Copyright ? 2008 by the Genetics Society of America
Dynamic Regulation of Single-Stranded Telomeres in
Stephanie Smith, Soma Banerjee, Regina Rilo1and Kyungjae Myung2
Genome Instability Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute,
National Institutes of Health, Bethesda, Maryland 20892
Manuscript received August 24, 2007
Accepted for publication December 12, 2007
The temperature-sensitive phenotypes of yku70D and yku80D have provided a useful tool for under-
standing telomere homeostasis. Mutating the helicase domain of the telomerase inhibitor Pif1 resulted in
the inactivation of cell cycle checkpoints and the subsequent rescue of temperature sensitivity of the
yku70D strain. The inactivation of Pif1 in yku70D increased overall telomere length. However, the long
G-rich, single-stranded overhangs at the telomeres, which are the major cause of temperature sensitivity,
were slightly increased. Interestingly, the rescue of temperature sensitivity in strains having both pif1-m2
and yku70D mutations depended on the homologous recombination pathway. Furthermore, the BLM/
WRN helicase yeast homolog Sgs1 exacerbated the temperature sensitivity of the yku70D strain. Therefore,
the yKu70-80 heterodimer and telomerase maintain telomere size, and the helicase activity of Pif1 likely
also helps to balance the overall size of telomeres and G-rich, single-stranded overhangs in wild-type cells
by regulating telomere protein homeostasis. However, the absence of yKu70 may provide other proteins
such as those involved in homologous recombination, Sgs1, or Pif1 additional access to G-rich, single-
stranded DNA and may determine telomere size, cell cycle checkpoint activation, and, ultimately,
disorders (Mimori and Hardin 1986; Reeves 1987).
Biochemical analyses of the Ku70-Ku80 heterodimer
protein demonstrated that it bound in a sequence-
nonspecific fashion to virtually all double-stranded DNA
ends, including 59- or 39-protruding ends, blunt ends
(Mimoriand Hardin1986),and duplexDNA ending in
stem-loop structures (Falzon et al. 1993). Ku’s DNA-
binding activity is believed to function by both holding
together broken double-stranded DNA, at least in
mammals through its interaction with DNA-PKcs (Downs
and Jackson 2004; Spagnolo et al. 2006), and protecting
against end-to-end fusions at telomeres (Riha et al. 2002;
Jaco et al. 2004; Myung et al. 2004). Inactivation ex-
periments in different species demonstrated that the
Ku70-Ku80 heterodimer is important for the pro-
tection of DNA ends during both DNA repair and
telomere maintenance (Nussenzweig et al. 1996; Gu
et al. 1997; Ouyang et al. 1997; Boulton and Jackson
1998; Nugent et al. 1998; Bailey et al. 1999; Hsu
u protein was originally identified as an autoantigen
recognized by sera from patients with rheumatic
et al. 1999; Samper et al. 2000; Friesland et al. 2003;
Downs and Jackson 2004; Jaco et al. 2004; Myung
et al. 2004).
Telomeres are specific DNA structures at the ends of
chromosomes that secure genetic information by pro-
tecting chromosomes from degradation with the help
of many telomere-associated proteins (Lingner and
Cech 1998; De Lange 2002). In Saccharomyces cerevisiae,
telomeric DNA is 250–400 bp long with a simple repeat
tract, C1-3A/TG1-3(Vega et al. 2003). A G-rich, single-
stranded tail is generated at the ends of the telomeres
in late S-phase, presumably by the nuclease activity
(Wellinger et al. 1993; Maringele and Lydall 2002;
Bertuch and Lundblad 2004). In wild-type cells, telo-
merase extends the G-rich strand followed by general
DNA replication that fills in the opposite C-strand and,
in other phases of the cell cycle. Telomere length is
affected by many factors, including DNA replication,
telomere synthesis by telomerase, and the level of
degradation protection provided by telomere mainte-
nance proteins (Lingner and Cech 1998; De Lange
2002; Vega et al. 2003). Mutations in yKU70 or yKU80
decreased overall telomere length and led to an
elongation of G-rich, single-stranded tails in all cell
cycles (Porter et al. 1996; Tsukamoto et al. 1997;
Boulton and Jackson 1998; Nugent et al. 1998). In
1Present address: University of California at Davis, Sacramento, CA
2Corresponding author: Genome Instability Section, Genetics and Mo-
lecular Biology Branch, National Human Genome Research Institute,
Genetics 178: 693–701 (February 2008)
mutation of yKU70 or yKU80 altered telomere position
effect, which was defined as a suppression of gene
expression in a subtelomeric region (Boulton and
Jackson 1998; Evans et al. 1998; Laroche et al. 1998;
Nugent et al. 1998).
yku70D or yku80D mutations cause a growth defect at
37? (Feldmann and Winnacker 1993; Boulton and
sensitivity is the result of telomere homeostasis defects
that activate cell cycle checkpoints, and not the result
of deficiencies in DNA double-strand break repair
pathways, such as nonhomologous end joining (NHEJ)
(Nugent et al. 1998; Teoand Jackson 2001; Lewis et al.
2002; Maringele and Lydall 2002). In these studies,
the temperature sensitivity of yku70D or yku80D strains
was rescued either by mutations in checkpoint genes or
in EXO1 or by the overexpression of telomerase sub-
units, including EST2, TLC1, or EST1. Interestingly, the
overexpression of telomerase subunits did not change
telomere length or the G-rich, single-stranded over-
hangs at telomeres, despite the suppression of cell cycle
checkpoint activationelicited by the yku mutation at 37?
(Nugent et al. 1998; Teoand Jackson 2001; Lewis et al.
2002). How cell cycle checkpoints are repressed by
telomerase overexpression remains unclear.
Mutations in yeast S. cerevisiae PIF1, which encodes a
DNA helicase, were identified in a screen to detect
telomere maintenance proteins (Schulz and Zakian
1994). Yeast PIF1 encodes a transcript that makes two
alternatively translated proteins controlled by two dis-
tinct initiating methionine codons (Lahaye et al. 1991;
Schulz and Zakian 1994). Both the nuclear and mi-
tochondrial Pif1 proteins have a 59-39 DNA helicase
activity (Lahaye et al. 1991; Schulz and Zakian 1994).
The pif1-m2 allele, which inactivates only the nuclear
function of Pif1, results in elongated telomeres, the
addition of telomeres at telomere seed sequences placed
at subtelomeric sites, and an increased rate of sponta-
neous gross chromosomal rearrangements (Schulz and
Zakian 1994; Zhou et al. 2000; Myung et al. 2001a). On
the other hand, the pif1-m1 allele, which abrogates the
mitochondrial Pif1, results in a petite phenotype.
The temperature-sensitive phenotype of yku70D has
been a useful tool for determining which proteins are
required for the maintenance of telomeric structures.
In this study, we found that the temperature-sensitive
phenotype of yku70D was suppressed by the mutation of
the telomerase inhibitor Pif1. The suppression of the
temperature-sensitive phenotype of yku70D was due to
the active role of homologous recombination (HR)-
dependent end protection.
MATERIALS AND METHODS
General genetic methods: Media for propagating yeast
strains used in this study are yeast extract–peptone–dextrose
(YPD) media containing 1% (w/v) yeast extract, 2% (w/v)
peptone, and 2% (w/v) dextrose, synthetic drop-out (SD)
media containing 0.67% (w/v) yeast nitrogen base without
amino acids, and 2% (w/v) dextrose with appropriate amino
acids depending on the strains used. To make solid media,
1.5% (w/v) agar for YPD plates and 2.3% (w/v) for SD plates
were added.All S. cerevisiae strains were propagated at 25?, 30?,
or 37? as indicated. All S. cerevisiae strains used in this study
ura3-52, leu2D1, trp1D63, his3D200, lys2DBgl, hom3-10, ade2D1,
ade8? and detail genotypes or strains used in this study are
listed in Table 1. Strains used in this study were generated by
tion methods, and correct gene disruptions were verified by
PCR as described (Myung et al. 2001c). The sequences of
primers used to generate disruption cassettes and confirm
transformation, yeast chromosomal DNA isolation, and PCR
were performed as previously described (Myung et al. 2001c;
Smith et al. 2004). The overexpression of the RAD52 gene was
achieved with a multicopy Yep13 plasmid carrying the RAD52
open reading frame with 4 kb upstream of the translation
Determination of length of telomeres and G-rich, single-
stranded DNA at telomeres: Telomere lengths were deter-
mined by XhoI digestion of chromosomal DNA, followed by
of single-stranded DNA at telomeres was determined by a
comparison of telomeric signals by Southern hybridization
with a TG repeat probe in nondenaturing in-gel hybridization
with signals from denaturing conditions.
Northern hybridization: RNA was prepared by using Gentra
Systems Purescript RNA purification system from exponential
1:50 dilution of overnight cultured yeast at 30?. RNA samples
were boiled in the presence of ethidium bromide and form-
aldehyde, cooled, and then loaded onto a 1.2% agarose, 13
MOPS, 1% formaldehyde gel in 13 RNA gel loading dye.
Electrophoresis was performed at 100 V for 1 hr. The RNA was
UV. Prehybridization was carried out for 2 hr at 68? in 0.5 m
sodium phosphate, 7% (w/v) SDS, 1 mm EDTA (pH 7.0),
followed by hybridization with radiolabeled probe. DNA for
making a probe to detect HUG1 expression was generated
by PCR with the primers PRKJM 1107 (59-GACCATGGAC
CAAGGCCTTAACCCAAAG-39) and PRKJM1108 (59-CAGAA
AGACCGCCGCGACGTTCGACGGC-39). The DNA for a con-
trol probe to detect ACT1 expression was made by PCR with
the primers PRKJM959 (59-CTCAATCCAAGAGAGGTATCTT
GAC-39) and PRKJM960 (59-GTGGTGGAGAAAGAGTAACCA
CGTTC-39). Both PCR products were then radiolabeled
with ½a-32P?dCTP by random priming as previously described
(Banerjee and Myung 2004).
Western blotting: Cell extracts were prepared from expo-
nential cultures grown for an additional 4 hr at either 30? or
37? after 1:50 dilution of overnight cultured yeast at 30?.
Cultures were harvested and washed with 20% trichloroacetic
acid, glass beads were used to break the cell walls, and the
2 m Tris. Samples were boiled and centrifuged before loading
onto a 7–12% SDS-PAGE (Bio-Rad, Hercules, CA). The pro-
tein was transferred to a PVDF membrane. The membranes
were blocked in 13 Western blocking reagent (Roche) and
then incubated with an anti-Rad53 antibody (Santa Cruz).
After washing and incubation in the secondary anti-goat HRP
antibody, detection was achieved using Western blocking
detection reagents (GE Healthcare).
694S. Smith et al.
of yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the
RNA component of telomerase). Genetics 160: 49–62.
Li, J. L., R. J. Harrison, A. P. Reszka, R. M. Brosh, Jr., V. A. Bohr
et al., 2001Inhibition of the Bloom’s and Werner’s syndrome
helicases by G-quadruplex interacting ligands. Biochemistry 40:
Lingner, J., and T. R. Cech, 1998
maintenance. Curr. Opin. Genet. Dev. 8: 226–232.
Lovett, S. T., and R. K. Mortimer, 1987
mutants of the RAD55 gene of Saccharomyces cerevisiae: effects
of temperature, osmotic strength and mating type. Genetics
Maringele, L., and D. Lydall, 2002
stranded DNA at telomeres activates subsets of DNA damage
and spindle checkpoint pathways in budding yeast yku70Delta
mutants. Genes Dev. 16: 1919–1933.
Mimori, T., and J. A. Hardin, 1986
tween Ku protein and DNA. J. Biol. Chem. 261: 10375–10379.
Myung, K., C. Chen and R. D. Kolodner, 2001a
cooperate in the suppression of genome instability in Saccharomy-
ces cerevisiae. Nature 411: 1073–1076.
Myung, K., A. Datta, C. Chen and R. D. Kolodner, 2001b
the Saccharomyces cerevisiae homologue of BLM and WRN, sup-
presses genome instability and homeologous recombination.
Nat. Genet. 27: 113–116.
Myung, K., A. Datta and R. D. Kolodner, 2001c
spontaneous chromosomal rearrangements by S phase check-
point functions in Saccharomyces cerevisiae. Cell 104: 397–408.
Myung, K., G. Ghosh, F. J. Fattah, G. Li, H. Kim et al., 2004
lation of telomere length and suppression of genomic instability
in human somatic cells by Ku86. Mol. Cell. Biol. 24: 5050–5059.
Nugent, C. I., G. Bosco, L. O. Ross, S. K. Evans, A. P. Salinger et al.,
1998 Telomere maintenance is dependent on activities required
for end repair of double-strand breaks. Curr. Biol. 8: 657–660.
Nussenzweig, A., C. Chen, V. da Costa Soares, M. Sanchez,
K. Sokol et al., 1996 Requirement for Ku80 in growth and im-
munoglobulin V(D)J recombination. Nature 382: 551–555.
Onoda, F., M. Seki, A. Miyajima and T. Enomoto, 2000
of sister chromatid exchange in Saccharomyces cerevisiae sgs1
disruptants and the relevance of the disruptants as a system to
evaluate mutations in Bloom’s syndrome gene. Mutat. Res.
Onoda, F., M. Seki, A. Miyajima and T. Enomoto, 2001
ment of SGS1 in DNA damage-induced heteroallelic recombina-
tion that requires RAD52 in Saccharomyces cerevisiae. Mol. Gen.
Genet. 264: 702–708.
Ouyang, H., A. Nussenzweig, A. Kurimasa, V. C. Soares, X. Li et al.,
1997Ku70 is required for DNA repair but not for Tcell antigen
receptor gene recombination in vivo. J. Exp. Med. 186: 921–929.
Polotnianka, R. M., J. Li and A. J. Lustig, 1998
erodimer is essential for protection of the telomere against nu-
cleolytic and recombinational activities. Curr. Biol. 8: 831–834.
Porter, S. E., P. W. Greenwell, K. B. Ritchie and T. D. Petes,
1996The DNA-binding protein Hdf1p (a putative Ku homo-
logue) is required for maintaining normal telomere length in
Saccharomyces cerevisiae. Nucleic Acids Res. 24: 582–585.
Reeves, W. H., 1987Antinuclear antibodies as probes to explore the
structural organization of the genome. J. Rheumatol. 14(Suppl.
Riha, K., J. M. Watson, J. Parkey and D. E. Shippen, 2002
mere length deregulation and enhanced sensitivity to genotoxic
Telomerase and chromosome end
Characterization of null
Mechanism of interaction be-
The yeast Ku het-
stress in Arabidopsis mutants deficient in Ku70. EMBO J. 21:
Samper, E., F. A. Goytisolo, P. Slijepcevic, P. P. van Buul and M. A.
Blasco, 2000Mammalian Ku86 protein prevents telomeric fu-
sions independently of the length of TTAGGG repeats and the
G-strand overhang. EMBO Rep. 1: 244–252.
Schulz, V., and V. A. Zakian, 1994
case inhibits telomere elongation and de novo telomere forma-
tion. Cell 76: 145–155.
Smith, S., J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta et al.,
2004 Mutator genes for suppression of gross chromosomal re-
arrangements identified by a genome-wide screening in Saccharo-
myces cerevisiae. Proc. Natl. Acad. Sci. USA 101: 9039–9044.
Spagnolo, L., A. Rivera-Calzada, L. H. Pearl and O. Llorca,
2006 Three-dimensional structure of the human DNA-PKcs/
Ku70/Ku80 complex assembled on DNA and its implications
for DNA DSB repair. Mol. Cell 22: 511–519.
Sugawara, N., X. Wang and J. E. Haber, 2003
Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombi-
nation. Mol. Cell 12: 209–219.
Sun, H., R. J. Bennett and N. Maizels, 1999
cerevisiaeSgs1 helicase efficiently unwinds G-G pairedDNAs. Nu-
cleic Acids Res. 27: 1978–1984.
Teo, S. H., and S. P. Jackson, 2001
sion suppresses telomere-specific checkpoint activation in the
yeast yku80 mutant. EMBO Rep. 2: 197–202.
Tsukamoto, Y., J. Kato and H. Ikeda, 1997
ipate in DNA repair and recombination in Saccharomyces cere-
visiae. Nature 388: 900–903.
Vega, L. R., M. K. Mateyak and V. A. Zakian, 2003
end: telomerase access in yeast and humans. Nat. Rev. Mol. Cell
Biol. 4: 948–959.
Vega, L. R., J. A. Phillips, B. R. Thornton, J. A. Benanti, M. T.
Onigbanjoet al., 2007 Sensitivity of yeast strainswithlongG-tails
to levels of telomere-bound telomerase. PLoS Genet. 3: e105.
Wagner, M., G. Price and R. Rothstein, 2006
Top3 reveals an interaction between the Sgs1 and Pif1 DNA hel-
icases in Saccharomyces cerevisiae. Genetics 174: 555–573.
Watt, P. M., E. J. Louis, R. H. Borts and I. D. Hickson, 1995
a eukaryotic homolog of E. coli RecQ that interacts with topoiso-
merase II in vivo and is required for faithful chromosome segre-
gation. Cell 81: 253–260.
Weinert, T. A., and L. H. Hartwell, 1993
mutants and specificity of the RAD9 checkpoint. Genetics 134:
Wellinger, R. J., A. J. Wolf and V. A. Zakian, 1993
tion and formation of single-strand TG1-3 tails occur sequentially
in late S phase ona yeast linearplasmid. Mol.Cell.Biol. 13: 4057–
Williamson, J. R., M. K. Raghuraman and T. R. Cech, 1989
valent cation-induced structure of telomeric DNA: the G-quartet
model. Cell 59: 871–880.
Wu, X., and N. Maizels, 2001Substrate-specific inhibition of RecQ
helicase. Nucleic Acids Res. 29: 1765–1771.
Zahler, A. M., J. R. Williamson, T. R. Cech and D. M. Prescott,
1991Inhibition of telomerase by G-quartet DNA structures. Na-
ture 350: 718–720.
Zhou, J., E. K. Monson, S. Teng, V. P. Schulz and V. A. Zakian,
2000Pif1p helicase, a catalytic inhibitor of telomerase in yeast.
Science 289: 771–774.
The Saccharomyces PIF1 DNA heli-
In vivo roles of
Telomerase subunit overexpres-
Silencing factors partic-
Getting to the
The absence of
Cell cycle arrest of cdc
Communicating editor: A. Nicolas
Regulation of Telomeres in yku