EXO1 Plays a Role in Generating Type I and Type II Survivors in Budding Yeast

Corresponding author: David Lydall, University of Newcastle, Henry Wellcome Laboratory for Biogerontology Research, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE, United Kingdom., d.a.lydall{at}ncl.ac.uk (E-mail)

Communicating editor: A. NICOLAS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 homologous recombination for telomere maintenance, when telomerase cannot be activated. Although recombination 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 that EXO1 is important for generating type I survivors in yku70 mre11 cells and type II survivors in tlc1 cells. Moreover, in tlc1 cells, EXO1 seems to contribute to the senescence process itself.

BUDDING yeast cells, like almost all immortal eukaryotic cells, use telomerase to maintain the end of their chromosomes. Cells lacking telomerase components, for example, est1, est2, or tlc1 mutants, have been engineered and studied for their ability to escape replicative senescence (a state of cell cycle arrest caused by short and/or defective telomeres) and survive indefinite periods of time using a secondary mechanism of telomere maintenance. This mechanism appears to be based on break-induced replication (BIR), a variant of the main DSB-repair mechanism in budding yeast, homologous recombination, and is associated with amplification of telomeric or subtelomeric structures (LUNDBLAD and BLACKBURN 1993 ; MCEACHERN and BLACKBURN 1995 ; TENG and ZAKIAN 1999 ). One major difference between BIR and the telomerase-based way of maintenance is that recombination can copy and amplify only preexistent structures, while telomerase can synthesize a telomere de novo.

Long-term survival without evidence of telomerase activity was found in 15% of malignant tumors, where it was termed alternative lengthening of telomeres (ALT; GROBELNY et al. 2001 ; HENSON et al. 2002 ). Recent data report a higher incidence of ALT (25–66%) in some types of cancer like glioblastoma and osteosarcoma (HAKIN-SMITH et al. 2003 ; ULANER et al. 2003 ). Because the mechanism of ALT is practically indistinguishable from BIR (REDDEL et al. 1997 ), recombination at telomeres in budding yeast is considered a good model for understanding the telomerase-independent survival in mammalian cells.

It is clear that genes involved in recombination at double-strand breaks are also responsible for amplification of telomeres in the absence of telomerase (LE et al. 1999 ; SUGAWARA et al. 2000 ; CHEN et al. 2001 ). But, perhaps with the exception of RAD52, which is essential for recombination, all other recombination genes seem to be divided into two distinct pathways, depending on what kind of structure is amplified. One pathway is responsible for amplification of subtelomeric Y' regions and is dependent on RAD51, RAD54, RAD55, RAD57, and RAD52 (CHEN et al. 2001 ). In yeast, approximately two-thirds of the chromosomes have one to four homeologous subtelomeric Y' regions, adjacent to telomeres (PRYDE and LOUIS 1997 ). Cells that escape senescence by amplifying Y' regions are termed type I survivors (LUNDBLAD and BLACKBURN 1993 ; TENG and ZAKIAN 1999 ; CHEN et al. 2001 ). Another pathway involves the RAD50/MRE11/XRS2 complex together with RAD52, RAD59, SRS2, SGS1, and TID1 and drives the amplification of TG-telomeric repeats to generate type II survivors (CHEN et al. 2001 ; SIGNON et al. 2001 ).

There are subtle differences between the recombination-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 is a relatively rare event, with approximately only one in a million cells able to complete this process; in contrast, double-strand breaks (DSBs) are highly efficiently repaired.

Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little was known about the nucleases that generate the substrate for recombination.

EXO1 encodes a 5' to 3' exonuclease with FLAP endonuclease activity (TRAN et al. 2002 ). Exo1p is involved in resection of meiotic DSBs (TSUBOUCHI and OGAWA 2000 ), mitotic DSBs (FIORENTINI et al. 1997 ), and unprotected telomeres (MARINGELE and LYDALL 2002 ; LYDALL 2003 ). In addition, EXO1 affects meiotic recombination (KHAZANEHDARI and BORTS 2000 ; KIRKPATRICK et al. 2000 ; SYMINGTON et al. 2000 ) and is part of a complex with mismatch repair proteins (TISHKOFF et al. 1997 ; SOKOLSKY and ALANI 2000 ; TRAN et al. 2001 ).

Here we present evidence that EXO1 is important for generating recombination-dependent, type I and II survivors, in two different types of senescent mutants: telomere capping defective (yku70 mre11) and telomerase-negative (tlc1) cells. We show that deletion of EXO1 delays the appearance of survivors for 30–40 generations. Therefore, EXO1 appears to be one of the "initiators" of the recombination process that allow rare cells to escape senescence and became immortal in the absence of telomerase.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains:
All strains used in this study are isogenic, in the W303 background, and RAD5⁺. To construct strains, standard genetic procedures of transformation and tetrad analysis were followed (ADAMS et al. 1997 ). Since W303 strains contain an ade2-1 mutation, YPD (yeast extract, peptone, and dextrose) medium was routinely supplemented with adenine at 50 mg/liter. All yku70 mre11 exo1 strains and their controls were made by crossing DLY1331 (mre11::hisG::URA3) with DLY1408 (yku70::HIS3 exo1::LEU2). All tlcl exo1 strains were made by crossing DLY1628 [tlc1::HIS3 pTLC1:URA3 (pSD120 from D. Gottschling)] with DLY1948 (exo1::LEU2 rad52::TRP1). Diploid cells that had lost the pTLC1 plasmid were sporulated, dissected, and germinated.

Growth rate assay:
Cells of the appropriate genotype (four independent strains for each genotype of interest) were isolated from a fresh germination plate and grown in YPD medium overnight. The following morning, cells were counted using a hemocytometer, diluted to 1 x 10⁵ cells/ml, and then incubated at 23°, with aeration. Every 23 hr, cell densities were measured and then the culture was diluted with fresh YPD liquid medium to a density of 10⁵ cells/ml. During counting, cells were put on ice. This cycle was repeated for several days. At indicated time points, samples were collected for telomere length analysis.

Streak assay:
Cells of the appropriate genotype were picked from a fresh germination plate and streaked onto YPD plates. After incubation for 4 days at 23°, similar amounts of cells (1 x 10⁷ cells) from several independent colonies were picked and restreaked on YPD plates. This restreaking was repeated several times to allow senescence and appearance of survivors.

Microcolony assays:
Colony-purified yeast strains were inoculated into 1 ml YPD and grown overnight with aeration at 23° until they reached a concentration of 8 x 10⁶ cells/ml. Cells were sonicated briefly and spread on plates. The plates were incubated at 23°. After an appropriate length of time, the colonies were photographed and the cell numbers in 100 colonies were counted/estimated.

Telomere and subtelomere Southern blots:
Southern blot analyses were performed to examine telomere and Y' lengths. Each time, cells from three or four independent mutants with the same genotype were pooled prior to DNA extraction, except for Southern blot analysis of single colonies. Yeast genomic DNA was isolated and 20 ng of DNA was digested with XhoI and separated on a 0.8% agarose gel. DNA was then transferred to a Magna Nylon membrane (Genetic Research Instrumentation) and UV crosslinked. The membrane was then hybridized with a Y'-TG probe (pHT128; TSUBOUCHI and OGAWA 2000 ) and hybridization was detected using a nonradioactive detection kit (Amersham, Arlington Heights, IL).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

EXO1 is important for generating survivors in yku70 mre11 senescent cells:
Initiation of (sub)telomeric amplification in the absence of telomerase is a rare event. Factors involved in the initiation of survivors were still unknown, but we speculated that EXO1 might be one, because of its role in degradation of unprotected telomeres (MARINGELE and LYDALL 2002 ). Our previous experiments have demonstrated that defective telomeres in cdc13-1 and yku70 mutants were targeted by the Exo1p exonuclease. EXO1 was important for resecting the 5' AC strand, from telomeres toward centromeres, leaving behind a detectable 3' overhang, up to 1 kb long in yku70 strains (MARINGELE and LYDALL 2002 ). One consequence of Exo1p activity at defective telomeres was arrest of the cell cycle before anaphase (MARINGELE and LYDALL 2002 ). The ability to degrade unprotected telomeres in vivo is not a common feature of all exonucleases (M. ZUBKO, unpublished data) suggesting that Exo1p has a special function at telomeres.

We, therefore, tested the role of EXO1 in senescence and survival of cells with telomere defects caused by the absence of telomere protective genes YKU70 and MRE11. This model of senescence and survival is based on the observation that yku70 mre11 double mutants senesce very rapidly, during the early growth of germinated spores (RITCHIE and PETES 2000 ; DUBOIS et al. 2002 ; MARINGELE and LYDALL 2002 ).

We monitored growth of yku70 mre11 double mutants and yku70 mre11 exo1 triple mutants. Cells were taken directly from the germination plate (four independent strains of each genotype) and incubated in liquid at 23° for several days. At 23°, wild-type strains would divide 10 times a day. Cell densities were scored every 23 hr, followed by dilution to 10⁵ cells/ml and continued incubation. At 23°, yku70 and yku70 exo1 cells reached and maintained densities of 0.5 x 10⁸ cells/ml, similar to the wild type, while other control mutants, mre11 and mre11 exo1, reached densities 5-fold lower than those of the wild type (Fig 1A). In contrast, yku70 mre11 and yku70 mre11 exo1 cells were in crisis during the early days of the experiment, when they had densities 200-fold lower than those of the wild type, consistent with early entry into senescence (Fig 1A).

View larger version (66K): In this window In a new window Download PPT slide   Figure 1. EXO1 is 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 every 23 hr, followed by dilution to 1 x 105 cells/ml and continued incubation. Each symbol represents 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 germination plate were spread on fresh YPD plates (passage 1), incubated 4 days at 23°, and then passaged to other plates (passage 2). Several passages were consecutively performed.

Both types of senescent mutants, yku70 mre11 (EXO1⁺) and yku70 mre11 exo1, were able to generate survivors, but with different dynamics. While EXO1⁺ survivors escaped senescence early (days 4–5) and grew, after 2 days, at growth rates (determined by cell density after 23 hr growth) similar to those of mre11 mutants, exo1 survivors appeared more slowly and gradually reached, after 15 days in liquid culture, a 5-fold lower cell density, compared with mre11 cells (Fig 1A).

The data above suggest that EXO1 plays a significant, but not essential, role in generating survivors. Another possibility would be that EXO1 is important for cell vitality in an mre11 background, even in the absence of senescence. When growth rates of mre11 exo1 cells are compared with those of mre11 mutants, it appears that exo1 deletion did indeed cause a kind of crisis during the first 4 days, when mre11 exo1 mutants daily reached only 25% of the cell density of mre11 strains (Fig 1A; MOREAU et al. 2001 ). However, growth of mre11 exo1 strains improved during the following generations, oscillating between 55 and 98% of the mre11 growth. Thus, the requirement for EXO1 was stronger in yku70 mre11 survivors than in mre11 mutants. Interestingly, deletion of EXO1 in yku70 cells shows an effect opposite to that in mre11 cells, because exo1 mutation improves the growth rate of yku70 mutants 15% at 23° (Fig 1A). At 37°, an exo1 even rescues the inviability of yku70 mutants (MARINGELE and LYDALL 2002 ).

The requirement for EXO1 in recovery from senescence was also tested by growth on plates, when comparable amounts of cells from germination plates were spread on fresh YPD plates (passage 1) and incubated at 23° for 4 days (Fig 1B). Several passages were performed consecutively. This experiment confirmed that early generations of yku70 mre11 (EXO1⁺) and yku70 mre11 exo1 mutants were in a state of minimal growth, presumably senescent (passage 1), and that EXO1⁺ strains generated survivors more rapidly (passage 2) compared with exo1 strains (passage 3). Also, EXO1⁺ survivors grew notably better than exo1 survivors (passage 3), and a difference in growth was maintained even after 15 passages, which is equivalent to 600 wild-type generations (Fig 1D, compare size of individual colonies; data not shown).

EXO1 is required for maintenance of survivors:
The continued growth defect in yku70 mre11 exo1 could have many possible explanations, such as slow metabolic rate, longer cell cycle, decreased viability, high rate of resenescence, and/or delay in reappearance of survivors. It is known that in telomerase-defective (est1) survivors, the senescence phenotype reappears in some subclones (LUNDBLAD and BLACKBURN 1993 ). We monitored the ability of "established" survivors (with a common history of 32 days in culture, therefore longer than 200 generations) to form colonies by microcolony assay, spreading similar amounts of cells on plates after a short sonication and then incubating them for 24 hr at 23° (Fig 2).

View larger version (152K): In this window In a new window Download PPT slide   Figure 2. EXO1 is required for the maintenance of survivors. 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.

Although a number of nondividing cells (single-cell "colonies") or cells with a large bud (two-cell colonies) were microscopically detected after 24 hr in all mutants with a mre11 background, the percentage of these one- or two-cell colonies in yku70 mre11 survivors was similar to that in mre11 or mre11 exo1 control strains and did not exceed 15% under normal conditions (Fig 2; data not shown). By contrast, yku70 mre11 exo1 survivors had a high fraction of one- or two-cell colonies (35%) after 24 hr, suggesting they were confronted with high levels of resenescence and/or difficulties in regeneration of survivors, which can explain the lower cell density reached by these mutants in liquid culture. Also, it is clear that some yku70 mre11 exo1 colonies were similar in size to yku70 mre11 EXO1⁺ colonies, and therefore a difference in growth caused by a slow metabolic rate or delayed cell cycle can be excluded (Fig 2).

EXO1 increases the rate of Y' amplification in yku70 mre11 survivors (type I survivors):
Some cells can escape senescence and generate survivors. In yeast, presumably also in mammalian cells, this occurs by correction of the telomeric defect. Repair proteins that belong to the recombination pathways are able to amplify subtelomeric regions (in yeast, Y' regions), generating type I survivors, or amplify terminal telomeric TG repeats, generating type II survivors. It is known that Rad50p, which acts in a complex with Mre11p, is required to maintain type II survivors, because in a rad50 background, type I survivors replace type II survivors very early (CHEN et al. 2001 ). Since Mre11p and Rad50p function together, type I survivors would be expected to predominate in an mre11 background as well.

Also, the fact that exo1 survivors appear late and maintain a growth defect, compared with the EXO1⁺ strains, raised the possibility that exo1 survivors were using a different survival mechanism. To address the mechanism, we performed Southern blots to detect Y' or TG amplifications (Fig 3). Cells were collected every 3 days and DNA was cut with XhoI and probed with a Y'-TG probe; that gave three fragments, 6.5, 5.5, and 1.3 kb in wild-type cells. The two larger fragments correspond to repetitive Y''s, while the shortest fragment is the terminal 1 kb of Y' and also contains 350 bp of telomeric TG repeats (Fig 3A).

View larger version (72K): In this window In a new window Download PPT slide   Figure 3. EXO1 increases the rate of Y' amplification in yku70 mre11 survivors (type I survivors). (a) Schematic representation of the end of a chromosome in budding yeast. The probe used binds to the telomeric end of the Y' repeat (1 kb) and to 120 bp from the telomeric TG sequences. (b) Y' amplification in different mutants grown in liquid culture and monitored by Southern blot. Cells were grown in liquid and collected on the day indicated above each lane of the gel. DNA was cut with XhoI and hybridized with the Y'-TG probe. The DNA loading control represents 10 ng of uncut DNA stained with ethidium bromide.

In mre11 control strains, the telomeric fragment was shorter than that in wild type (Fig 3B), consistent with previous data (BOULTON and JACKSON 1998 ; CHAMANKHAH et al. 2000 ). Deletion of EXO1 from the control mre11 strains, maintained by telomerase, apparently does not influence the length or intensity of (sub)-telomeric fragments, consistent with earlier observations (Fig 3B; TSUBOUCHI and OGAWA 2000 ; MOREAU et al. 2001 ). We observed that growth of mre11 exo1 strains improved after several days in liquid culture (Fig 1A), but this growth improvement did not correlate with any notable change at the telomeres of these strains (Fig 3B).

However, in senescent yku70 mre11 cells, the effect of exo1 was obvious. During the first 3 days in culture, yku70 mre11 cells were senescent and the larger fragments corresponding to Y' looked similar to wild type, while the short fragment appeared less intense, presumably due to loss of telomeric sequences in many of these cells (Fig 3B). But after 6 days, yku70 mre11 cells had already amplified the Y' repeats (type I survivors), and this amplification apparently remained constant afterward (days 6–12; Fig 3B). Deletion of EXO1 from yku70 mre11 cells considerably slowed the rate of Y' amplification. Amplification occurred slowly, but progressively during 3–12 days in culture; however, even after 12 days, the Y' amplification was less pronounced in yku70 mre11 exo1 cells than in yku70 mre11 cells after 6 days in culture (Fig 3B). Therefore, cells lacking EXO1 had difficulty in generating type I survivors. The difference in dynamics of Y' amplification, in exo1 vs. EXO1⁺ survivors, is not a consequence of poor cell growth, because similar amounts of DNA were analyzed in Southern blot assays (see DNA loading control, Fig 3B) and, initially, Y' amplifications occur during senescence. Rather, the growth defect might be explained by difficulties and delays in amplification of Y' regions in yku70 mre11 exo1 strains due to the absence of EXO1.

YKU70 inhibits the maintenance of type I survivors:
We have shown that Exo1 degrades telomeres in yku70 mutants (MARINGELE and LYDALL 2002 ). To see if Exo1 plays a role in generating type I survivors in the presence of YKU70, we analyzed tlc1 mre11 strains. These strains senesce due to the absence of the telomerase RNA template, Tlc1, while deletion of Mre11 ensures that only type I survivors can be generated. We monitored senescence and survival in experiments similar to those described in Fig 1. In this experiment, an exo1 decreased the density of tlc1 mre11 cells before and after senescence (Fig 4A). This is partially due to the synergic effect of exo1 and mre11 deletions in decreasing the cell viability, as previously mentioned. A second phenomenon was noted: during the early postsenescence period, tlc1 mre11 survivors grew poorly compared with yku70 mre11 survivors and did not reach the growth levels of mre11 single mutants, as yku70 mre11 survivors did (Fig 4A vs. Fig 1A). Also, the Y' amplification in tlc1 mre11 survivors was inferior to the Y' amplification in yku70 mre11 survivors (Fig 4B vs. Fig 3B). The data suggest that the presence of Yku70 makes the maintenance of type I survivors difficult and might explain why tlc1 cells preferentially maintain type II survivors.

View larger version (48K): In this window In a new window Download PPT slide   Figure 4. EXO1 opposes adaptation during senescence. (a) Strains as indicated in the legend were grown as described in Fig 1A. (b) Y' amplification was monitored as in Fig 3B.

EXO1 opposes adaptation and plays a role in generating type I survivors in tlc1 mre11 cells:
We next addressed the question of whether EXO1 plays a role in generating type I survivors, in the presence of YKU70, in tlc1 mre11 mutants. Fig 4A shows that tlc1 mre11 exo1 survivors grew about fivefold less than tlc1 mre11 survivors, during the early days of the postsenescence period (days 9–12). This may be due to the synthetic effect of mre11 and exo1 in decreasing the cell viability. However, was the escape from senescence and early postsenescence growth of tlc1 mre11 exo1 strains due to recombination events that generated type I survivors? To test this, we deleted RAD52, which is essential for recombination. It is clear from Fig 4A that RAD52-recombination defective, tlc1 mre11 exo1 rad52 strains also escape senescence and their postsenescent growth curve almost overlaps with the growth curve of tlc1 mre11 exo1 mutants, during the first 17 days in culture. By contrast, tlc1 mre11 rad52 cells did not escape senescence. Thus, tlc1 mre11 exo1 rad52 cells appeared to be adapting to the telomere defect, rather than using recombination to overcome senescence.

Thus, early postsenescence growth of tlc1 mre11 exo1 cells was RAD52 independent, while growth of tlc1 mre11 cells during the same period was strictly RAD52 dependent. Later on, RAD52⁺ strains amplified the Y' repeats, tlc1 mre11 exo1 mutants to a lesser extent than tlc1 mre11 mutants (Fig 4B), while rad52 survivors showed no amplification of subtelomeric regions (data not shown). Adaptation is defined as escape from the cell cycle arrest without repair of the DNA damage (TOCZYSKI et al. 1997 ; LEE et al. 1998 ). In this case it means to escape senescence with short telomeric regions.

Our interpretation is that EXO1 is, indeed, required to generate type I survivors in tlc1 mre11 mutants, but also to suppress the adaptation to telomeric damage and the recombination-independent escape from senescence.

EXO1 plays a role in generating type II survivors in tlc1 cells:
If EXO1 plays a role in the amplification of subtelomeric repeats, presumably by generating long 3' overhangs that would invade hom(e)ologous structures, what about its role in type II survivors? Type II survivors amplify the TG-telomeric repeats, with help from a nuclease/helicase complex (Rad50p/Mre11p/Xrs2p), a helicase (Srs2p or Sgs1p), and recombination proteins (Rad52p and Rad59p; CHEN et al. 2001 ). We wondered if there was any requirement for Exo1p in generating type II survivors.

In experiments similar to those described in Fig 1, we monitored the senescence and survival of tlc1 exo1 cells vs. tlc1 cells (Fig 5). The strains were germinated from spores that had inherited the normal telomere length from their parents. During the presenescent period, tlc1 exo1 strains grew 1.5-fold better than tlc1 EXO1⁺ single mutants each day (Fig 5A; note the lack of overlap of error bars on days 3 and 4). This suggests that in the absence of telomerase, EXO1 contributes to telomere shortening and senescence. However, this phenomenon was not observed in strains that lack MRE11 (Fig 4A). It is possible that the synthetic effect of mre11 and exo1 mutations in decreasing cell viability (presumably for reasons other than telomere damage, because mre11 exo1 and mre11 cells have similarly short telomeres; Fig 3B) counteracts the effect of exo1 in presenescent mre11 cells.

View larger version (88K): In this window In a new window Download PPT slide   Figure 5. EXO1 is important for generating type II survivors in tlc1 cells. (a) Strains as indicated in the legend were grown as described in Fig 1A. (b) Telomeric length was monitored by Southern blot as in Fig 3B. On day 0 cells were taken directly from germination plates and grown for 4 hr in liquid culture, and DNA was prepared. (c) Y' and TG amplification was monitored as in Fig 3B. (d) Germinated spores were grown and mass passaged daily on plates for 6 days. On the sixth day the majority of cells had entered senescence. By day 8 we were able to pick 12 colonies (A–L) that were still growing. These colonies were grown overnight in liquid culture before DNA was prepared. Y' and TG amplification was monitored as in Fig 3B.

As telomeres progressively shortened, cells became senescent. The vast majority of cells stopped cell division after 6 days in liquid culture, when tlc1 and tlc1 exo1 cells had 20- to 25-fold lower densities, compared with their growth rate from day 1 (Fig 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 of tlc1 or exo1 cells (Fig 5A). This indicates that EXO1 plays a role in generating survivors in a tlc1 background.

Since telomerase-defective tlc1 cells entered senescence more slowly than yku70 mre11 cells (compare Fig 1A and Fig 5A) we were able to monitor the effect of EXO1 on telomere degradation as cells entered senescence. We purified DNA from cells as early as possible after they had germinated and every day thereafter. The effect of EXO1 on telomere shortening in presenescent cells was clear. In EXO1⁺ 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 of 10⁸ cells). After this, during days 1–7, telomere shortening appeared to stop. In contrast, tlc1 exo1 cells contained significantly longer telomeres at day 0 that slowly declined over the next 6 days, to reach a length similar to that in tlc1 cells by day 7 (Fig 5B).

To understand the role of Exo1p during recovery from senescence, Southern blots were used to analyze the dynamics of telomere amplification in tlc1 exo1 double mutants vs. tlc1 single mutants (Fig 5C). After 6 days in culture, both types of mutants had short telomeres. After 8 days in culture, tlc1 cells had amplified the telomeric TG sequences, generating type II survivors, while the short telomeres from tlc1 exo1 cells looked unchanged (Fig 5C). After 10 days, tlc1 exo1 cells showed little evidence of telomere amplification, and also their Y' repeats were indistinguishable from wild-type Y' repeats (Fig 5C). The tlc1 exo1 cells significantly amplified their telomeres after 12 days in culture. The pattern of telomere amplification in tlc1 exo1 survivors was also different from that in tlc1 mutants: they seemed to maintain a fraction of short telomeres, together with amplified telomeric TG sequences (Fig 5C).

Cells containing short Y' telomeres and extensive TG repeats are not commonly observed. We wondered if this pattern represented the initial phase in the generation of type II survivors. To test this we cloned 12 colonies from cells that had been passaged on agar plates for 8 days. At this point very few cells formed colonies. Four of 6 tlc1 colonies were survivors: 2 (C and E) were type I, and 2 (A and F) were type II (Fig 5D). Both of these early type II survivors still contained a lower terminal Y' fragment, similar to the tlc1 exo1 strains at day 12 (Fig 5C). When we examined DNA from the tlc1 exo1 cells, only 1 colony (G) had generated a type I survivor at this point, while the other colonies showed little (I and L have a weak 1.3-kb band) or no evidence for amplification and were, possibly, late senescent colonies (i.e., colonies derived from presenescent cells that have previously spent a long time in G₁, before Start, due to the caloric restriction caused by a large number of cells competing for nutrients on a limited agar surface).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

With respect to its activity at defective telomeres, EXO1 encodes an exonuclease able to attack, degrade, and alert checkpoint pathways in yku70 and cdc13-1 mutants (MARINGELE and LYDALL 2002 ). Here we have shown that Exo1p activity at telomeres has consequences beyond damage generation and cell cycle arrest. Our experiments suggest that Exo1p is one of the initiators of a recombination process that allows only a "one in a million" cell to amplify its telomeres or its subtelomeric regions, to escape senescence and become immortal in the absence of telomerase.

We show that EXO1 plays three roles in cells that enter senescence. First, the presence of EXO1 in mre11 yku70 or tlc1 cells appears to contribute to entry into senescence. Second, the presence of EXO1 in the same cells contributes to rapid escape from senescence by generating type I and type II survivors. Thus, EXO1 accelerates entry into and exit from senescence. Third, EXO1 inhibits adaptation to senescence. All these effects of Exo1 can be explained by its 5' to 3' nuclease activity that generates single-stranded DNA overhangs at telomeres.

In yku70 mre11 cells, Exo1p is able to generate long 3' overhangs in subtelomeric regions (MARINGELE and LYDALL 2002 ). The experiments we report here further suggest that the long overhangs generated by Exo1p might serve as the invading strands in a recombination process that involves Rad52p and Rad51p. As a consequence, type I survivors are generated (Fig 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 activities.

View larger version (36K): In this window In a new window Download PPT slide   Figure 6. A model for EXO1 in generating type I and II survivors. (a) Exo1p initiates recombination in Y' subtelomeric regions, processing the ends of the chromosomes in senescent cells. It degrades the 5' strand, generating long 3' overhangs, which invade homologous regions from other chromosomes. Subsequently, type I survivors are generated. (b) Type II survivors might represent the product of a disrupted recombination pathway that had difficulty in progressing into the Y', due to the protective presence/activity of both Yku70p/Yku80p and MRE11/RAD50/XRS2 complexes and that specializes in amplification of TG repeats. Exo1p activity is presumably required to degrade the telomeric 5' strand produced by a helicase (Srs2/Sgs1). Degradation of the 5' strand allows the 3' strand to invade other homologous substrates. Subsequently, type II survivors are generated.

In tlc1 cells, EXO1 is also important for generating type II survivors. Type II survivors might represent the product of a "disrupted" recombination pathway that had difficulty in progressing into the Y' regions, due to the protective presence/activity of both Yku70p/Yku80p and MRE11/RAD50/XRS2 complexes, and that specializes in amplification of TG repeats. Exo1p activity is presumably required to degrade the telomeric 5' strand produced by a helicase. It is known that Sgs1 and Srs2 are required for BIR and/or generation of type II survivors (COHEN and SINCLAIR 2001 ; HUANG et al. 2001 ; JOHNSON et al. 2001 ; SIGNON et al. 2001 ). Degradation of the 5' strand enables the 3' strand to invade other homologous and homeologous substrates. Subsequently, type II survivors are generated (Fig 6B).

In previous studies, an antirecombination activity has been attributed to the mismatch repair proteins in Escherichia coli (PETIT et al. 1991 ) and Saccharomyces cerevisiae (DATTA et al. 1996 ). Rizki and Lundblad showed that defects in mismatch repair, caused by deletion of MSH2, increased the survival rates of telomerase-defective cells. This was suggested to be due to increased telomeric recombination in msh2 strains (RIZKI and LUNDBLAD 2001 ; LUNDBLAD 2002 ). Exo1p co-immunoprecipitates with Msh2p and is considered a mismatch repair exonuclease (TISHKOFF et al. 1997 ; SOKOLSKY and ALANI 2000 ). Interestingly, our experiments suggested that EXO1 promoted subtelomeric recombination, rather than opposed it. How is it possible that two interacting mismatch repair proteins have opposite effects toward recombination? Our previous studies demonstrated that Exo1p generates single-stranded DNA in subtelomeric regions (MARINGELE and LYDALL 2002 ), but this activity did not require MSH2 (L. MARINGELE, unpublished data; M. ZUBKO, unpublished data). It is possible that a mismatch repair complex between Exo1p and Msh2p decreased the number of Exo1p proteins available to initiate homologous recombination. Thus, the positive effect of msh2 deletion on telomere recombination would be indirect, by increasing the amount of (unbound) Exo1p.

In conclusion, EXO1 is important for generating type I survivors in yku70 mre11 cells and type II survivors in tlc1 cells. Moreover, EXO1 seems to contribute to the senescence process itself, in tlc1 cells. We speculate that in human cells also, interference with EXO1 or other nucleases active at uncapped telomeres might slow down both the entry into senescence and the speed of generating recombination-dependent survivors.

	FOOTNOTES

¹ Present address: School of Clinical Medical Sciences-Gerontology, University of Newcastle, Henry Wellcome Laboratory for Biogerontology Research, Newcastle General Hospital, Newcastle upon Tyne, NE4 6BE, United Kingdom.

	ACKNOWLEDGMENTS

We thank A. Bertuch and V. Lundblad for sharing unpublished data, R. Blankley for a tlc1 deletion strain, D. Gottschling for the TLC1 plasmids, and H. Tsubouchi and H. Ogawa for the Y'-TG plasmid. The Wellcome Trust funded this work.

Manuscript received June 26, 2003; Accepted for publication November 24, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ADAMS, A., D. E. GOTTSCHLING, C. A. KAISER and T. STEARNS, 1997 Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BOULTON, S. J. and S. P. JACKSON, 1998  Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 17:1819-1828.[CrossRef][Medline]

CHAMANKHAH, M., T. FONTANIE, and W. XIAO, 2000  The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions. Genetics 155:569-576.[Abstract/Free Full Text]

CHEN, Q., A. IJPMA, and C. W. GREIDER, 2001  Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol. Cell. Biol. 21:1819-1827.[Abstract/Free Full Text]

COHEN, H. and D. A. SINCLAIR, 2001  Recombination-mediated lengthening of terminal telomeric repeats requires the Sgs1 DNA helicase. Proc. Natl. Acad. Sci. USA 98:3174-3179.[Abstract/Free Full Text]

DATTA, A., A. ADJIRI, L. NEW, G. F. CROUSE, and S. JINKS ROBERTSON, 1996  Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:1085-1093.[Abstract]

DUBOIS, M. L., Z. W. HAIMBERGER, M. W. MCINTOSH, and D. E. GOTTSCHLING, 2002  A quantitative assay for telomere protection in Saccharomyces cerevisiae. Genetics 161:995-1013.[Abstract/Free Full Text]

FIORENTINI, P., K. N. HUANG, D. X. TISHKOFF, R. D. KOLODNER, and L. S. SYMINGTON, 1997  Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro. Mol. Cell. Biol. 17:2764-2773.[Abstract]

GROBELNY, J. V., M. KULP-MCELIECE, and D. BROCCOLI, 2001  Effects of reconstitution of telomerase activity on telomere maintenance by the alternative lengthening of telomeres (ALT) pathway. Hum. Mol. Genet. 10:1953-1961.[Abstract/Free Full Text]

HAKIN-SMITH, V., D. A. JELLINEK, D. LEVY, T. CARROLL, and M. TEO et al., 2003  Alternative lengthening of telomeres and survival in patients with glioblastoma multiforme. Lancet 361:836-838.[CrossRef][Medline]

HENSON, J. D., A. A. NEUMANN, T. R. YEAGER, and R. R. REDDEL, 2002  Alternative lengthening of telomeres in mammalian cells. Oncogene 21:598-610.[CrossRef][Medline]

HUANG, P., F. E. PRYDE, D. LESTER, R. L. MADDISON, and R. H. BORTS et al., 2001  SGS1 is required for telomere elongation in the absence of telomerase. Curr. Biol. 11:125-129.[CrossRef][Medline]

JOHNSON, F. B., R. A. MARCINIAK, M. MCVEY, S. A. STEWART, and W. C. HAHN et al., 2001  The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase. EMBO J. 20:905-913.[CrossRef][Medline]

KHAZANEHDARI, K. A. and R. H. BORTS, 2000  EXO1 and MSH4 differentially affect crossing-over and segregation. Chromosoma 109:94-102.[CrossRef][Medline]

KIRKPATRICK, D. T., J. R. FERGUSON, T. D. PETES, and L. S. SYMINGTON, 2000  Decreased meiotic intergenic recombination and increased meiosis I nondisjunction in exo1 mutants of Saccharomyces cerevisiae. Genetics 156:1549-1557.[Abstract/Free Full Text]

LE, S., J. K. MOORE, J. E. HABER, and C. W. GREIDER, 1999  RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase. Genetics 152:143-152.[Abstract/Free Full Text]

LEE, S. E., J. K. MOORE, A. HOLMES, K. UMEZU, and R. D. KOLODNER et al., 1998  Saccharomyces Ku70, Mre11/Rad50, and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94:399-409.[CrossRef][Medline]

LUNDBLAD, V., 2002  Telomere maintenance without telomerase. Oncogene 21:522-531.[CrossRef][Medline]

LUNDBLAD, V. and E. H. BLACKBURN, 1993  An alternative pathway for yeast telomere maintenance rescues est1-senescence. Cell 73:347-360.[CrossRef][Medline]

LYDALL, D., 2003  Hiding at the ends of yeast chromosomes: telomeres, nucleases and checkpoint pathways. J. Cell Sci. 116:4057-4065.[Abstract/Free Full Text]

MARINGELE, L. and D. LYDALL, 2002  EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70 mutants. Genes Dev. 16:1919-1933.[Abstract/Free Full Text]

MCEACHERN, M. J. and E. H. BLACKBURN, 1995  Runaway telomere elongation caused by telomerase RNA gene mutations. Nature 376:403-409.[CrossRef][Medline]

MOREAU, S., E. A. MORGAN, and L. S. SYMINGTON, 2001  Overlapping functions of the Saccharomyces cerevisiae Mre11, Exo1 and Rad27 nucleases in DNA metabolism. Genetics 159:1423-1433.[Abstract/Free Full Text]

PETIT, M. A., J. DIMPFL, M. RADMAN, and H. ECHOLS, 1991  Control of large chromosomal duplications in Escherichia coli by the mismatch repair system. Genetics 129:327-332.[Abstract]

PRYDE, F. E. and E. J. LOUIS, 1997  Saccharomyces cerevisiae telomeres. A review. Biochemistry 62:1232-1241.[Medline]

REDDEL, R. R., T. M. BRYAN, and J. P. MURNANE, 1997  Immortalized cells with no detectable telomerase activity. A review. Biochemistry 62:1254-1262.[Medline]

RITCHIE, K. B. and T. D. PETES, 2000  The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast. Genetics 155:475-479.[Abstract/Free Full Text]

RIZKI, A. and V. LUNDBLAD, 2001  Defects in mismatch repair promote telomerase-independent proliferation. Nature 411:713-716.[CrossRef][Medline]

SIGNON, L., A. MALKOVA, M. L. NAYLOR, H. KLEIN, and J. E. HABER, 2001  Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break. Mol. Cell. Biol. 21:2048-2056.[Abstract/Free Full Text]

SOKOLSKY, T. and E. ALANI, 2000  EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae. Genetics 155:589-599.[Abstract/Free Full Text]

SUGAWARA, N., G. IRA, and J. E. HABER, 2000  DNA length dependence of the single-strand annealing pathway and the role of Saccharomyces cerevisiae RAD59 in double-strand break repair. Mol. Cell. Biol. 20:5300-5309.[Abstract/Free Full Text]

SYMINGTON, L. S., L. E. KANG, and S. MOREAU, 2000  Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae. Nucleic Acids Res. 28:4649-4656.[Abstract/Free Full Text]

TENG, S. C. and V. A. ZAKIAN, 1999  Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol. Cell. Biol. 19:8083-8093.[Abstract/Free Full Text]

TISHKOFF, D. X., A. L. BOERGER, P. BERTRAND, N. FILOSI, and G. M. GAIDA et al., 1997  Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2. Proc. Natl. Acad. Sci. USA 94:7487-7492.[Abstract/Free Full Text]

TOCZYSKI, D. P., D. J. GALGOCZY, and L. H. HARTWELL, 1997  CDC5 and CKII control adaptation to the yeast DNA damage checkpoint. Cell 90:1097-1106.[CrossRef][Medline]

TRAN, P. T., J. A. SIMON, and R. M. LISKAY, 2001  Interactions of Exo1p with components of MutLalpha in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 98:9760-9765.[Abstract/Free Full Text]

TRAN, P. T., N. ERDENEZ, S. DUDLEY, and R. M. LISKAY, 2002  Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. DNA Repair 1:895-912.[CrossRef][Medline]

TSUBOUCHI, H. and H. OGAWA, 2000  Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae. Mol. Biol. Cell 11:2221-2233.[Abstract/Free Full Text]

ULANER, G. A., H. Y. HUANG, J. OTERO, Z. ZHAO, and L. BEN-PORAT et al., 2003  Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma. Cancer Res. 63:1759-1763.[Abstract/Free Full Text]

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