo1 and Rap1-Rif2

inhibiting MRX activity (Bonetti et al., 2010a, b). Rif1

and Rif2 bind the C-terminus of Rap1, but seem to have

opposite effects on telomere capping: defects caused by

inactivation of the CST complex or Ku are exacerbated

by loss of Rif1 but alleviated by loss of Rif2 (Addinall

et al., 2011; Anbalagan et al., 2011). Mutations in Rap1

result in increased levels of both NHEJ and resection at

the telomeres, suggesting that Rap1 binding is essential to

prevent any telomere processing (Pardo & Marcand,

2005; Marcand et al., 2008). Moreover, it has been shown

that Rif2, but not Rif1, prevents the association of MRX/

Tel1 to telomeres (Hirano et al., 2009; Bonetti et al.,

2010a, b).

Finally, it is possible that telomerase may have a cap-

ping function, in addition to its DNA synthesis activity.

Although no increased end degradation is observed in

cells recruiting lower levels of telomerase (e.g. in cells car-

rying mutations in the TLC1 RNA gene), combining

these mutations with cdc13-1 leads to increased tempera-

ture sensitivity, suggesting some protective role for telo-

merase (Vega et al., 2007; Addinall et al., 2011).

Finally, situations exist in which telomere capping is

attained by alternative, Cdc13-independent mechanisms.

The Tbf1 binding protein, for example, has been shown

to cap yeast telomeres carrying human telomeric repeats

(Fukunaga et al., 2012; Ribaud et al., 2012; Di Domenico

et al., 2013). Strains deleted for the CDC13 gene can be

created by inactivating the resection machinery (e.g.

Exo1) together with the Rad9 checkpoint or the Pif1 heli-

case (Zubko & Lydall, 2006; Dewar & Lydall, 2010; Ngo

& Lydall, 2010). Similarly, yeast cells without Cdc13 are

able to grow if they overexpress a truncated version of

Stn1 and Ten1 (Petreaca et al., 2006). The telomeres in

these strains are still maintained by the activity of telo-

merase. In the absence of both telomerase and HR, cells

senesce, but can be kept alive by deleting the EXO1 gene.

In these strains, large palindromic structures cap the telo-

meres and preserve viability (Maringele & Lydall,

2004a, b).

Comparison to mammals

Similar to the yeast telomeres, mammalian telomeres con-

tain specialized dsDNA and ssDNA binding proteins (‘shel-

terin’, reviewed in Palm & de Lange, 2008). However, the

specific complexes involved are slightly different, as one

would expect from cells facing different environments and

dissimilar biologic backgrounds. A Rap1 ortholog is pres-

ent and was originally described as unable of binding DNA,

although such ability has been recently shown for the

human protein (Arat & Griffith, 2012). Rap1 binds to the

TRF2 protein, which, together with TRF1, covers the telo-

meric dsDNA. The ssDNA overhang is bound by POT1,

which is linked to the TRF1-TRF2-RAP1 complex by the

TIN2 and TPP1 proteins. In addition, a CST complex is


Yeast telomeres 151

present too; it contains orthologs of the Stn1 and Ten1

yeast proteins, but Cdc13 is replaced by CTC1 (Palm & de

Lange, 2008; Giraud-Panis et al., 2010; Sfeir & de Lange,

2012). Although its precise role is still unknown, similar to

the yeast CST, it resembles RPA and its removal results in

telomere uncapping (Bryan et al., 2013).

Experiments in which shelterin proteins were removed

in mammalian cells demonstrated that, like in yeast, cap-

ping requires the repression of the ATM and ATR

branches of the DDR, and the inhibition of both NHEJ

and HR. TRF2 seems to play a more active role than

TRF1, which is mainly involved in telomere replication

and length regulation (Ohki & Ishikawa, 2004; Sfeir et al.,

2009). In a recent tour de force, Sfeir and de Lange (2012)

removed shelterin completely in mouse cells and analyzed

the mechanisms at action in its total absence. Their

results suggest six different pathways that impinge on the

end-protection problem: the ATM (yeast Tel1) pathway

and classical NHEJ repair are repressed by the activity of

TRF2, whereas the ATR (yeast Mec1) pathway and HR

are inhibited by ssDNA-bound POT1. As a second line of

defense, Ku and 53BP1 (yeast Rad9) prevent alternative

NHEJ and hyper-resection (as well as HR; Sfeir & de

Lange, 2012). The integration of the CST complex into

this framework awaits a better characterization of its roles

in DNA replication and end-processing (Stewart et al.,

2012a, b; Wang et al., 2012). Thus, the basic mechanisms

that maintain telomere length and structure seem to be

universal, despite clear differences in protein composition

and cellular regulation.

Telomere DNA replication

A second important role played by telomeres is to solve

the end-replication problem: due to the nature of DNA

synthesis, which requires an RNA primer, information is

lost at the telomeres with each cell division (Olovnikov,

1971; Watson, 1972). However, it should be noted that

the main loss of telomere repeats with each replication

cycle is due to the leading-strand replication of the

resected telomere end (e.g. Fig. 2d). Telomere repeat

addition, carried out by the specialized reverse transcrip-

tase telomerase, brings a solution to the problem. How-

ever, telomerase activity must be coordinated with the

replication of the rest of the genome. Replication origins

located close to telomeres replicate very late in the S

Fig. 2. Replication of telomeres. (a) In G1 cells, telomeres are unavailable for elongation. Est2, the catalytic subunit of telomerase is present at

the telomeres, but inactive. Ku and the CST are present too. (b) End resection is carried out by a combination of nucleases and helicases,

controlled by the kinases Tel1 and CDK1. Resection creates ssDNA, which may bind RPA. (c) Telomerase is recruited through interactions

between Est1 and the CST. It is still unclear whether this depends, or not, on the arrival of the replication fork, and how resection is terminated.

The amount of Cdc13 at the telomeres increases (and possibly that of RPA, not shown). (d) Upon activation, telomerase elongates the TG-rich

strand. Pola-primase, recruited by the CST, completes lagging-strand replication. It is not clear whether telomerase and Pola-primase activities are

concomitant, or even whether they depend on each other. Okazaki fragments at telomeres are eventually ligated to the Okazaki fragments

created by the moving replication fork. Note that the leading-strand synthesis leaves a short, blunt-ended telomere that needs to be resected to

allow telomerase activity. Note also that the amount of resection will determine the rate of telomere attrition in the absence of telomerase

activity (the longer the resection, the shorter the leading telomere). RPA is assumed to be present at ssDNA between Okazaki fragments.


152 M. Kupiec

phase of the cell cycle (Raghuraman et al., 2001); this

phenomenon is independent of the replication origin

sequence: any origin located near telomeres is fired late

(Ferguson & Fangman, 1992). The mechanism by which

proximity to telomeres affects origin firing is still

unknown, but it is apparently independent of telomeric

silencing, as mutations in the SIR complex do not affect

replication timing (Stevenson & Gottschling, 1999).

Whether this effect is related to perinuclear tethering has

not been investigated; mutations in Ku, which also affect

telomere tethering, cause earlier firing of origins close to

telomeres (Cosgrove et al., 2002; Lian et al., 2011). It has

been suggested that this effect is telomere-length depen-

dent and particularly Rif1 dependent (Lian et al., 2011).

Indeed, short telomeres fire earlier in S than those of nor-

mal size, implying that telomere length (or the amount of

telomeric proteins bound) plays a role in dictating repli-

cation timing (Bianchi & Shore, 2007a, b).

As expected from the obligatory coordination between

telomerase activity and genome-wide replication, muta-

tions in DNA polymerases or replication factors affect

telomere length (Carson & Hartwell, 1985; Askree et al.,

2004; Grossi et al., 2004; Gatbonton et al., 2006). How-

ever, this effect is still telomerase dependent (Adams

Martin et al., 2000; Grossi et al., 2004) implying that lack

of coordination between replication and telomerase,

rather than a direct role replacing telomerase, is responsi-

ble for the phenotypes observed. Indeed, the CST compo-

nents interact physically with subunits of DNA

polymerase alpha/primase (Qi & Zakian, 2000; Grossi

et al., 2004; Sun et al., 2011). As Cdc13 also interacts

with telomerase through its Est1 subunit (Qi & Zakian,

2000), the CST is in an excellent position to serve as

coordinator. The G-strand overhang is created postrepli-

cationally (Dionne & Wellinger, 1996, 1998) by degrada-

tion of the C-strand. Note that after removal of the

primer RNAs, the strand synthesized by the leading DNA

polymerase should have a 3′ overhang, whereas the other

end should be blunt (Fig. 2d). However, both ends

undergo C-strand degradation to generate G-rich pro-

truding overhangs (Dionne & Wellinger, 1996; Wellinger

et al., 1996). Thus, importantly, it is the extent of resec-

tion of the telomeres, rather than the length of the RNA

primer, the main factor determining the Hayflick limit

(the number of generations a cell lacking telomerase can

undergo before senescence). Importantly, the two types of

telomeres are treated differently. For example, the MRX

complex can be found at the leading telomere, but not at

the lagging one (Faure et al., 2010). As telomerase cannot

work on blunt-ended DNA, C-strand degradation is

essential for its activity. The presence of MRX at

telomeres replicated by the leading-strand polymerase

(Faure et al., 2010) suggests that these are preferentially

elongated by telomerase, as MRX is required to recruit

Tel1 and telomerase. In addition, MRX preferentially

binds short telomeres (Negrini et al., 2007; McGee et al.,

2010). The conclusion is thus that telomerase should

preferentially elongate short leading-strand telomeres.

Indeed, short telomeres have been shown to be preferen-

tially elongated, in a Tel1-dependent fashion (see below;

Teixeira et al., 2004; Chang et al., 2007).

Cell cycle dependency

As mentioned before, a fusion between Cdc13 and the

telomerase subunit Est1 can elongate telomeres, even in

the presence of cdc13-2 mutations or Est1 alleles unable

to interact with Est2, the catalytic subunit of telomerase

(Evans & Lundblad, 1999). In addition, a Cdc13–Est2fusion allows the cells to replicate telomeres in the

absence of the essential Est1 subunit. Thus, the role of

the Cdc13–Est1 interaction is to recruit telomerase (Est2).

Using either cells with an inducible DSB (Diede &

Gottschling, 1999) or with a critically short telomere

(Marcand et al., 2000), it was possible to demonstrate

that telomerase is not active in cells at the G1 phase of

the cell cycle. ChIP experiments were used to determine

the cell cycle dependency for the recruitment of the vari-

ous telomere replication factors to the telomeres (Fig. 2).

Cdc13 was found to be associated with telomeric DNA

throughout the cell cycle, but its levels increase in late S

phase, together with the appearance of the long G-over-

hangs (Taggart et al., 2002; Fig. 2a and b). Similarly, Est1

and Est3 are only observed at this cell cycle stage. Sur-

prisingly, however, Est2, which encodes the catalytic sub-

unit of telomerase, was found telomere-associated

throughout the cell cycle, although its levels also increase

in late S/G2 (Taggart et al., 2002). These results reflect

the fact that there are two different pathways of telomer-

ase recruitment (remarkably, both totally dependent on

the integrity of the TLC1 RNA): Recruitment of Est2 in

G1 requires an interaction between Yku80 and a specific

stem-loop structure in TLC1 (Fisher et al., 2004). The

recruitment in late S/G2 is Est1 and Cdc13 dependent

(Chan et al., 2008; Fig. 2c).

It is still unclear what is the significance of the presence

of Est2 at the telomeres in G1, as at this time, telomerase

cannot add nucleotides to the unprocessed telomeres.

Mutations that eliminate the interaction between Yku80

and TLC1 have only a very modest effect in the recruit-

ment of Est2 (Peterson et al., 2001; Fisher et al., 2004). It

is remarkable that Ku plays a dual role as a DNA binding

protein that recognizes the dsDNA at the end of

chromosomes, but it also functions as a specific RNA

binding protein. Recent work has shown that these

two activities are mutually exclusive, suggesting a new


Yeast telomeres 153

recruitment model in which Ku is responsible for import-

ing telomerase into the nucleus and retaining it there via

its interactions with TLC1 (Pfingsten et al., 2012). Once

in the nucleus, Ku may be handed off from TLC1 to the

telomeric DNA, where Cdc13 interacts with Est1 to secure

telomerase to the telomere. Sir4, which binds Ku, may

also play some role in this mechanism, as it also interacts

with Cdc13, and both Est1 and Sir4 interact with the

nuclear envelope protein Mps3 (Pfingsten et al., 2012),

suggesting a role in perinuclear tethering. This model,

however, does not relate to the cell cycle phase at which

this recruitment may occur. The Cdc13 interaction with

Est1 may need activation to promote telomerase recruit-

ment and may also activate the new or extant telomerase

at late S (Evans & Lundblad, 2002; Fig. 2c).

Microscopic observation of fluorescently labeled TLC1

in single living cells (Gallardo et al., 2011) showed that

telomerase is mobile throughout the cell cycle, but its

movement decreases, and its intensity increases, at late S

phase. The surge in Est1 and Est3 at this stage of the cell

cycle suggests a model in which telomerase is assembled

in situ when all the subunits converge on the telomeric

DNA. The recruitment of Est3 to telomerase was shown

to be Est1 dependent (Tuzon et al., 2011). However,

expression of Est1 in G1, which results in Est1 and Est3

recruitment to the extant Est2-TLC1, does not allow telo-

merase activation at that phase of the cell cycle, indicating

that additional conditions must be met (Osterhage et al.,

2006). These could be CDK1-related (e.g. phosphoryla-

tion of one of the proteins by the activated CDK1 may be

a prerequisite), or the telomeric DNA may need to be in

a particular molecular configuration (e.g. C-strand

resected) for telomerase to act. Interestingly, the Rif1 and

Rif2 proteins may also restrict G1 activation (Gallardo

et al., 2011).

To summarize, the current model for telomere replica-

tion (Fig. 2) assumes that telomere elongation is coordi-

nated with chromosomal replication (Shore & Bianchi,

2009). However, precise details of the timing are lacking.

Late in S phase, the C-strand is processed, creating

G-overhangs (Dionne & Wellinger, 1996, 1998; Frank

et al., 2006; Fig. 2b). The MRX-Tel1 pathway plays a role

in this event (Ritchie & Petes, 2000; Faure et al., 2010;

Gao et al., 2010), which seems to be controlled/affected

also by the Tel1 and CDK1 kinases and the Rap1/Rif pro-

teins (Gardner et al., 1999; Craven & Petes, 2000; Frank

et al., 2006). The newly formed G-overhang is covered by

CST. This could be promoted by phosphorylation of

Cdc13 or other telomeric proteins by the Tel1 (or Mec1)

kinase (Tseng et al., 2006, but see Gao et al., 2010). The

CST in turn recruits telomerase by interactions between

Cdc13 and Est1 (Fig. 2c). Telomerase adds TG repeats to

the G-rich strand (Fig. 2d). When does the moving fork

interact with telomerase is not clear. Interaction of the

CST with the DNA polymerase alpha complex (approach-

ing the telomere with the moving fork) may bring to an

end the TG-strand extension and promote lagging-strand

synthesis of the CA-rich strand by polymerase delta

(Diede & Gottschling, 1999; Shore & Bianchi, 2009).

Alternatively, the CST may recruit polymerase alpha/

primase independently of the moving fork, and the telo-

meric Okazaki fragments may ligate to those created by

the moving lagging strand (Fig. 2d). A confounding factor

is that short telomeres seem to affect the timing of firing

of the distal origins of replication, with short telomeres

replicating earlier in S phase (Bianchi & Shore, 2007a, b).

The role of the RPA complex at telomeres is still

controversial: the CST has been defined as a ‘telomere-

specific RPA-like complex’. Although RPA has been

shown to be present at late S at telomeres (Schramke

et al., 2004), its presence could just reflect the presence of

the chromosomal lagging-strand replication machinery.

However, recent evidence supports a more direct role of

RPA in lagging-strand synthesis at telomeres (Luciano

et al., 2012). RPA binds to the two daughter telomeres

during telomere replication but depends on the MRX

complex for its binding to the leading-strand telomere.

Moreover, RPA specifically co-precipitates with Ku and

seems to associate with Cdc13 and Est1 (Luciano et al.,

2012). Another issue that has not been sufficiently

explored is the order and timing of chromatin remodel-

ing once the telomere has been replicated: the newly

replicated telomere must regain its special telomere chro-

matin composition and structure.

Telomere localization and transcription

Telomere tethering

Yeast chromosomes in the nucleus are not randomly

located: all centromeres are clustered around the spindle

pole body (yeast spindle organizer), whereas all telomeres

are embedded in the nuclear envelope (Therizols et al.,

2010). Telomeres play a central role in determining this

nuclear configuration (reviewed in Taddei & Gasser,

2012). Several microscopic techniques have demonstrated

that telomeres are found in clusters around the nuclear

periphery (Palladino et al., 1993; Gotta & Gasser, 1996).

Interestingly, the number of clusters is far lower than the

number of telomeres: the 32 telomeres of an haploid yeast

cell are usually seen in 3–6 clusters (Gotta et al., 1996).

These foci move with a constant random motion that is

more constrained than that of a nontelomeric locus

(Schober et al., 2008; Therizols et al., 2010).

Many results suggested a correlation between perinucle-

ar position and silencing in yeast. The telomeric clusters


154 M. Kupiec

at the nuclear envelope are enriched for SIR proteins

(Gotta et al., 1996). Mutations in Ku or SIR components

partially affect telomere position (Laroche et al., 1998;

Hediger et al., 2002). However, only a double mutant

sir4D yku70D shows completely delocalized telomeres,

demonstrating that redundant anchoring mechanisms are

at play (Hediger et al., 2002). The association of telo-

meres to the nuclear periphery requires at least two

nuclear envelope proteins, Esc1 and Mps3. Esc1 interacts

with the C-terminus of Sir4, competing with the Yku80

protein, which binds the same region (Andrulis et al.,

2002; Taddei et al., 2004). Mps3 resides in the nuclear

periphery and has an N-terminal acidic extension that

protrudes toward the nuclear interior and is also capable

of binding both Ku and Sir4; deleting this extension pre-

vents telomere tethering, although the cells remain viable

(Bupp et al., 2007). Some results suggest that the

Ku-dependent pathway tends to dominate during G1,

while the Sir4/Esc1-dependent tethering pathway is pre-

ponderant during S phase (Hediger et al., 2002). As cells

prepare to enter mitosis during G2, the telomeres lose

their peripheral localization; perinuclear positioning is

re-established in early G1 phase (Smith et al., 2003).

Interestingly, it is possible that different telomeres differ

in their dependence on these two pathways: during G1,

for example, the tethering of the telomere VI-right

depends primarily on the Ku pathway and that of the

telomere VI-left primarily on the Sir4/Esc1 pathway

(Bystricky et al., 2005).

SUMOylation of Yku80 and of Sir4 may play a regula-

tory role in telomere length maintenance and tethering,

although the details are still unclear (Ferreira et al., 2011;

Hang et al., 2011). Loss of SUMOylation abolishes tether-

ing without affecting TPE (Ferreira et al., 2011), demon-

strating that tethering and TPE are separable.

It is very likely that the Ku pathway for tethering involves

additional components. Identifying these proteins is compli-

cated by the fact that Ku plays additional roles in transcrip-

tional silencing (Gravel et al., 1998), telomerase recruitment

(Stellwagen et al., 2003), and specification of replication

timing (Cosgrove et al., 2002). In addition, Mps3 is also able

to interact directly with telomerase via its connections with

Est1 (Antoniacci et al., 2007) and is required for the tether-

ing of Yku80 and TLC1 (Schober et al., 2009).

Interestingly, a deletion of YKU80 shows increased

recombination between interstitial and telomeric regions,

suggesting that Ku80 tethering sequesters this region and

prevents recombination (Marvin et al., 2009a, b). Tethering

of telomeres to the nuclear envelope also reduces the effi-

ciency of the homology search when a DSB is created close

to a telomere (Therizols et al., 2006; Agmon et al., 2013).

Establishing a causal relationship between subnuclear

organization and transcriptional repression has been

difficult, because all the mutants that alter the position of

silent domains also affect silencing. In many cases,

peripheral localization of DNA within the yeast nucleus

has been shown to reinforce transcriptional silencing. For

example, artificial localization to the periphery enhances

transcriptional repression at a compromised silencer

(Andrulis et al., 1998). However, positioning and silenc-

ing can be separated: for example, repression can be

maintained at an intact silencer that is released from the

nuclear periphery (Gartenberg et al., 2004). In a series of

clever experiments, Taddei et al. (2004) showed that peri-

nuclear chromatin anchoring can occur prior to or inde-

pendently of transcriptional repression. Moreover, as

explained above, SUMOylation seems to be required for

tethering but not for TPE (Ferreira et al., 2011). Silencing

and anchoring, however, are carried out by the same set

of proteins (Ku and SIR) and reinforce each other:

Ku-mediated anchoring of Rap1-bound telomeres allows

Sir4 recruitment, which in turn increases the recruitment

of Sir2 and Sir3, spreading the silencing along the chro-

matin. As silent chromatin spreads, the interactions of

Sir4 to the nuclear enveloped are reinforced (Taddei

et al., 2004).

Telomere transcription: TERRA

In addition to the telomeric DNA repeats and the telo-

mere-associated proteins, it is possible to detect noncod-

ing telomeric repeat-containing RNA (TERRA) at the

telomeres (Fig. 3). TERRA is conserved from yeast to

humans; it encompasses both telomeric and subtelomeric

regions and is transcribed by the RNA polymerase II

(Luke et al., 2008; Bell et al., 2010; Schoeftner & Blasco,

2010; Arora et al., 2011; Iglesias et al., 2011). In yeast,

telomeric RNA ranges from a few hundred nucleotides to

as much as 1200 nt and is rapidly degraded by the Rat1

exonuclease, which is in charge of degrading all mRNAs

(Luke et al., 2008). The TERRA transcripts overlap the Y′elements, which are able to encode a helicase that is

expressed in cells exposed to stress or upon loss of telo-

merase activity (Yamada et al., 1998). Interestingly, a sim-

ilar protein is expressed from subtelomeric regions in

S. pombe (Hansen et al., 2006). In telomerase-negative

cells, Y′ transcripts have been shown to be amplified by a

retrotransposon-mediated mechanism (Maxwell et al.,

2004). However, the possible interactions between Y′transcripts, Y′-encoded helicases and TERRA remain

unexplored.

The role of TERRA in telomere biology remains enig-

matic. It has been proposed that TERRA levels may regu-

late telomere length; for example, increased levels of

TERRA (e.g. in rat1-1 cells grown at semi-permissive con-

ditions) lead to shorter telomere length (Luke et al.,


Yeast telomeres 155

2008). Similarly, expression of a strong inducible pro-

moter at the subtelomeric region leads to telomere short-

ening (Sandell et al., 1994). On the other hand, there are

reports of telomere defects caused by reduced TERRA lev-

els (Azzalin et al., 2007; Deng et al., 2009). A complicat-

ing issue is that TERRA expression, and thus probably

telomere regulation, appears to be differently affected by

the subtelomeric structure of each individual chromo-

somal arm. TERRA expression in both X and XY′ typesof repeats is repressed by a Rap1-based pathway, but only

the first type is also repressed by SIR proteins (Iglesias

et al., 2011).

In humans, TERRA is tightly regulated by the non-

sense-mediated decay machinery, which degrades mRNA

molecules with mutations (Chawla & Azzalin, 2008), and

the DNA methyltransferases DNMT3b and DNMT1

methylate TERRA promoters within CpG islands and thus

downregulate its expression (Nergadze et al., 2009).

TERRA transcription is promoted by the MLL histone

methyltransferase (Caslini et al., 2009).

The potential mechanism of action of TERRA is not

yet clear: TERRA-like RNA oligonucleotides inhibit telo-

merase activity in vitro (Schoeftner & Blasco, 2008; Redon

et al., 2010), suggesting a direct regulation of telomerase,

perhaps through inhibition of the polymerization reaction

of telomerase. However, RNase H inhibits TERRA overex-

pression effect, suggesting that TERRA anneals with the

telomeric DNA (Luke et al., 2008). Recent work has

shown that in cells lacking telomerase activity, TERRA

plays also a role in delaying senescence by promoting

alternative lengthening of telomeres (ALT; elongation of

telomeres by HR), whereas senescence is accelerated in

cells unable to recombine (Balk et al., 2013).

A recent publication by the Chartrand group followed

endogenous TERRA expression in single yeast cells using

RNA fluorescence in situ hybridization (FISH). They

found that TERRA expression is induced by telomere

shortening, leading to the accumulation of TERRA mole-

cules into a single perinuclear focus. Simultaneous time-

lapse imaging of telomerase RNA and TERRA revealed

telomerase nucleation on TERRA foci in early S phase.

Their results suggest that the TERRA foci may act as scaf-

folds for the recruitment of telomerase molecules and

trigger the formation of telomerase clusters (which they

call T-Recs). The TERRA–telomerase cluster is subse-

quently recruited to the short telomere from which

TERRA molecules originate, suggesting that TERRA plays

a role in the recruitment of telomerase to short telomeres

(Cusanelli et al., 2013).

Regulation of telomere length

Telomere length is remarkably variable between organ-

isms, from a few hundred nucleotides in S. cerevisiae to

tens of kb in mice. Despite this variability, cells express-

ing telomerase keep telomeres at a very constant length.

Due to the ease with which it can be genetically

manipulated and its rapid growth, yeast has greatly

contributed to our understanding of telomere length

homeostasis.

CSTRif2Rif1 Rap1

Cdc13Stn1Ten1

Telomerase

RNA PolII

RAT1

LONG TELOMERE

SHORT TELOMERE

RNA PolII

?? ??

TERRA focus

Telomeraserecruitment

Fig. 3. Proposed regulation of telomere

length by TERRA. TERRA is expressed from a

telomere-located promoter. At telomeres of

normal length, Rat1 constantly degrades the

newly made RNA. At short telomeres, in

contrast, TERRA is highly expressed. The

mechanism controlling this fact remains

enigmatic. It is still unclear, for example,

whether transcription requires removal of

telomeric proteins (“??”). Recent work has

shown that TERRA forms foci together with

telomerase and are jointly recruited, by a still

unknown mechanism, to the same short

telomere that expressed TERRA.


156 M. Kupiec

Uniform telomere length homeostasis is achieved by a

balance between shortening and elongating mechanisms

within the cell. Telomere shortening takes place naturally

by the ‘end-replication problem’ (the inability to fill-in

gaps left at the telomere ends by removal of the RNA pri-

mer) and by end resection or partial uncapping episodes.

In cells with extremely long telomeres, however, an addi-

tional mechanism exists that can shorten telomeres to the

wt length within one or just a few generations. This

mechanism was termed TRD, or telomeric rapid deletion

(Li & Lustig, 1996; Bucholc et al., 2001), and is the result

of HR between telomeric repeats, which generates chro-

mosomes with short telomeres and a telomeric DNA cir-

cle (Bucholc et al., 2001). Two elongating mechanisms

exist for telomeres: HR (also known as ALT) and telo-

merase-mediated telomere elongation. Telomerase-driven

telomere elongation must be regulated to attain the uni-

form wild-type telomere size.

Early work suggested a model in which telomerase

activity is regulated in such a way that it slows down with

telomere size (Marcand et al., 1999). Sophisticated experi-

ments carried out by the Lingner laboratory, in which

telomere elongation events could be followed in individ-

ual cells during a single cell cycle, showed that not all

telomeres are elongated in each cell cycle. Rather, telo-

meres with short TG tracts tend to be preferentially cho-

sen for elongation (Teixeira et al., 2004). However, the

extent of elongation is independent of the original TG

tract length. Moreover, if a strain with two telomerases

that differ in their RNA templates is used, it is possible to

show that multiple rounds of association and dissociation

can take place on a single telomere on a single cell cycle

(Chang et al., 2007). Tel1 (the yeast ATM orthologue, or

Mec1/ATM in its absence) plays a pivotal role in increas-

ing telomerase processivity at very short telomeres (Chang

et al., 2007), which are preferentially chosen with the help

of the kinase(s) (Arneric & Lingner, 2007). In the absence

of the Rif proteins, the frequency (but not the extent) of

elongation events is increased (Teixeira et al., 2004).

Based on these experiments, Lingner et al. proposed that

the telomere may exist in either an extendible or a nonex-

tendible state. Three models have been put forward for

molecular mechanisms of the extendible/nonextendible

states (Teixeira et al., 2004; Shore & Bianchi, 2009):

(1) The resection model: Resection of the C-strand may

be more extensive if the TG tracts are short. This

would generate a longer G-overhang to which more

CST molecules would bind, thus increasing the

chances of telomerase recruitment. Support for this

model is supplied by experiments in which a single

DSB is flanked by either short (80 bp) or a long

(250 bp) TG tract: ChIP of Cdc13 showed higher lev-

els in the short construct (Negrini et al., 2007).

(2) The activation model: Telomerase activity may

become activated by short TG tracts. Support for this

model comes from alleles of CDC13 (a member of the

CST complex) that show stable, capped, and short

telomeres, indicating that, as in tel1 mutants, telomer-

ase is able to elongate extremely short telomeres (i.e.

no Est phenotype is detected), but not those with sizes

closer to that of wt cells (Meier et al., 2001). This type

of mutants suggests that interactions with Est1 (Evans

& Lundblad, 2002; DeZwaan & Freeman, 2009) may

be present all the time but activated at the right cir-

cumstances.

(3) The recruitment model: According to this third pos-

sibility, the TG tract length regulates the association of

telomerase with the telomere end. It is not clear what

mechanism would preferentially recruit more telomer-

ase to those telomeres having short TG tracts. Direct

measurement by ChIP of protein levels at short vs

long telomeres (Bianchi & Shore, 2007a, b; Sabourin

et al., 2007) supports the last model, as Est1, Est2, and

Tel1, but not Cdc13, were present at higher levels at

short telomeres. The presence of Tel1 at short telo-

meres also suggests that phosphorylation of some of

the proteins involved may help in the recruitment.

Cdc13 is phosphorylated by both Tel1 (and Mec1 in

its absence; Tseng et al., 2006) and by CDK1 (Li et al.,

2009). In both cases, it has been reported that

phosphorylation promotes Est1 binding and thus telo-

merase recruitment. However, genetic analysis of site-

specific mutants that abolish phosphorylation argues

against this simple model (Gao et al., 2010). It has

also been proposed that phosphorylation by Mec1 is

intended to prevent telomere addition at spurious

DSBs (Zhang & Durocher, 2010).

Tel1 activity thus appears to be critical for elongating

short telomeres. In tel1D mutants, the telomeres are

extremely short, but stable (Lustig & Petes, 1986), yet

they do not bind Est1 or Est2 (Goudsouzian et al., 2006).

It has been shown that Tel1 recruitment to telomeres

takes place by interactions with the C-terminus of Xrs2

(Sabourin et al., 2007). But why would this interaction be

favored at telomeres with short TG tracts? Marcand et al.

(2008) suggested that the Rif proteins, and particularly

Rif2, may inhibit the MRX–Tel1 pathway. In short telo-

meres, this inhibition would be reduced, allowing Tel1

recruitment. Supporting information to this idea comes

from the fact that by ChIP, Rif2 levels, but not Rif1 lev-

els, are lower in strains with short telomeres and that

Tel1 no longer binds to a short telomere better than to

one of normal size in rif2D cells (McGee et al., 2010).

Additional support for this model comes from work by

Hirano et al. (2009), showing that Rif2, and to a lower


Yeast telomeres 157

extent Rif1, blocks Tel1 (but not MRX) association at

longer TG tracts by competing for Xrs2 binding. Telo-

meres are extremely long in rif1D and rif2D mutants

(Hardy et al., 1992a, b; Wotton & Shore, 1997). The dou-

ble mutant, however, exhibits even longer telomeres (sim-

ilar in size to those seen in mutants of the RAP1 gene

lacking the C-terminal domain that interacts with both

Rif proteins; Kyrion et al., 1992; Wotton & Shore,

1997).This is consistent with two separate mechanisms

that control the activity of telomerase. It should be noted,

however, that the molecular mechanism of action of Rif1

is unknown and that Rap1-dependent, Rif-independent

mechanisms of telomere length control have been sug-

gested in the past too (Negrini et al., 2007; Marcand

et al., 2008).

Another negative regulator of telomerase is Pif1, a 5′–3′helicase that seems to work by a mechanism that is inde-

pendent of Rif1 and Rif2 (Schulz & Zakian, 1994). The

helicase motif of Pif1 is required to prevent telomerase

activity in vivo and in vitro, suggesting that Pif1 acts cata-

lytically to prevent telomere elongation (Zhou et al.,

2000; Boule & Zakian, 2006). In the absence of Pif1, Est1

levels increase at the telomeres, whereas its overexpression

reduces the association of both Est1 and Est2 with the

telomeres (Boule & Zakian, 2006). It has been thus pro-

posed that the role of Pif1 is to actively displace telomer-

ase from its substrate. However, the role of Pif1 is more

complex: The protein is cell cycle regulated, peaking at

late S/G2 and is preferentially recruited to short telomeres

by an unknown mechanism. In its absence, Est2 binds

equally well to short and normal-sized telomeres (Vega

et al., 2007), suggesting that it participates in telomere

length homeostasis. In addition, Pif1 prevents the addi-

tion of telomeres at chromosomal DSBs (Schulz &

Zakian, 1994; Myung et al., 2001) thus avoiding gross

chromosomal rearrangements and seems to be preferen-

tially located at regions of the genome with G-quadru-

plex-forming potential, where it may facilitate DNA

replication (Paeschke et al., 2013). Thus, Pif1 may have

roles in general DNA replication (e.g. helping replicate

secondary structures), as well as direct involvement in

telomere length regulation.

Life in the absence of telomerase

Yeast strains that lack telomerase activity exhibit an ‘ever

shortening telomere’ (Est) phenotype: with each genera-

tion, more and more cells stop dividing and senesce, until

no further growth is observed (Lundblad & Szostak,

1989). Senescence can occur in cells in which the average

telomere length is close to that of the wild type, indicat-

ing that a massive shortening of telomeres is not a prere-

quisite for senescence (Lundblad & Blackburn, 1993).

Indeed, it is enough to have a single short telomere that

cannot be elongated by telomerase to elicit the growth

arrest, even in telomerase-proficient cells (Abdallah et al.,

2009; Khadaroo et al., 2009).

From a population of senescing cells, rare survivors arise

by a recombination-based mechanism, also known as ALT.

Survivors are usually of two types: Type I, which amplify

internal Y′ repeats, and Type II, which have enlarged TG

repeats. Whereas Type I survivors are more common, they

grow slowly and in liquid cultures are usually overgrown

by Type II cells (Teng & Zakian, 1999; Grandin &

Charbonneau, 2007). The central recombination protein

Rad52 is essential for both classes, and usually no survivors

are observed in its absence (Lundblad & Blackburn, 1993).

Another essential protein is Pol32, a subunit of DNA poly-

merase Delta also involved in break-induced replication, a

process in which a broken chromosomal end invades a dif-

ferent chromosome and copies its content until its end

(Lydeard et al., 2007). This dependency suggests that both

types of telomere maintenance pathways occur by recom-

bination-dependent DNA replication. Interestingly, a

Rad52- and Pol32-independent mechanism can be found

at low frequency in survivors with extremely long telo-

meres (Grandin & Charbonneau, 2009; Lebel et al., 2009).

The mechanism at action in these cells is still enigmatic.

Type I survivors grow slowly and maintain short telo-

meres with normal G-overhangs. The cells show massive

amplification of their Y′ sequences in a tandem repeat

array. Extra-chromosomal Y′ arrays can also be detected;

these have been proposed to be intermediates in the

process of recombination leading to Y′ amplification

(Larrivee & Wellinger, 2006). In addition to Rad52 and

Pol32, the appearance of Type I survivors requires the

Rad51, Rad54, Rad55, and Rad57 recombination proteins

(Le et al., 1999; Chen et al., 2001).

Type II survivors show an increase in the telomeric TG

repeats; the length distribution is extremely variable, with

some telomeres being very short and others extending for

more than 10 kb. In these cells, TG circles are detected,

presumably created by a rolling-circle mechanism

followed by recombination of circles into the genome

(Larrivee & Wellinger, 2006). Long telomeres in Type II

survivors progressively shorten with cell growth and

require constant recombination events to maintain cell

growth. The genetic requirements of these cells differ

from those of Type I survivors: they require the MRX

complex, the Rad52 paralog Rad59, and the yeast ortho-

log of the WRN and BLM helicases, Sgs1 (Diede &

Gottschling, 1999; Chen et al., 2001; Huang et al., 2001;

Johnson et al., 2001). The extrachromosomal telomeric

circles observed in Type II survivors could be created by

the same mechanism that generates TRD (Li & Lustig,

1996; Natarajan & McEachern, 2002).


158 M. Kupiec

The choice of recombination mechanism to generate

survivors is affected by the DDR: the Mec1 and 9-1-1

pathways control Type II survivor formation, by a mecha-

nism that is independent of the Rad53 and Chk1 kinases

(Grandin & Charbonneau, 2007). Interestingly, cdc13-1

cells lacking the Ku complex survive by a Type II mecha-

nism independent of the MRX and Rad59, but dependent

on Rad51 (Grandin & Charbonneau, 2003).

Systems biology of telomere biology

In the last decade, a revolution has taken place in biology,

which turned the traditional reductionist approach of

molecular biology upside down, to attempt a whole-

encompassing, gestalt view of the cell. A flurry of

genome-wide approaches were launched, in which a

systematic approach was taken to try to map all the genes

(genomics), RNA molecules (transcriptomics), proteins

(proteomics), and metabolites (metabolomics) in a given

organism. This ‘omics’ approach was driven by the

sophisticated genetics of yeast, which allowed the con-

struction of mutant collections, fusion protein collections,

and many other tools. The systems biology revolution is

still ongoing, and an effort is being made to map, for

example, all the genetic and physical interactions in the

yeast cells.

Telomere biology is benefitting from this approach,

which greatly enlarged our knowledge about the regula-

tion of telomere-related processes. The yeast genome has

close to 6000 recognized genes. A collection of 4700

mutants was constructed by systematically deleting each

individual nonessential gene in yeast (nonessential yeast

mutant collection; Winzeler et al., 1999). This collection

was later complemented by two additional libraries of

mutants of all the essential genes (yeast has c. 1300 essen-

tial genes), in which either hypomorphic (Breslow et al.,

2008) or temperature-sensitive alleles (Ben-Aroya et al.,

2008) of the genes were created. These mutant collections

allow researchers to carry out systematic mutant screens

even if the phenotype of interest is not selectable. For

example, three publications reported the systematic

screening of the mutant collections, looking for those

mutants that affect telomere length (telomere length

maintenance or tlm mutants). In this brute-force

approach, DNA was extracted from each individual yeast

strain, and telomere length was measured by Southern

blot (Askree et al., 2004; Gatbonton et al., 2006; Ungar

et al., 2009). Together, these papers identified c. 400

genes affecting telomere length. This list starkly contrasts

with the 30 or so genes known to do so at the time the

screens were carried out (Askree et al., 2004) and stresses

the central role played by telomere biology in the yeast

life cycle, as c. 7% of the genome affects telomere biology.

Moreover, it also demonstrate the complexity of the

challenge: mutation in any of the TLM genes changes the

final telomere size; as this size is determined by mecha-

nisms that elongate or shorten telomeres (mechanisms

that are positively and negatively regulated), this means

that each of the 400 genes participates in determining the

equilibrium between the two types of activity. Remark-

ably, however, in each genetic background, wt cells

exhibit always telomeres of the same size; thus, in the

tug-of-war between elongating and shortening mecha-

nisms, the equilibrium is attained at the same telomere

length. The genes uncovered in these screens, as expected,

include those affecting DNA and chromatin metabolism,

but almost all functions in the cell are also represented,

including RNA and protein synthesis, traffic and

modification, metabolic pathways, mitochondrial func-

tions, etc. The challenge ahead, of course, is to determine

how all these genes impinge on the telomere length deter-

mination.

The fact that a near-complete list of TLM genes is

available opens the door for further exploration of telo-

mere biology. Using computational approaches and the

vast amount of information about protein–protein and

genetic interactions in yeast, for example, network models

of the telomere biology have been established, allowing

their study (Rog et al., 2005; Shachar et al., 2008; Yosef

et al., 2009).

Secondary screens were also carried out on the tlm

mutant collection. In one of these, TLC1 RNA levels were

measured in all tlm mutants, and 24 were found to affect

telomere length via their effect on TLC1 levels (Mozdy

et al., 2008). These results suggest that the level of telo-

merase RNA may be limiting in telomere length mainte-

nance. A second screen explored the effect of starvation

on telomere length (Ungar et al., 2011). Starved cells, or

those exposed to the TorC1 inhibitor rapamycin, respond

by dramatically shortening their telomeres. By screening

the tlm mutants for those that do not respond to the

starvation signals, it was found that the Ku heterodimer

plays a central role in the starvation response. When cells

are starved, Ku protein levels are reduced, affecting telo-

mere length. This finding is particularly interesting in

light of studies suggesting that calorie restriction may

lengthen life span, whereas telomere attrition leads to cel-

lular senescence (Ungar et al., 2011). In another study

that followed the response of yeast telomeres to environ-

mental stimuli, it was found that exposure to ethanol

elongates telomeres, whereas caffeine and high tempera-

ture reduce telomere length (Romano et al., 2013). Again,

a systematic screen of tlm mutants revealed that the

Rap1/Rif1 pathway is necessary for the transduction of

three different environmental signals. Interestingly, rif2Dstrains and mutants of the MRX/Tel1 pathway exhibited


Yeast telomeres 159

a stronger-than-expected response to ethanol, which

causes elongation, indicating that these pathways have a

role in preventing length-independent elongation of telo-

meres. Finally, Jin-Qiu Zhou et al. explored the tlm col-

lection looking for mutants that affect the survival

pathways in the absence of telomerase activity. The TLC1

gene was knocked out in 280 tlm mutants, and the pat-

terns of senescence and survival were monitored. New

functional roles were found for 10 genes that affect the

emerging ratio of Type I vs. Type II survivors and 22

genes that are required for Type II survivor generation.

For example, the Pif1 helicase and the INO80 chromatin

remodeling complex reduced the frequency of Type I sur-

vivors, whereas the kinase, endopeptidase, and other pro-

teins of small size (KEOPS) complex is required for Type

II recombination (Hu et al., 2013).

The KEOPS complex is a good example of a group of

proteins with a central role in telomere biology identified

in several genome-wide screens. The components of the

KEOPS complex include Kae1, a putative endopeptidase

with an unknown role which is absolutely conserved in

Archaea, Bacteria, and Eukarya; Bud32, a serine/threonine

kinase; Pcc1, the yeast homolog of the two human cancer

testis antigens that are specifically expressed in different

tumors but also in normal testes and ovaries (Kisseleva-

Romanova et al., 2006); Gon7 (also referred to as Pcc2),

a small protein with no known functional domains,

found only in fungi; and Cgi121, whose human homolog

binds the human Bud32, also known as the p53-related

protein kinase (Miyoshi et al., 2003). For example, Dow-

ney et al. screened the nonessential mutant collection for

mutants that partially suppress the temperature sensitivity

of cdc13-1, caused by telomere uncapping. Among other

suppressors, they identified KEOPS members and demon-

strated that this complex is required for telomere capping

and maintenance (Downey et al., 2006).

An additional genome-wide screen looked for mutants

affecting the telomere three-dimensional configuration: A

construct carrying a TATA-less galactose-inducible

upstream activating sequence downstream of the URA3

gene is able to transcribe the URA3 gene only if folded

back on itself. This is indeed the case of the construct

integrated close to a telomere. A genome-wide screen for

mutants affecting telomere fold-back identified 112 genes.

Among various biologic processes uncovered, lysine

deacetylation was found to be essential for the fold-back,

through Rif2-dependent recruitment of the Rpd3L com-

plex to telomeres. Absence of Rpd3 function generates

increased susceptibility to nucleolytic degradation and the

initiation of premature senescence, suggesting a protective

role for Rpd3 deacetylation activity (Poschke et al., 2012).

As explained, the Lydall laboratory conducted a system-

atic screen for mutants that affect growth of a cdc13-1

allele. This screen identified 369 gene deletions that could

be divided into eight different phenotypic classes. The

results included many of the checkpoint-affecting genes

expected, but also genes in a variety of unexpected catego-

ries, such as RNA metabolism, phosphate and iron homeo-

stasis, etc. In addition, the screen identified a number of

genes of previously unknown function renamed restriction

of telomere capping (RTC) or maintenance of telomere

capping (MTC; Downey et al., 2006; Addinall et al., 2008).

This screen was extended, by systematically looking for

suppressors or enhancers of the yku70D mutation and

comparing them to the results obtained for cdc13-1. By

developing a sophisticated analysis, named quantitative

fitness analysis, a detailed map of the genetic interactions

of both capping proteins could be observed. In this analy-

sis, mutations in some genes, such as those the nonsense-

mediated decay pathway, were shown to suppress cdc13-1

but to enhance the phenotype of yku70D. The response to

telomere uncapping was shown to be genetically complex,

with many genes involved in a variety of processes affect-

ing the outcome (Addinall et al., 2011).

Another genome-wide screen examined, in a systematic

fashion, the kinetics of senescence, by crossing the

est1D mutation to the nonessential mutant collection. As

expected, the vast majority of gene deletions showed no

strong effects on entry into/exit from senescence. How-

ever, c. 200 gene deletions (among them the well-charac-

terized rad52D mutant) accelerated entry into senescence,

and such cells often could not recover growth. A smaller

number of strains (among them rif1D) accelerated both

entry into senescence and subsequent recovery (Chang

et al., 2011).

A screen for enhancers of the MMS sensitivity of tel1Duncovered a small number (13) of genes. These included

Yku70, members of the 9-1-1 pathway, the CCR4-NOT

deadenylase complex, nuclear pore components, and sev-

eral histone deacetylases. Most of these mutants caused

the MMS sensitivity due to their effects on telomeres

(Piening et al., 2013).

The systems biology revolution is at its infancy; we can

summarize at this stage that the genome-wide studies

have vastly extended our view of telomere biology. The

number of cellular processes affecting various aspects of

telomere integrity, replication, length regulation, and

structure is remarkable. Most of the genes uncovered in

these screens are evolutionarily conserved and likely to

act similarly in other organisms, including humans.

A better understanding of the mechanisms regulating

telomere biology will have significant medical implica-

tions, especially in the fields of aging and cancer. The

yeast S. cerevisiae, as an easily manipulated organism that

grows fast and has superb genetics and molecular biology,

has contributed tremendously (and continues to do so)


160 M. Kupiec

to our understanding of the basic mechanisms of the

cells, including telomere biology.

Acknowledgements

I would like to thank all members of the Kupiec labora-

tory for encouragement and support and Tom Petes for

comments on the manuscript. Research in the laboratory

is supported by grants from the Israel Cancer Research

Fund, the US-Israel Bi-national Fund, the Israeli Ministry

of Science and Technology, and the Israel Science Foun-

dation. We apologize to all our colleagues whose work

was not quoted due to length constraints.

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Biology of Telomeres, Lessons From Budding Yeast