EXO1
Plays a Role in Generating Type I and Type II Survivors in Budding Yeast
Laura Maringele
1and David Lydall
2School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Manuscript received June 26, 2003
Accepted for publication November 24, 2003
ABSTRACT
Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures. Similarly, human cells that enter senescence can use homolo-gous recombination for telomere maintenance, when telomerase cannot be activated. Although recombina-tion proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination. Here we demonstrate that the Exo1 exonuclease is an initiator of the recombination process that allows cells to escape senescence and become immortal in the absence of telomerase. We show thatEXO1is important for generating type I survivors inyku70⌬mre11⌬cells and type II survivors intlc1⌬cells. Moreover, intlc1⌬cells,EXO1seems to contribute to the senescence process itself.
B
UDDING yeast cells, like almost all immortal eukary- guishable from BIR (Reddelet al.1997), recombination otic cells, use telomerase to maintain the end of at telomeres in budding yeast is considered a good their chromosomes. Cells lacking telomerase compo- model for understanding the telomerase-independent nents, for example,est1⌬,est2⌬, ortlc1⌬mutants, have survival in mammalian cells.been engineered and studied for their ability to escape It is clear that genes involved in recombination at replicative senescence (a state of cell cycle arrest caused double-strand breaks are also responsible for amplifica-by short and/or defective telomeres) and survive in- tion of telomeres in the absence of telomerase (Leet definite periods of time using a secondary mechanism al.1999;Sugawaraet al.2000;Chenet al. 2001). But, of telomere maintenance. This mechanism appears to perhaps with the exception ofRAD52, which is essential be based on break-induced replication (BIR), a variant for recombination, all other recombination genes seem of the main DSB-repair mechanism in budding yeast, to be divided into two distinct pathways, depending homologous recombination, and is associated with on what kind of structure is amplified. One pathway is amplification of telomeric or subtelomeric structures responsible for amplification of subtelomeric Y⬘regions (Lundblad and Blackburn 1993; McEachern and and is dependent onRAD51, RAD54, RAD55, RAD57, Blackburn1995;TengandZakian1999). One major andRAD52(Chenet al.2001). In yeast, approximately difference between BIR and the telomerase-based way two-thirds of the chromosomes have one to four ho-of maintenance is that recombination can copy and meologous subtelomeric Y⬘ regions, adjacent to telo-amplify only preexistent structures, while telomerase meres (PrydeandLouis1997). Cells that escape
senes-can synthesize a telomerede novo. cence by amplifying Y⬘ regions are termed type I
Long-term survival without evidence of telomerase survivors (LundbladandBlackburn1993;Tengand activity was found inⵑ15% of malignant tumors, where Zakian 1999; Chenet al. 2001). Another pathway in-it was termed alternative lengthening of telomeres volves theRAD50/MRE11/XRS2complex together with (ALT;Grobelnyet al.2001;Hensonet al.2002). Recent RAD52, RAD59, SRS2, SGS1, and TID1 and drives the data report a higher incidence of ALT (25–66%) in amplification of TG-telomeric repeats to generate type some types of cancer like glioblastoma and
osteosar-II survivors (Chenet al.2001;Signonet al.2001). coma (Hakin-Smith et al. 2003; Ulaner et al. 2003).
There are subtle differences between the recombina-Because the mechanism of ALT is practically
indistin-tion-amplification induced by telomere shortening and the recombination-repair induced by a double-strand break, despite the fact that they involve the same genes. For example, initiation of (sub) telomeric amplification 1Present address:School of Clinical Medical Sciences-Gerontology,
is a relatively rare event, with approximately only one in
University of Newcastle, Henry Wellcome Laboratory for
Biogerontol-a million cells Biogerontol-able to complete this process; in contrBiogerontol-ast,
ogy Research, Newcastle General Hospital, Newcastle upon Tyne, NE4
6BE, United Kingdom. double-strand breaks (DSBs) are highly efficiently re-2Corresponding author:School of Clinical Medical
Sciences-Gerontol-paired.
ogy, University of Newcastle, Henry Wellcome Laboratory for
Bioger-Although recombination proteins required to
gener-ontology Research, Newcastle General Hospital, Newcastle upon Tyne
NE4 6BE, United Kingdom. E-mail: [email protected] ate telomerase-independent survivors have been
nuclease activity (Tranet al. 2002). Exo1p is involved tion, except for Southern blot analysis of single colonies. in resection of meiotic DSBs (TsubouchiandOgawa Yeast genomic DNA was isolated and ⵑ20 ng of DNA was digested withXhoI and separated on a 0.8% agarose gel. DNA
2000), mitotic DSBs (Fiorentini et al. 1997), and
un-was then transferred to a Magna Nylon membrane (Genetic
protected telomeres (Maringele and Lydall 2002;
Research Instrumentation) and UV crosslinked. The mem-Lydall2003). In addition,EXO1affects meiotic recom- brane was then hybridized with a Y⬘-TG probe (pHT128; Tsu-bination (Khazanehdari and Borts 2000; Kirkpat- bouchi and Ogawa 2000) and hybridization was detected ricket al.2000;Symingtonet al.2000) and is part of using a nonradioactive detection kit (Amersham, Arlington
Heights, IL).
a complex with mismatch repair proteins (Tishkoffet al.1997;SokolskyandAlani2000;Tranet al.2001). Here we present evidence that EXO1 is important
for generating recombination-dependent, type I and II RESULTS
survivors, in two different types of senescent mutants:
EXO1is important for generating survivors inyku70⌬
telomere capping defective (yku70⌬ mre11⌬) and
telo-mre11⌬senescent cells:Initiation of (sub)telomeric am-merase-negative (tlc1⌬) cells. We show that deletion of
plification in the absence of telomerase is a rare event. EXO1 delays the appearance of survivors for ⵑ30–40
Factors involved in the initiation of survivors were still generations. Therefore,EXO1appears to be one of the
unknown, but we speculated thatEXO1might be one, “initiators” of the recombination process that allow rare
because of its role in degradation of unprotected telo-cells to escape senescence and became immortal in the
meres (Maringele and Lydall 2002). Our previous absence of telomerase.
experiments have demonstrated that defective telo-meres incdc13-1andyku70⌬mutants were targeted by the Exo1p exonuclease.EXO1was important for
resect-MATERIALS AND METHODS
ing the 5⬘ AC strand, from telomeres toward centro-Yeast strains: All strains used in this study are isogenic,
meres, leaving behind a detectable 3⬘overhang, up to
in the W303 background, andRAD5⫹. To construct strains,
1 kb long in yku70⌬ strains (Maringele andLydall standard genetic procedures of transformation and tetrad
2002). One consequence of Exo1p activity at defective
analysis were followed (Adamset al.1997). Since W303 strains
contain anade2-1mutation, YPD (yeast extract, peptone, and telomeres was arrest of the cell cycle before anaphase dextrose) medium was routinely supplemented with adenine (MaringeleandLydall2002). The ability to degrade at 50 mg/liter. All yku70⌬ mre11⌬ exo1⌬ strains and their
unprotected telomeresin vivois not a common feature
controls were made by crossing DLY1331 (mre11⌬::hisG::
of all exonucleases (M. Zubko, unpublished data) sug-URA3) with DLY1408 (yku70⌬::HIS3 exo1⌬::LEU2). All tlcl⌬
exo1⌬ strains were made by crossing DLY1628 [tlc1⌬::HIS3 gesting that Exo1p has a special function at telomeres. pTLC1:URA3 (pSD120 from D. Gottschling)] with DLY1948 We, therefore, tested the role ofEXO1in senescence (exo1⌬::LEU2 rad52⌬::TRP1). Diploid cells that had lost the and survival of cells with telomere defects caused by pTLC1 plasmid were sporulated, dissected, and germinated.
the absence of telomere protective genes YKU70 and Growth rate assay:Cells of the appropriate genotype (four
MRE11. This model of senescence and survival is based
independent strains for each genotype of interest) were
iso-lated from a fresh germination plate and grown in YPD me- on the observation thatyku70⌬mre11⌬double mutants
dium overnight. The following morning, cells were counted senesce very rapidly, during the early growth of germi-using a hemocytometer, diluted to 1⫻105cells/ml, and then
nated spores (Ritchie and Petes 2000;DuBois et al.
incubated at 23⬚, with aeration. Every 23 hr, cell densities were
2002;MaringeleandLydall2002).
measured and then the culture was diluted with fresh YPD
We monitored growth ofyku70⌬mre11⌬double
mu-liquid medium to a density of 105cells/ml. During counting,
cells were put on ice. This cycle was repeated for several days. tants and yku70⌬ mre11⌬ exo1⌬ triple mutants. Cells
At indicated time points, samples were collected for telomere were taken directly from the germination plate (four length analysis.
independent strains of each genotype) and incubated Streak assay:Cells of the appropriate genotype were picked
in liquid at 23⬚for several days. At 23⬚, wild-type strains
from a fresh germination plate and streaked onto YPD plates.
After incubation for 4 days at 23⬚, similar amounts of cells would divideⵑ10 times a day. Cell densities were scored
(ⵑ1 ⫻ 107 cells) from several independent colonies were
every 23 hr, followed by dilution to 105 cells/ml and
picked and restreaked on YPD plates. This restreaking was continued incubation. At 23⬚,yku70⌬andyku70⌬exo1⌬ repeated several times to allow senescence and appearance
cells reached and maintained densities of ⵑ0.5 ⫻ 108
of survivors.
cells/ml, similar to the wild type, while other control Microcolony assays:Colony-purified yeast strains were
inoc-ulated into 1 ml YPD and grown overnight with aeration at mutants, mre11⌬ andmre11⌬ exo1⌬, reached densities
23⬚until they reached a concentration ofⵑ8⫻106cells/ml.
5-fold lower than those of the wild type (Figure 1a). In
Cells were sonicated briefly and spread on plates. The plates
contrast,yku70⌬mre11⌬andyku70⌬mre11⌬exo1⌬cells
were incubated at 23⬚. After an appropriate length of time,
were in crisis during the early days of the experiment,
the colonies were photographed and the cell numbers in 100
Figure 1.—EXO1is important for generating survivors in yku70⌬ mre11⌬ senescent cells. (a) Four independent strains of each genotype were taken directly from germination plates and grown in liquid culture. Cell densities were counted ev-ery 23 hr, followed by dilution to 1⫻105cells/
ml and continued incubation. Each symbol repre-sents the average cell density reached by cells with the same genotype after 23 hr and the error bars represent the standard deviations. Error bars are shown only for the yku70⌬mre11⌬ and yku70⌬ mre11⌬exo1⌬mutants. (b) Cells from a germina-tion plate were spread on fresh YPD plates (pas-sage 1), incubated 4 days at 23⬚, and then pas(pas-saged to other plates (passage 2). Several passages were consecutively performed.
the wild type, consistent with early entry into senescence rable amounts of cells from germination plates were spread on fresh YPD plates (passage 1) and incubated (Figure 1a).
Both types of senescent mutants, yku70⌬ mre11⌬ at 23⬚ for 4 days (Figure 1b). Several passages were performed consecutively. This experiment confirmed (EXO1⫹) andyku70⌬mre11⌬exo1⌬, were able to
gener-ate survivors, but with different dynamics. WhileEXO1⫹ that early generations of yku70⌬mre11⌬(EXO1⫹) and yku70⌬mre11⌬exo1⌬mutants were in a state of minimal survivors escaped senescence early (days 4–5) and grew,
after 2 days, at growth rates (determined by cell density growth, presumably senescent (passage 1), and that EXO1⫹ strains generated survivors more rapidly (pas-after 23 hr growth) similar to those ofmre11⌬mutants,
exo1⌬ survivors appeared more slowly and gradually sage 2) compared with exo1⌬strains (passage 3). Also, EXO1⫹ survivors grew notably better thanexo1⌬ survi-reached, after 15 days in liquid culture, a 5-fold lower
cell density, compared withmre11⌬cells (Figure 1a). vors (passage 3), and a difference in growth was main-tained even after 15 passages, which is equivalent to The data above suggest thatEXO1plays a significant,
but not essential, role in generating survivors. Another 600 wild-type generations (Figure 1d, compare size of individual colonies; data not shown).
possibility would be thatEXO1is important for cell
vital-ity in an mre11⌬ background, even in the absence of EXO1is required for maintenance of survivors:The continued growth defect inyku70⌬mre11⌬exo1⌬could senescence. When growth rates of mre11⌬ exo1⌬ cells
are compared with those ofmre11⌬mutants, it appears have many possible explanations, such as slow metabolic rate, longer cell cycle, decreased viability, high rate of that exo1⌬ deletion did indeed cause a kind of crisis
during the first 4 days, whenmre11⌬exo1⌬mutants daily resenescence, and/or delay in reappearance of survi-vors. It is known that in telomerase-defective (est1⌬) reached onlyⵑ25% of the cell density ofmre11⌬strains
(Figure 1a;Moreau et al. 2001). However, growth of survivors, the senescence phenotype reappears in some subclones (LundbladandBlackburn1993). We moni-mre11⌬exo1⌬strains improved during the following
gen-erations, oscillating between 55 and 98% of themre11⌬ tored the ability of “established” survivors (with a com-mon history of 32 days in culture, therefore longer than growth. Thus, the requirement forEXO1 was stronger
in yku70⌬ mre11⌬ survivors than in mre11⌬ mutants. 200 generations) to form colonies by microcolony assay, spreading similar amounts of cells on plates after a short Interestingly, deletion ofEXO1inyku70⌬cells shows an
effect opposite to that in mre11⌬ cells, because exo1⌬ sonication and then incubating them for 24 hr at 23⬚ (Figure 2).
mutation improves the growth rate ofyku70⌬mutants
ⵑ15% at 23⬚(Figure 1a). At 37⬚, anexo1⌬even rescues Although a number of nondividing cells (single-cell “colonies”) or cells with a large bud (two-cell colonies) the inviability of yku70⌬ mutants (Maringele and
Lydall2002). were microscopically detected after 24 hr in all mutants
with amre11⌬background, the percentage of these one-The requirement for EXO1 in recovery from
simi-Figure2.—EXO1is required for the maintenance of survi-vors. Small amounts of cells from the same passage (eight, corresponding to 32 days in culture) were diluted to the same concentration, briefly sonicated, spread onto a YPD plate, and then incubated for 23 hr at 23⬚before being photographed.
lar to that inmre11⌬ormre11⌬exo1⌬control strains and did not exceed 15% under normal conditions (Figure 2; data not shown). By contrast, yku70⌬mre11⌬exo1⌬
survivors had a high fraction of one- or two-cell colonies Figure3.—EXO1increases the rate of Y⬘amplification in (ⵑ35%) after 24 hr, suggesting they were confronted yku70⌬mre11⌬survivors (type I survivors). (a) Schematic rep-resentation of the end of a chromosome in budding yeast.
with high levels of resenescence and/or difficulties in
The probe used binds to the telomeric end of the Y⬘repeat
regeneration of survivors, which can explain the lower
(ⵑ1 kb) and toⵑ120 bp from the telomeric TG sequences.
cell density reached by these mutants in liquid culture. (b) Y⬘amplification in different mutants grown in liquid cul-Also, it is clear that someyku70⌬mre11⌬exo1⌬colonies ture and monitored by Southern blot. Cells were grown in were similar in size toyku70⌬mre11⌬EXO1⫹ colonies, liquid and collected on the day indicated above each lane of the gel. DNA was cut with XhoI and hybridized with the
and therefore a difference in growth caused by a slow
Y⬘-TG probe. The DNA loading control representsⵑ10 ng of
metabolic rate or delayed cell cycle can be excluded
uncut DNA stained with ethidium bromide.
(Figure 2).
EXO1increases the rate of Yⴕamplification inyku70⌬
mre11⌬survivors (type I survivors):Some cells can es- with a Y⬘-TG probe; that gave three fragments,ⵑ6.5, 5.5, cape senescence and generate survivors. In yeast, pre- and 1.3 kb in wild-type cells. The two larger fragments sumably also in mammalian cells, this occurs by correc- correspond to repetitive Y⬘’s, while the shortest frag-tion of the telomeric defect. Repair proteins that belong ment is the terminal 1 kb of Y⬘and also containsⵑ350 to the recombination pathways are able to amplify sub- bp of telomeric TG repeats (Figure 3a).
telomeric regions (in yeast, Y⬘regions), generating type Inmre11⌬control strains, the telomeric fragment was I survivors, or amplify terminal telomeric TG repeats, shorter than that in wild type (Figure 3b), consistent generating type II survivors. It is known that Rad50p, with previous data (BoultonandJackson1998; Cha-which acts in a complex with Mre11p, is required to mankhahet al.2000). Deletion ofEXO1from the control maintain type II survivors, because in a rad50⌬ back- mre11⌬ strains, maintained by telomerase, apparently ground, type I survivors replace type II survivors very does not influence the length or intensity of (sub)-telo-early (Chen et al. 2001). Since Mre11p and Rad50p meric fragments, consistent with earlier observations function together, type I survivors would be expected (Figure 3b; Tsubouchi and Ogawa2000; Moreau et to predominate in anmre11⌬background as well. al. 2001). We observed that growth of mre11⌬ exo1⌬ Also, the fact that exo1⌬ survivors appear late and strains improved after several days in liquid culture (Fig-maintain a growth defect, compared with the EXO1⫹ ure 1a), but this growth improvement did not correlate strains, raised the possibility that exo1⌬ survivors were with any notable change at the telomeres of these strains using a different survival mechanism. To address the (Figure 3b).
Figure 4.—EXO1 op-poses adaptation during se-nescence. (a) Strains as in-dicated in the legend were grown as described in Fig-ure 1a. (b) Y⬘amplification was monitored as in Fig-ure 3b.
ments corresponding to Y⬘looked similar to wild type, those described in Figure 1. In this experiment, anexo1⌬ decreased the density oftlc1⌬mre11⌬cells before and while the short fragment appeared less intense,
presum-ably due to loss of telomeric sequences in many of these after senescence (Figure 4a). This is partially due to the synergic effect of exo1⌬ and mre11⌬ deletions in cells (Figure 3b). But after 6 days,yku70⌬mre11⌬cells
had already amplified the Y⬘repeats (type I survivors), decreasing the cell viability, as previously mentioned. A second phenomenon was noted: during the early postse-and this amplification apparently remained constant
afterward (days 6–12; Figure 3b). Deletion ofEXO1from nescence period, tlc1⌬ mre11⌬ survivors grew poorly compared with yku70⌬ mre11⌬ survivors and did not yku70⌬mre11⌬cells considerably slowed the rate of Y⬘
amplification. Amplification occurred slowly, but pro- reach the growth levels of mre11⌬ single mutants, as yku70⌬mre11⌬survivors did (Figure 4avs.Figure 1a). gressively during 3–12 days in culture; however, even
after 12 days, the Y⬘amplification was less pronounced Also, the Y⬘amplification intlc1⌬mre11⌬survivors was inferior to the Y⬘amplification inyku70⌬mre11⌬ survi-in yku70⌬ mre11⌬ exo1⌬ cells than in yku70⌬ mre11⌬
cells after 6 days in culture (Figure 3b). Therefore, cells vors (Figure 4b vs. Figure 3b). The data suggest that the presence of Yku70 makes the maintenance of type lackingEXO1had difficulty in generating type I
survi-vors. The difference in dynamics of Y⬘amplification, in I survivors difficult and might explain why tlc1⌬ cells preferentially maintain type II survivors.
exo1⌬vs. EXO1⫹survivors, is not a consequence of poor
cell growth, because similar amounts of DNA were ana- EXO1opposes adaptation and plays a role in generat-ing type I survivors intlc1⌬mre11⌬cells:We next ad-lyzed in Southern blot assays (see DNA loading control,
Figure 3b) and, initially, Y⬘amplifications occur during dressed the question of whether EXO1 plays a role in generating type I survivors, in the presence ofYKU70, senescence. Rather, the growth defect might be
ex-plained by difficulties and delays in amplification of Y⬘ in tlc1⌬ mre11⌬ mutants. Figure 4a shows that tlc1⌬ mre11⌬ exo1⌬ survivors grew about fivefold less than regions inyku70⌬mre11⌬ exo1⌬strains due to the
ab-sence ofEXO1. tlc1⌬mre11⌬survivors, during the early days of the
post-senescence period (days 9–12). This may be due to the
YKU70inhibits the maintenance of type I survivors:
We have shown that Exo1 degrades telomeres inyku70⌬ synthetic effect ofmre11⌬andexo1⌬ in decreasing the cell viability. However, was the escape from senescence mutants (MaringeleandLydall2002). To see if Exo1
plays a role in generating type I survivors in the presence and early postsenescence growth oftlc1⌬mre11⌬exo1⌬ strains due to recombination events that generated type of YKU70, we analyzed tlc1⌬ mre11⌬ strains. These
strains senesce due to the absence of the telomerase I survivors? To test this, we deleted RAD52, which is essential for recombination. It is clear from Figure 4a RNA template, Tlc1, while deletion ofMre11 ensures
portant for generating type II survivors intlc1⌬cells. (a) Strains as indicated in the legend were grown as de-scribed in Figure 1a. (b) Telomeric length was moni-tored by Southern blot as in Figure 3b. On day 0 cells were taken directly from germination plates and grown for 4 hr in liquid cul-ture, and DNA was pre-pared. (c) Y⬘and TG ampli-fication was monitored as in Figure 3b. (d) Germinated spores were grown and mass passaged daily on plates for 6 days. On the sixth day the majority of cells had en-tered senescence. By day 8 we were able to pick 12 colo-nies (A–L) that were still growing. These colonies were grown overnight in liq-uid culture before DNA was prepared. Y⬘and TG ampli-fication was monitored as in Figure 3b.
nescent growth curve almost overlaps with the growth subtelomeric repeats, presumably by generating long 3⬘ overhangs that would invade hom(e)ologous structures, curve oftlc1⌬mre11⌬exo1⌬mutants, during the first 17
days in culture. By contrast, tlc1⌬mre11⌬rad52⌬cells what about its role in type II survivors? Type II survivors amplify the TG-telomeric repeats, with help from a did not escape senescence. Thus, tlc1⌬ mre11⌬ exo1⌬
rad52⌬cells appeared to be adapting to the telomere nuclease/helicase complex (Rad50p/Mre11p/Xrs2p), a helicase (Srs2p or Sgs1p), and recombination proteins defect, rather than using recombination to overcome
senescence. (Rad52p and Rad59p;Chenet al.2001). We wondered
if there was any requirement for Exo1p in generating Thus, early postsenescence growth of tlc1⌬ mre11⌬
exo1cells wasRAD52independent, while growth oftlc1⌬ type II survivors.
In experiments similar to those described in Figure mre11⌬cells during the same period was strictlyRAD52
dependent. Later on, RAD52⫹ strains amplified the Y⬘ 1, we monitored the senescence and survival of tlc1⌬ exo1⌬ cells vs. tlc1⌬ cells (Figure 5). The strains were repeats,tlc1⌬mre11⌬ exo1⌬mutants to a lesser extent
than tlc1⌬ mre11⌬ mutants (Figure 4b), while rad52⌬ germinated from spores that had inherited the normal telomere length from their parents. During the prese-survivors showed no amplification of subtelomeric
re-gions (data not shown). Adaptation is defined as escape nescent period,tlc1⌬exo1⌬strains grewⵑ1.5-fold better thantlc1⌬ EXO1⫹single mutants each day (Figure 5a; from the cell cycle arrest without repair of the DNA
damage (Toczyskiet al.1997;Leeet al.1998). In this note the lack of overlap of error bars on days 3 and 4). This suggests that in the absence of telomerase,EXO1 case it means to escape senescence with short telomeric
regions. contributes to telomere shortening and senescence.
However, this phenomenon was not observed in strains Our interpretation is thatEXO1is, indeed, required
to generate type I survivors in tlc1⌬ mre11⌬ mutants, that lackMRE11(Figure 4a). It is possible that the syn-thetic effect ofmre11⌬andexo1⌬mutations in decreas-but also to suppress the adaptation to telomeric damage
and the recombination-independent escape from senes- ing cell viability (presumably for reasons other than telomere damage, because mre11⌬ exo1⌬ and mre11⌬ cence.
EXO1 plays a role in generating type II survivors in cells have similarly short telomeres; Figure 3b) counter-acts the effect ofexo1⌬in presenescentmre11⌬cells.
As telomeres progressively shortened, cells became senescent. The vast majority of cells stopped cell division after 6 days in liquid culture, whentlc1⌬andtlc1⌬exo1⌬ cells had 20- to 25-fold lower densities, compared with their growth rate from day 1 (Figure 5a). Soon after, tlc1⌬ cells generated survivors that reached their full growth potential within 2 days (day 8). By contrast,tlc1⌬ exo1⌬cells grew poorly and needed more time (11–12 days total) to achieve a better growth rate, still 1.6-fold less than that oftlc1⌬or exo1⌬ cells (Figure 5a). This indicates thatEXO1plays a role in generating survivors in atlc1⌬background.
Since telomerase-defective tlc1⌬ cells entered senes-cence more slowly thanyku70⌬mre11⌬cells (compare Figures 1A and 5A) we were able to monitor the effect ofEXO1on telomere degradation as cells entered senes-cence. We purified DNA from cells as early as possible after they had germinated and every day thereafter. The effect ofEXO1on telomere shortening in presenescent cells was clear. InEXO1⫹tlc1⌬cells significant telomere shortening had occurred by the time we were first able to purify DNA (as the spores germinated and grew from a single cell to a colony ofⵑ108cells). After this, during days 1–7, telomere shortening appeared to stop. In con-trast, tlc1⌬ exo1⌬ cells contained significantly longer telomeres at day 0 that slowly declined over the next 6
Figure6.—A model forEXO1in generating type I and II
days, to reach a length similar to that intlc1⌬ cells by survivors. (a) Exo1p initiates recombination in Y⬘subtelomeric
day 7 (Figure 5b). regions, processing the ends of the chromosomes in senescent
cells. It degrades the 5⬘strand, generating long 3⬘overhangs,
To understand the role of Exo1p during recovery
which invade homologous regions from other chromosomes.
from senescence, Southern blots were used to analyze
Subsequently, type I survivors are generated. (b) Type II
survi-the dynamics of telomere amplification intlc1⌬ exo1⌬
vors might represent the product of a disrupted
recombina-double mutants vs. tlc1⌬ single mutants (Figure 5c). tion pathway that had difficulty in progressing into the Y⬘, After 6 days in culture, both types of mutants had short due to the protective presence/activity of both Yku70p/
Yku80p andMRE11/RAD50/XRS2complexes and that
special-telomeres. After 8 days in culture,tlc1⌬cells had
ampli-izes in amplification of TG repeats. Exo1p activity is
presum-fied the telomeric TG sequences, generating type II
ably required to degrade the telomeric 5⬘ strand produced
survivors, while the short telomeres from tlc1⌬ exo1⌬ by a helicase (Srs2/Sgs1). Degradation of the 5⬘
strand allows
cells looked unchanged (Figure 5c). After 10 days,tlc1⌬ the 3⬘ strand to invade other homologous substrates. Subse-exo1⌬cells showed little evidence of telomere amplifica- quently, type II survivors are generated.
tion, and also their Y⬘ repeats were indistinguishable from wild-type Y⬘repeats (Figure 5c). Thetlc1⌬exo1⌬ cells significantly amplified their telomeres after 12 days
from thetlc1⌬exo1⌬cells, only 1 colony (G) had gener-in culture. The pattern of telomere amplification gener-in
ated a type I survivor at this point, while the other tlc1⌬exo1⌬survivors was also different from that intlc1⌬
colonies showed little (I and L have a weak 1.3-kb band) mutants: they seemed to maintain a fraction of short
or no evidence for amplification and were, possibly, late telomeres, together with amplified telomeric TG
se-senescent colonies (i.e., colonies derived from presenes-quences (Figure 5c).
cent cells that have previously spent a long time in G1, Cells containing short Y⬘telomeres and extensive TG
before Start, due to the caloric restriction caused by a repeats are not commonly observed. We wondered if
large number of cells competing for nutrients on a this pattern represented the initial phase in the
genera-limited agar surface). tion of type II survivors. To test this we cloned 12
colo-nies from cells that had been passaged on agar plates for 8 days. At this point very few cells formed colonies.
DISCUSSION
Four of 6 tlc1⌬ colonies were survivors: 2 (C and E)
were type I, and 2 (A and F) were type II (Figure 5d). With respect to its activity at defective telomeres, EXO1encodes an exonuclease able to attack, degrade, Both of these early type II survivors still contained a
Exo1p generates single-stranded DNA in subtelomeric of a recombination process that allows only a “one in
a million” cell to amplify its telomeres or its subtelomeric regions (MaringeleandLydall2002), but this activity did not require MSH2 (L. Maringele, unpublished regions, to escape senescence and become immortal in
the absence of telomerase. data;M. Zubko, unpublished data). It is possible that
a mismatch repair complex between Exo1p and Msh2p We show that EXO1 plays three roles in cells that
enter senescence. First, the presence ofEXO1inmre11⌬ decreased the number of Exo1p proteins available to initiate homologous recombination. Thus, the positive yku70⌬ortlc1⌬cells appears to contribute to entry into
senescence. Second, the presence ofEXO1in the same effect of msh2⌬ deletion on telomere recombination would be indirect, by increasing the amount of (un-cells contributes to rapid escape from senescence by
generating type I and type II survivors. Thus, EXO1 bound) Exo1p.
In conclusion,EXO1is important for generating type accelerates entry into and exit from senescence. Third,
EXO1inhibits adaptation to senescence. All these effects I survivors inyku70⌬mre11⌬cells and type II survivors in tlc1⌬ cells. Moreover, EXO1 seems to contribute to of Exo1 can be explained by its 5⬘to 3⬘nuclease activity
that generates single-stranded DNA overhangs at telo- the senescence process itself, intlc1⌬cells. We speculate that in human cells also, interference with EXO1 or meres.
In yku70⌬ mre11⌬ cells, Exo1p is able to generate other nucleases active at uncapped telomeres might slow down both the entry into senescence and the speed long 3⬘overhangs in subtelomeric regions (Maringele
and Lydall 2002). The experiments we report here of generating recombination-dependent survivors. further suggest that the long overhangs generated by We thank A. Bertuch and V. Lundblad for sharing unpublished Exo1p might serve as the invading strands in a recombi- data, R. Blankley for atlc1⌬deletion strain, D. Gottschling for the
TLC1plasmids, and H. Tsubouchi and H. Ogawa for the Y⬘-TG
plas-nation process that involves Rad52p and Rad51p. As a
mid. The Wellcome Trust funded this work.
consequence, type I survivors are generated (Figure 6a). Although EXO1 is important for this process, it is not essential, because type I survivors are slowly produced
in exo1⌬cells, presumably due to redundant nuclease LITERATURE CITED
activities. Adams, A., D. E. Gottschling, C. A. KaiserandT. Stearns, 1997
Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press,
Intlc1⌬ cells,EXO1 is also important for generating
Cold Spring Harbor, NY.
type II survivors. Type II survivors might represent the
Boulton, S. J., andS. P. Jackson, 1998 Components of the
Ku-product of a “disrupted” recombination pathway that dependent non-homologous end-joining pathway are involved in
telomeric length maintenance and telomeric silencing. EMBO
had difficulty in progressing into the Y⬘regions, due to
J.17:1819–1828.
the protective presence/activity of both
Yku70p/Yku-Chamankhah, M., T. FontanieandW. Xiao, 2000 The Saccharo-80p andMRE11/RAD50/XRS2complexes, and that spe- myces cerevisiae mre11(ts)allele confers a separation of DNA repair
and telomere maintenance functions. Genetics155:569–576.
cializes in amplification of TG repeats. Exo1p activity is
Chen, Q., A. IjpmaandC. W. Greider, 2001 Two survivor pathways
presumably required to degrade the telomeric 5⬘strand
that allow growth in the absence of telomerase are generated
produced by a helicase. It is known that Sgs1 and Srs2 by distinct telomere recombination events. Mol. Cell. Biol.21:
1819–1827.
are required for BIR and/or generation of type II
survi-Cohen, H., and D. A. Sinclair, 2001 Recombination-mediated
vors (Cohen and Sinclair 2001; Huang et al. 2001;
lengthening of terminal telomeric repeats requires the Sgs1 DNA
Johnson et al.2001; Signonet al. 2001). Degradation helicase. Proc. Natl. Acad. Sci. USA98:3174–3179.
Datta, A., A. Adjiri, L. New, G. F. CrouseandS. Jinks Robertson,
of the 5⬘ strand enables the 3⬘strand to invade other
1996 Mitotic crossovers between diverged sequences are
regu-homologous and homeologous substrates. Subsequently,
lated by mismatch repair proteins in Saccharomyces cerevisiae.
type II survivors are generated (Figure 6b). Mol. Cell. Biol.16:1085–1093.
DuBois, M. L., Z. W. Haimberger, M. W. McIntosh and D. E. In previous studies, an antirecombination activity has
Gottschling, 2002 A quantitative assay for telomere
protec-been attributed to the mismatch repair proteins in
Esche-tion inSaccharomyces cerevisiae. Genetics161:995–1013.
richia coli(Petitet al.1991) andSaccharomyces cerevisiae Fiorentini, P., K. N. Huang, D. X. Tishkoff, R. D. Kolodnerand L. S. Symington, 1997 Exonuclease I of Saccharomyces
cerevis-(Datta et al. 1996). Rizki and Lundblad showed that
iae functions in mitotic recombination in vivo and in vitro. Mol.
defects in mismatch repair, caused by deletion ofMSH2,
Cell. Biol.17:2764–2773.
increased the survival rates of telomerase-defective cells. Grobelny, J. V., M. Kulp-McElieceandD. Broccoli, 2001 Effects
of reconstitution of telomerase activity on telomere maintenance
This was suggested to be due to increased telomeric
by the alternative lengthening of telomeres (ALT) pathway. Hum.
recombination inmsh2⌬strains (RizkiandLundblad
Mol. Genet.10:1953–1961.
2001;Lundblad2002). Exo1p co-immunoprecipitates Hakin-Smith, V., D. A. Jellinek, D. Levy, T. Carroll, M. Teo et al., 2003 Alternative lengthening of telomeres and survival in
with Msh2p and is considered a mismatch repair
exo-patients with glioblastoma multiforme. Lancet361:836–838.
nuclease (Tishkoff et al. 1997; Sokolsky and Alani
Henson, J. D., A. A. Neumann, T. R. Yeagerand R. R. Reddel,
2000). Interestingly, our experiments suggested that 2002 Alternative lengthening of telomeres in mammalian cells.
Oncogene21:598–610.
Huang, P., F. E. Pryde, D. Lester, R. L. Maddison, R. H. Bortset al., Ritchie, K. B., andT. D. Petes, 2000 The Mre11p/Rad50p/Xrs2p 2001 SGS1 is required for telomere elongation in the absence of complex and the Tel1p function in a single pathway for telomere telomerase. Curr. Biol.11:125–129. maintenance in yeast. Genetics155:475–479.
Johnson, F. B., R. A. Marciniak, M. McVey, S. A. Stewart, W. C. Rizki, A., andV. Lundblad, 2001 Defects in mismatch repair
pro-Hahnet al., 2001 The Saccharomyces cerevisiae WRN homolog mote telomerase-independent proliferation. Nature 411: 713– Sgs1p participates in telomere maintenance in cells lacking te- 716.
lomerase. EMBO J.20:905–913. Signon, L., A. Malkova, M. L. Naylor, H. KleinandJ. E. Haber,
Khazanehdari, K. A., andR. H. Borts, 2000 EXO1 and MSH4 2001 Genetic requirements for RAD51- and RAD54-indepen-differentially affect crossing-over and segregation. Chromosoma dent break-induced replication repair of a chromosomal
double-109:94–102. strand break. Mol. Cell. Biol.21:2048–2056.
Kirkpatrick, D. T., J. R. Ferguson, T. D. PetesandL. S. Symington, Sokolsky, T., andE. Alani, 2000 EXO1andMSH6are high-copy 2000 Decreased meiotic intergenic recombination and in- suppressors of conditional mutations in theMSH2mismatch re-creased meiosis I nondisjunction inexo1mutants ofSaccharomyces pair gene ofSaccharomyces cerevisiae. Genetics155:589–599. cerevisiae. Genetics156:1549–1557. Sugawara, N., G. IraandJ. E. Haber, 2000 DNA length
depen-Le, S., J. K. Moore, J. E. HaberandC. W. Greider, 1999 RAD50 dence of the single-strand annealing pathway and the role of and RAD51 define two pathways that collaborate to maintain Saccharomyces cerevisiae RAD59 in double-strand break repair. telomeres in the absence of telomerase. Genetics152:143–152. Mol. Cell. Biol.20:5300–5309.
Lee, S. E., J. K. Moore, A. Holmes, K. Umezu, R. D. Kolodneret al., Symington, L. S., L. E. KangandS. Moreau, 2000 Alteration of 1998 Saccharomyces Ku70, Mre11/Rad50, and RPA proteins gene conversion tract length and associated crossing over during regulate adaptation to G2/M arrest after DNA damage. Cell94: plasmid gap repair in nuclease-deficient strains of Saccharomyces
399–409. cerevisiae. Nucleic Acids Res.28:4649–4656.
Lundblad, V., 2002 Telomere maintenance without telomerase.
Teng, S. C., andV. A. Zakian, 1999 Telomere-telomere recombina-Oncogene21:522–531. tion is an efficient bypass pathway for telomere maintenance in
Lundblad, V., andE. H. Blackburn, 1993 An alternative pathway
Saccharomyces cerevisiae. Mol. Cell. Biol.19:8083–8093. for yeast telomere maintenance rescues est1-senescence. Cell73:
Tishkoff, D. X., A. L. Boerger, P. Bertrand, N. Filosi, G. M. Gaida
347–360.
et al., 1997 Identification and characterization of
Saccharo-Lydall, D., 2003 Hiding at the ends of yeast chromosomes:
telo-myces cerevisiae EXO1, a gene encoding an exonuclease that meres, nucleases and checkpoint pathways. J. Cell Sci.116:4057–
interacts with MSH2. Proc. Natl. Acad. Sci. USA94:7487–7492. 4065.
Toczyski, D. P., D. J. GalgoczyandL. H. Hartwell, 1997 CDC5
Maringele, L., and D. Lydall, 2002 EXO1-dependent
single-and CKII control adaptation to the yeast DNA damage check-stranded DNA at telomeres activates subsets of DNA damage and
point. Cell90:1097–1106. spindle checkpoint pathways in budding yeastyku70⌬mutants.
Tran, P. T., J. A. SimonandR. M. Liskay, 2001 Interactions of Genes Dev.16:1919–1933.
Exo1p with components of MutLalpha in Saccharomyces
cerevis-McEachern, M. J., andE. H. Blackburn, 1995 Runaway telomere
iae. Proc. Natl. Acad. Sci. USA98:9760–9765. elongation caused by telomerase RNA gene mutations. Nature
Tran, P. T., N. Erdenez, S. DudleyandR. M. Liskay, 2002
Charac-376:403–409.
terization of nuclease-dependent functions of Exo1p in
Saccharo-Moreau, S., E. A. MorganandL. S. Symington, 2001 Overlapping
myces cerevisiae. DNA Repair1:895–912. functions of theSaccharomyces cerevisiaeMre11, Exo1 and Rad27
Tsubouchi, H., andH. Ogawa, 2000 Exo1 roles for repair of DNA nucleases in DNA metabolism. Genetics159:1423–1433.
Petit, M. A., J. Dimpfl, M. RadmanandH. Echols, 1991 Control double-strand breaks and meiotic crossing over in Saccharomyces of large chromosomal duplications in Escherichia coli by the cerevisiae. Mol. Biol. Cell11:2221–2233.
mismatch repair system. Genetics129:327–332. Ulaner, G. A., H. Y. Huang, J. Otero, Z. Zhao, L. Ben-Poratet
Pryde, F. E., andE. J. Louis, 1997 Saccharomyces cerevisiae telo- al., 2003 Absence of a telomere maintenance mechanism as a meres. A review. Biochemistry62:1232–1241. favorable prognostic factor in patients with osteosarcoma. Cancer
Reddel, R. R., T. M. BryanandJ. P. Murnane, 1997 Immortalized Res.63:1759–1763. cells with no detectable telomerase activity. A review.
