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Pif1- and Exo1-dependent nucleases coordinate
checkpoint activation following telomere
uncapping
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James M Dewar1 and David Lydall1,2,*
1Centre for Integrated Systems Biology of Ageing and Nutrition, Institute
for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK and
2Institute for Cell and Molecular Biosciences, Newcastle University,
Newcastle upon Tyne, Tyne-and-Wear, UK
Essential telomere ‘capping’ proteins act as a safeguard
against ageing and cancer by inhibiting the DNA damage
response (DDR) and regulating telomerase recruitment,
thus distinguishing telomeres from double-strand breaks
(DSBs). Uncapped telomeres and unrepaired DSBs can
both stimulate a potent DDR, leading to cell cycle arrest
and cell death. Using the cdc13-1 mutation to conditionally
‘uncap’ telomeres in budding yeast, we show that the
telomere capping protein Cdc13 protects telomeres from
the activity of the helicase Pif1 and the exonuclease Exo1.
Our data support a two-stage model for the DDR at
uncapped telomeres; Pif1 and Exo1 resect telomeric DNA
o5kb from the chromosome end, stimulating weak
checkpoint activation; resection is extended 45kb by
Exo1 and full checkpoint activation occurs. Cdc13 is also
crucial for telomerase recruitment. However, cells lacking
Cdc13, Pif1 and Exo1, do not senesce and maintain their
telomeres in a manner dependent upon telomerase, Ku
and homologous recombination. Thus, attenuation of the
DDR at uncapped telomeres can circumvent the need for
otherwise-essential telomere capping proteins.
The EMBO Journal (2010) 29, 4020–4034. doi:10.1038/
emboj.2010.267; Published online 2 November 2010
Subject Categories: genome stability & dynamics
Keywords: Cdc13; DNA damage response; Exo1; Pif1;
uncapped telomeres
Introduction
Telomeres consist of double-stranded DNA (dsDNA) and
single-stranded DNA (ssDNA), bound by dsDNA- and
ssDNA-binding proteins (Blackburn et al, 2006; Lydall,
2009). This nucleoprotein ‘cap’ has at least two functions:
to shield the telomeric DNA from stimulating the DNA
damage response (DDR) and to regulate elongation of
the telomere by telomerase. In human senescent cells,
dysfunctional telomeres induce a sustained DDR (d’Adda di
Fagagna et al, 2003). In both budding yeast and mice, nuclease
activities that attack dysfunctional telomeres contribute to
telomere-driven senescence (Maringele and Lydall, 2004;
Schaetzlein et al, 2007). Therefore, understanding the regula-
tion of nuclease activities at dysfunctional telomeres in yeast is
likely to be informative about similar processes occurring at
mammalian telomeres and the human ageing process.
dsDNA-binding proteins and accessory factors are required
at both human telomeres (TRF1, TRF2, TIN2, TPP1, RAP1)
and budding yeast telomeres (Rap1, Rif1, Rif2) to prevent
DDRs (Wotton and Shore, 1997; de Lange, 2005; Celli and
de Lange, 2005; Marcand et al, 2008; Bonetti et al, 2010;
Vodenicharov et al, 2010). In budding yeast, telomeric ssDNA
is bound by Cdc13 with accessory proteins Stn1 and Ten1,
whereas in human cells, it is bound by POT1 (de Lange, 2005;
Gao et al, 2007). Cdc13–Stn1–Ten1 forms an evolutionarily
conserved complex (the CST complex) that has telomeric
roles in most organisms studied so far (Miyake et al, 2009;
Surovtseva et al, 2009). POT1 binds telomeric ssDNA and is
connected to the dsDNA-binding proteins of the telomere cap
by TPP1 and TIN2 (de Lange, 2009). Inactivation of POT1 or
Cdc13 induces ‘telomere uncapping’ and has similar conse-
quences—initiation of a DDR and resection of the telomeric
DNA by nuclease activities (Garvik et al, 1995; Baumann and
Cech, 2001; Pitt and Cooper, 2010).
The response to telomere uncapping is readily studied in
budding yeast by inactivation of Cdc13 using the thermo-
sensitive allele cdc13-1 (Garvik et al, 1995). Following Cdc13
inactivation, a potent DDR is initiated; telomeric DNA is
resected by nucleases, which degrade the AC (50) strand to
generate extensive TG (30) ssDNA that stimulates activation
of the DNA damage checkpoint, in a manner analogous to
that at DNA double-strand breaks (DSBs) (Figure 1A) (Garvik
et al, 1995; Lydall and Weinert, 1995; Vodenicharov and
Wellinger, 2006). There is relatively little understanding of
the nuclease activities responsible for generating ssDNA at
uncapped telomeres (Zubko et al, 2004). In contrast, there
has been much recent progress identifying nuclease activities
that function at DSBs (Gravel et al, 2008; Mimitou and
Symington, 2008; Zhu et al, 2008).
Exo1 is the only nuclease known to generate ssDNA at
uncapped telomeres in budding yeast (Maringele and Lydall,
2002). Exo1 is a 50 to 30 dsDNA exonuclease involved in DSB
resection and in mismatch repair (Tsubouchi and Ogawa,
2000; Gravel et al, 2008; Mimitou and Symington, 2008;
Zhu et al, 2008). In the absence of Exo1, ssDNA is still
generated following Cdc13 inactivation, demonstrating that
Received: 27 April 2010; accepted: 29 September 2010; published
online: 2 November 2010
*Corresponding author. Institute for Cell and Molecular Biosciences,
Newcastle University, Newcastle upon Tyne, Tyne and Wear NE2 4HH,
UK. Tel.: þ 44 191 222 5318; Fax: þ 44 191 222 7424;
E-mail: d.a.lydall@ncl.ac.uk
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other nuclease activities must also function at uncapped
telomeres. The determinant(s) of this Exo1-independent
ssDNA generation have not so far been identified, but at
least two hypothetical nuclease activities have been proposed
(ExoX and ExoY) (Zubko et al, 2004).
We sought to identify additional nuclease activities func-
tioning at uncapped telomeres following inactivation of
Cdc13. Bioinformatic analysis of genetic interactions found
the helicase Pif1 to be a candidate for contributing to nucle-
ase activity. Consistent with this hypothesis, we found
that Pif1 and Exo1 are required for different nuclease acti-
vities that generate ssDNA and activate the DNA damage
checkpoint following Cdc13 inactivation. Furthermore, deletion
of both PIF1 and EXO1 permits yeast cells to tolerate complete
loss of the essential telomere capping protein Cdc13.
Results
PIF1 and EXO1 define parallel pathways that inhibit
growth of cdc13-1 mutants
To identify potential nuclease(s) active in cdc13-1 mutants, we
reasoned that genes responsible for such activities would
interact with similar genes to those that EXO1 interacts with.
We used the BioGRID database to create a ranked list of genes
Deletion increases
telomere length
Deletion decreases
telomere length
Deletion not known to
affect telomere length
Shared genetic
interactions
Deletion suppresses cdc13-1
growth defect
Deletion enhances cdc13-1
growth defect
Deletion not known to affect
cdc13-1 growth defect
RAD50
ASF1 NUP84 SRS2
ELG1
POL30
POL32
RAD51
RAD1
RAD52
EXO1
RAD27
MRC1
SGS1
TOF1
RAD53
MRE11
RAD9
RAD24
PIF1
30°C27°C
cdc13-1
cdc13-1
pif1Δ cdc13-1
pif1Δ cdc13-1
exo1Δ cdc13-1
exo1Δ cdc13-1
pif1Δ exo1Δ cdc13-1
pif1Δ exo1Δ cdc13-1
B
C
1108
1195
4874
4875
1296
1297
5323
5324
36°C23°C
5′
5′
5′ TGGTGGTGGTGGTGGTGGTGG
3′ ACCACCACCACCACCACCACC
3′5′ GGTGGTGGTGGTGGTGGTGGTGGTG
Cell cycle
arrest
3′
3′ ACCACC 5′
3′
ACCACCACC3′ 5′
Cdc13-1
Nuclease(s)
Exo1
A
Mec1
Rad53
Dun1
Rad9
Chk1
Pds1
Checkpoint
36°C
23°CCappedtelomeres
Uncapped
telomeres
ssDNA
TGGTG
Figure 1 Pif1 and Exo1 inhibit growth of cdc13-1 mutants. (A) Inactivation of Cdc13 by use of the temperature-sensitive allele cdc13-1 leads to
telomere uncapping. Exo1 and additional nuclease(s) generate ssDNA at uncapped telomeres, which is the stimulus for Mec1-dependent
checkpoint activation and cell cycle arrest. (B) Ranked diagram of genes that share genetic interactions with EXO1 and are important in the
context of telomeres. (Thicker lines mean more shared genetic interactions). (C) Strains of the genotypes shown were serially diluted across
agar plates and grown at the temperatures indicated for 3 days. In this and other figures, strain numbers (DLYs) are shown adjacent.
Pif1 and Exo1 resect uncapped telomeres
JM Dewar and D Lydall
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