EXO1
Contributes to Telomere Maintenance in Both Telomerase-Proficient and
Telomerase-Deficient
Saccharomyces cerevisiae
Alison A. Bertuch*
,†,1and Victoria Lundblad*
*Department of Molecular and Human Genetics and†Department of Pediatrics, Hematology/Oncology Section,
Baylor College of Medicine, Houston, Texas 77030 Manuscript received August 4, 2003 Accepted for publication November 28, 2003
ABSTRACT
Previous work in budding yeast has indicated that telomeres are protected, at least in part, from the action of Exo1, which degrades the C-rich strand of partially uncapped telomeres. To explore this further, we examined the consequences of Exo1-mediated activity in strains that lacked Ku, telomerase, or both. Loss of Exo1 partially rescued the telomere length defect in ayku80⌬strain, demonstrating that exonuclease action can directly contribute to telomere shortening. The rapid loss of inviability displayed by ayku80⌬ est2⌬strain was also partially alleviated by anexo1⌬mutation, further supporting the proposal that Exo1 is one target of the activities that normally protect wild-type telomeres. Conversely, however, Exo1 activity was also capable of enhancing telomere function and consequently cell proliferation, by contributing to a telomerase-independent pathway for telomere maintenance. The recovery of recombination-dependent survivors that arose in ayku80⌬est2⌬strain was partially dependent on Exo1 activity. Furthermore, the types of recombination events that facilitate telomerase-independent survival were influenced by Exo1 activity, in bothest2⌬andyku80⌬est2⌬strains. These data demonstrate that Exo1 can make either positive or negative contributions to telomere function and cell viability, depending on whether telomerase or recombination is utilized to maintain telomere function.
T
HE proteins that associate with telomeric DNA, the severe telomere replication defect and an accompa-nying senescence phenotype (Lendvayet al.1996). short G-rich repetitive sequences present at theends of linear chromosomes, serve two essential roles Despite the proliferation defect that is brought about by a telomerase deficiency, rare populations of cells are (reviewed inMcEachernet al.2000). First, they mediate
replication of telomeric DNA, thereby preventing the able to acquire the ability to maintain their chromosome termini via a recombination mechanism. In budding terminal shortening that would otherwise accompany
yeast, two pathways have been described to generate semiconservative replication of linear molecules.
Sec-these survivors. Both pathways requireRAD52but other-ond, they play a critical role in preventing telomeres
wise have distinct genetic requirements and are there-from being recognized as DNA breaks or other forms
fore thought to utilize different recombination sub-of DNA damage.
strates (LundbladandBlackburn1993;Leet al.1999; Consequently, there are two general mechanisms by
Tengand Zakian1999;Chenet al. 2001). Telomeres which dysfunctional telomeres can arise. One is via
de-in one class of survivors (often called type I) are charac-fects in telomere replication, which can occur due to
terized by extensive amplification of both subtelomeric alterations in the enzyme telomerase or factors that
Y⬘ elements and flanking short G-rich stretches, al-regulate its activity. In either yeast or human cells in
though the terminal G-rich tracts remain short. A sec-which telomerase is not expressed, there is a gradual
ond class of survivors (called type II) maintain their loss of telomeric DNA, until a point is reached at which
telomeres via recombination of the terminal TG1-3 telo-telomeres can no longer sustain proper end protection
meric repeats, giving rise to very long and hetero-function, and further proliferation is blocked. In
bud-geneous terminal G-rich tracts. In both cases, recom-ding yeast, the catalytic core of the enzyme is composed
bination, instead of telomerase, replenishes telomeric of the Est2 reverse transcriptase protein and the TLC1
G-rich sequences, thereby permitting continued viabil-RNA subunit, while Est1p and Est3p are additional
sub-ity (reviewed inLundblad2002). Telomerase-indepen-units of the holoenzyme that contribute toin vivo
regula-dent telomere maintenance can also occur in human tion of enzyme function (reviewed in Dubrana et al.
cells, and increasing evidence indicates that this also 2001). Defects in any one of these subunits result in a
occurs via a recombination-based mechanism (Dunham
et al.2000;Varleyet al.2002).
The second mechanism by which dysfunctional telo-1Corresponding author:Department of Pediatrics, Baylor College of
meres can be generated is via defects in the telomeric
Medicine, 1 Baylor Plaza, Houston, TX 77030.
E-mail: abertuch@bcm.tmc.edu nucleoprotein complex that protects the natural
mosome ends. Uncapped telomeres become subject to telomerase-independent survivors, presumably by pro-viding a substrate for telomere recombination. These DNA degradative activities and are also sensed as DNA
damage, thereby triggering a DNA damage response observations indicate that one task of telomere end pro-tection is to prevent illegitimate access of Exo1 to telo-(Garvik et al. 1995; Barnes and Rio 1997; van
Steenselet al.1998;Karlsederet al.1999;Maringele meres and may provide insights into the mechanism by which an alternative pathway(s) for telomere mainte-and Lydall 2002). Defects in this essential telomere
function result in immediate effects on cell viability, nance is engaged by telomerase-negative cancer cells. in contrast to the delayed phenotypes displayed by a
telomerase deficiency. For example, in budding yeast,
MATERIALS AND METHODS
loss of the single-strand telomere DNA-binding protein
Cdc13 results in rapid and extensive resection of the Yeast strains and plasmids:AllSaccharomyces cerevisiaestrains
used in this work are isogenic derivatives of YPH275. The
C-rich telomeric strand, leading to cell cycle arrest and,
yku80⌬::kanR,est2⌬::URA3,tlc1⌬::LEU2, andrad52⌬::LYS2
mu-ultimately, irreparable DNA damage and cell death
tations have been previously described (Lendvayet al.1996;
(Garviket al.1995;Boothet al.2001). The Ku
hetero-Nugent et al. 1998; Bertuch and Lundblad 2003). The
dimer (which is composed ofⵑ70- and 80-kD subunits, exo1⌬::kanRdisruption removes amino acids 6–698 of the
702-encoded by YKU70andYKU80, respectively) also pro- amino-acid EXO1 open reading frame (ORF), and the
yku80⌬::LEU2disruption deletes the entireYKU80ORF plus
tects the C strand from resection. Unlikecdc13⌬strains,
78 and 76 bp of upstream and downstream sequences,
respec-which are inviable,yku70⌬andyku80⌬strains are viable
tively. Diploid strains YVL234 (MATa/␣ tlc1⌬::LEU2/TLC1
at 30⬚, although the length of the duplex telomeric tract,
yku80⌬::kanR/YKU80), YVL1068 (MATa/␣est2⌬::URA3/EST2
as well as the extent of the terminal single-stranded rad52⌬::LYS2/RAD52 yku80⌬::kanR/YKU80 CF [ura3::TRP1
G-rich overhang, is perturbed (BoultonandJackson SUP11 CEN4 D8B]), YVL2303 (MATa/␣est2⌬::URA/EST2 exo1⌬:: kanR/EXO1 yku80⌬::LEU2/YKU80), and YVL2359 (MATa/␣
1996; Porter et al. 1996; Gravel et al. 1998;
Polot-est2⌬::URA3/EST2 exo1⌬::kanR/EXO1 yku80⌬::LEU2/YUK80
niankaet al.1998). This altered terminal structure has
chk1⌬::HIS3/CHK1) were constructed by standard techniques
suggested that Ku also regulates the access and/or
activ-(by introducing the relevant gene deletions by one-step gene
ity of a telomere-processing entity. disruption or by mating isogenic freshly generated haploid Accumulating evidence indicates that one activity that strains of the appropriate genotype). All diploid strains have
the following isogenic genetic background:ura3-52/ura3-52
is restricted by the Cdc13 and Ku end protection factors
lys2-801/lys2-801 ade2-101/ade2-101 trp1⌬1/trp1⌬ his3⌬200/
is the 5⬘ to 3⬘ exonuclease, Exo1 (Maringele and
his3⌬200 leu2⌬1/leu2⌬1. Lydall2002). Exo1 has been previously characterized
Genetic methods:All incubations were performed at 28⬚,
for its role in a variety of DNA repair processes, includ- except where otherwise noted. Diploid strains were sporulated ing meiotic recombination, double-strand-break repair, at room temperature. Yeast genomic DNA preps and telomere
Southern blots were performed as previously described ( Lend-and repair of UV-damaged DNA (Moreau et al.2001
vayet al.1996). The serial dilution plate assay was performed
and references therein). Recent observations suggest
by resuspending dissection plate colonies in their entirety into
that Exo1 also functions at telomeres. When yku70⌬
200l of water. Tenfold serial dilutions were plated on YPAD
strains are propagated at high temperatures, there is and incubated for 2 days at 28⬚. Serial liquid culture experi-enhanced resection of the C strand of the telomere, ments were performed similarly to methods previously
de-scribed (Leet al.1999;RizkiandLundblad2001). Each spore
which leads to inviability withinⵑ10 generations.
Strik-colony in its entirety was inoculated into YPAD and grown for
ingly, both resection and inviability are rescued by a
1 day at 28⬚. Cell counts were determined; the cultures were
null mutation inEXO1(MaringeleandLydall2002).
subsequently diluted into fresh media, to 1 ⫻105 cells/ml, Similarly, the sequence loss and resulting inviability that and incubated for 22 hr; and cell counts were performed. occur in response to loss of Cdc13 function are also at The protocol was repeated every 22 hr. Several independent
isolates for each genotype were analyzed.
least partially Exo1 dependent (MaringeleandLydall
Telomeric G-strand overhang analysis:The extent of
single-2002; E.Pennock, E.Mandelland V.Lundblad,
un-stranded G-rich terminal sequences was determined as
pre-published observations).
viously described (Bertuch and Lundblad 2003). Briefly,
These observations suggest that Exo1 might be a gen- the strains were grown to midlog phase in rich media. Geno-eral mediator of telomere dysfunction. To address this mic DNA was isolated as previously described (Hoffmanand Winston1987), and equivalent amounts (10g) were
XhoI-idea further, we examined whether an Exo1 deficiency
digested and hybridized to a 5⬘32P-end-labeled dCCCACCACA affected telomere maintenance in both
telomerase-pro-CACACCCACACCC probe. The samples were resolved by
aga-ficient strains and recombination-dependent survivors
rose gel electrophoresis, and the gel was subsequently dried
from telomerase-deficient strains. Analysis of the conse- and exposed to detect single-strand telomeric DNA (native quences of an EXO1 deficiency in strains that lack gel in Figure 1B). To detect the full complement of telomeric
DNA (denatured gel in Figure 1B), the gel was denatured
YKU80, telomerase, or both activities indicates that Exo1
and rehybridized with the same32P-labeled dC
1-3A probe, as action is detrimental to cells with partially uncapped
previously described (DionneandWellinger1996). The
av-telomeres, presumably due to increased production of
erage single-strand telomeric DNA content for two
indepen-single-stranded DNA. Conversely, however, Exo1 activity dent isolates of each strain was quantified by normalizing, positively contributes to telomere maintenance in the relative to the average for theYKU80isolates, the ratio of the
native gel signal to the denatured gel signal for the Y⬘-containing
Figure 1.—Exo1 contributes to telomere length maintenance in a yku80⌬ strain. (A) Southern blot ofXhoI-digested genomic DNA isolated from wild-type, exo1⌬, yku80⌬, and exo1⌬ yku80⌬ strains probed to detect telo-meric sequences. Bracket represents the termi-nal Y⬘telomeric restriction fragments, which are present atⵑ2/3 of the yeast telomeres. (B) Detection of telomeric G-strand overhangs, in the same set of strains, by hybridization of XhoI-digested genomic DNA with an oligomeric probe specific for the G-rich strand, using na-tive gel electrophoresis (left), followed by in-gel denaturation and rehybridization with the same oligomeric probe (right). Indicated be-low the native gel is the average native telo-meric restriction fragment (TRF) signal/dena-tured TRF signal ratio normalized to wild type for each genotype.
telomeric restriction fragment. The relative signal strengths contributes to the formation of these abnormal termini, were determined by PhosphorImager and ImageQuant analy- the extent of the single-stranded G-rich overhang in sis. The increase in single-stranded TG1-3 DNA observed in
yku80⌬andexo1⌬yku80⌬strains was assessed by native
yku80⌬ strains was shown to be sensitive to Escherichia coli
gel analysis. Strikingly, the readily detectable increase
exonuclease I, a 3⬘- to 5⬘-specific single-strand exonuclease
(data not shown; see also Bertuch and Lundblad 2003), in terminal single-strandedness that is observed in a
indicating that the signal arises from terminal 3⬘ single- yku80⌬strain was substantially diminished in anexo1⌬ stranded overhangs. yku80⌬strain (Figure 1B). Quantitation of the extent of
single-strandedness indicated that Exo1 was responsible for most, although not all, of the increased G-rich
single-RESULTS
strand signal detected in yku80⌬ strains (Figure 1B). Therefore, the combined effects of anEXO1deficiency
EXO1contributes to telomere shortening in ayku80⌬
on telomere length and the terminal overhang in strain:To determine whetherEXO1contributes to
telo-yku80⌬strains argue that the loss of end protection that mere length homeostasis, telomere length inexo1⌬,
yku-occurs when Ku is absent also affects telomere length
80⌬, andexo1⌬yku80⌬telomerase-proficient strains was
maintenance. These observations about the conse-analyzed. Consistent with a previous report (Moreauet
quence of Exo1 action in the absence of Ku function
al.2001), no detectable alteration in telomere length
are also concordant with prior results reported by Mar-was observed inexo1⌬ mutants (Figure 1A). However,
ingeleandLydall(2002), who demonstrated that the when the telomere length of EXO1 yku80⌬and exo1⌬
much more extensive resection of chromosome termini
yku80⌬strains was compared, a small but reproducible
that occurs whenyku70⌬strains are propagated at high
EXO1-dependent effect was observed. Loss of YKU80
temperatures is similarly Exo1 dependent. results in substantial telomere shortening (Boulton
The lethality of a yku80⌬est2⌬strain is partially re-andJackson1996). Notably, the telomere length defect
lieved by loss of EXO1: Previous work has shown that observed in anexo1⌬ yku80⌬ strain, however, was not
loss of Ku function confers rapid lethality in a strain as severe as that of ayku80⌬strain (Figure 1A; see also
that also lacks telomerase (Gravelet al.1998;Nugent the denatured gel in Figure 1B). Thus, Exo1 partially
et al.1998). In contrast to anest2⌬strain, which initially contributes to the telomere maintenance defect that
exhibits healthy growth immediately upon sporulation occurs when Ku function is absent, presumably due
of a heterozygous diploid, a similarly generatedyku80⌬
to a skew in the balance between telomerase-mediated
est2⌬strain gives rise to a colony that consists largely of telomere-elongation and telomere-shortening activities.
dead cells, incapable of further propagation (Nugent Strains that are deficient for YKU80 or YKU70 also
et al.1998; Figure 2A). This synthetic lethality has been exhibit altered regulation of the single-stranded
over-proposed to be due to a severe end protection defect, hang, such that extended single-stranded termini are
whereby the accelerated telomere shortening—due to detectable throughout the cell cycle (Gravelet al.1998;
Figure2.—Exo1 contrib-utes to the lethal phenotype of ayku80⌬est2⌬strain. (A) Haploid strains of the indi-cated genotype were gener-ated by sporulation of the diploid strain YVL2359. Spore colonies were grown for 4 days at 28⬚ and resus-pended in their entirety and 10-fold serial dilutions were incubated for 2 days at 28⬚. (B) Single colonies of freshly generatedEST2 EXO1, est2⌬ EXO1, est2⌬ exo1⌬, and yku80⌬ est2⌬ exo1⌬ haploid strains were resuspended in their entirety, inoculated into YPAD, and grown for 24 hr at 28⬚. Cell counts and serial dilutions were performed as described inmaterials and methods. ThreeEST2 EXO1 isolates and seven isolates each of the est2⌬ EXO1, est2⌬ exo1⌬, and yku80⌬ est2⌬exo1⌬strains were ex-amined. (C) Diploid strain YVL2303 was sporulated to generateyku80⌬est2⌬exo1⌬ isolates. Isolates were propa-gated by single-colony isola-tion, for up to eight streak-outs. Each streak-out was incubated at 28⬚for 2 days and then stored at 4⬚. Colo-nies from the stored streak-outs were restreaked, as shown, and incubated for 3 days at 28⬚.
rapidly leads to telomeres that are incapable of binding exo1⌬mutation rescued some, but not all, of the block in growth potential. Loss of EXO1 function similarly end protection factors.
As discussed above, the data shown in Figure 1 indi- rescued ayku80⌬est1⌬strain, deleted for the Est1 com-ponent of the telomerase holoenzyme (data not shown). cate that anexo1⌬mutation partially alleviates the
telo-mere shortening observed in a yku80⌬ strain, as well In contrast, loss ofEXO1activity did not have a notable effect on the growth phenotype of a strain that lacks as rescuing the Ku-specific end protection defect. We
therefore asked whether loss of Exo1 would similarly only telomerase. To assess this, est2⌬ and est2⌬ exo1⌬
mutant strains were compared in a liquid growth assay. influence the phenotype of a yku80⌬ est2⌬
double-mutant strain. Freshly generatedyku80⌬est2⌬andyku- Both strains exhibited a comparable loss in growth po-tential after 5–6 days of liquid propagation, as a
conse-80⌬ est2⌬ exo1⌬ spore colonies were resuspended in
their entirety and assayed for viability by plating serial quence of critical telomere shortening and the resulting proliferation defect (Figure 2B). Following this growth dilutions and examining growth after 2 days of
incuba-tion. Figure 2A shows that anexo1⌬mutation partially nadir, however, the proliferative potential increased for both strains, such that both cultures were eventually rescued the lethality displayed by theyku80⌬est2⌬
be slightly delayed (see the last two time points in Figure 2B), suggesting that loss ofEXO1 might have a slight impact when telomeres become critically short. How-ever, this difference did not appear to be highly signifi-cant, arguing that loss ofEXO1function does not have a robust effect on the growth phenotype of a telomerase-defective strain that retainsYKU80function.
Recombination-dependent survivors obtained from a yku80⌬ est2⌬ strain are promoted byEXO1: Although theyku80⌬est2⌬exo1⌬strain did not grow as well as a
YKU80 EST2 EXO1strain, this triple-mutant strain was nevertheless capable of continuous long-term propaga-tion, although growth at successive time points was still relatively poor. Successive platings by propagation on solid media were characterized by microcolony forma-tion and poor plating efficiency, although there was a gradual increase in colony size at later time points (Fig-ure 2C). Liquid serial cult(Fig-ure propagation gave similar results: the yku80⌬ est2⌬ exo1⌬ strain could be stably and continuously propagated, albeit with an extremely low doubling rate, although at later time points, recom-bination-dependent survivors overtook the culture (Fig-ure 2B and see below). Therefore, loss of EXO1 was sufficient to allow long-term growth of ayku80⌬ est2⌬
mutant strain, consistent with the premise that the se-vere end protection defect characteristic of this double-mutant strain had been partially rescued.
However, although a yku80⌬est2⌬strain initially ex-hibited a much higher degree of cell death, when com-pared to ayku80⌬est2⌬exo1⌬strain (Figure 2A), survi-vors with a healthy growth characteristic could be recovered from theyku80⌬est2⌬strain, even after only limited propagation (Figure 3;Grandinand
Charbon-neau 2003). For this analysis, both yku80⌬ tlc1⌬ and Figure 3.—Stable RAD52-dependent survivors arise in
yku80⌬est2⌬strains were examined. When freshly gen- yku80⌬ tlc1⌬ and yku80⌬ est2⌬ strains. (A) Diploid strain
YVL234 was sporulated and dissected, and freshly isolated
erated yku80⌬ tlc1⌬ spores were restreaked for single
spore colonies were restreaked for single colonies. The top
colonies, no growth was initially observed after 2 days
photograph was taken after 2 days of growth at 30⬚, and the
of incubation, a time period that was sufficient to allow
bottom photograph was taken after an additional 4 days of
bothTLC1andtlc1⌬strains to form full-sized colonies growth at room temperature. (B) A haploidyku80⌬est2⌬strain (Figure 3A). However, following an additional 4-day was isolated from diploid strain YVL2303 and processed as described in Figure 2. Four additional isolates, analyzed in
incubation, a small number of heterogeneously sized
parallel, gave comparable results. (C) Single-colony
streak-colonies appeared on these yku80⌬ tlc1⌬ streak-outs.
outs of haploid strains of the indicated genotype (recovered
These colonies, as well as similarly obtainedyku80⌬est2⌬
from diploid strain YVL1068) were incubated for 4 days at 30⬚.
colonies, were capable of subsequent long-term propa-gation, with a growth phenotype that was comparable to that of survivors recovered from aYKU80 est2⌬strain
(Figure 3B and data not shown). Appearance of these conversely promotes the appearance of survivors in this same strain. Exo1 could contribute to this process by survivor colonies was also dependent onRAD52, because
they failed to arise inyku80⌬est2⌬rad52⌬mutants (Fig- increasing the degree of single-strandedness at telo-meres, thereby providing a substrate for recombination ure 3C). This suggests that theseyku80⌬est2⌬survivors
arise by the same well-characterized recombination- between telomeres. This also suggests that Exo1 action at telomeres might influence the types of dependent mechanisms that give rise to
telomerase-defective survivors (LundbladandBlackburn1993). independent survivors recovered. To address this possi-bility, the types of survivors that were recovered inEXO1
EXO1alters the pattern of survivors recovered from
telomerase-defective strains:The results shown in Fig- vs. exo1⌬strains were determined.
Examination of the telomeres ofyku80⌬est2⌬ survi-ures 2 and 3 demonstrate that, althoughEXO1
pat-Figure 4.—Exo1 influ-ences survivor formation in bothyku80⌬est2⌬andest2⌬ strains. Southern blot of XhoI-digested genomic DNA isolated from independent survivors of the designated genotypes obtained follow-ing sfollow-ingle-colony propaga-tion for 200 generapropaga-tions (nine successive streak-outs; A and B, left) or serial cul-turing for 9 days followed by single-colony isolation (ⵑ100–120 generations; B, right and C) probed to de-tect telomeric sequences is shown. Arrows represent in-ternal Y⬘elements and brack-ets indicate the terminal Y⬘ element telomere restriction fragment.
tern that was roughly reminiscent of type II recombina- the telomere profile of type II survivors, but additional survivors isolated from this triple-mutant strain exhib-tion (Figure 4A; see alsoGrandinandCharbonneau
2003). Theseyku80⌬est2⌬survivors differed from those ited the characteristic features of type I recombination, with extensive Y⬘amplification and a short terminal TG1-3 recovered from est2⌬ strains in one notable fashion.
Only type II-like survivors could be recovered from a tract (Figure 4B, lanes 1, 2, 5, and 6). This bias held up even if yku80⌬ est2⌬ exo1⌬ survivors were isolated
yku80⌬ est2⌬ strain, whereas both type I and type II
survivors were recovered from anest2⌬strain. Over 20 following serial liquid culturing. Previous work has shown that when anest2⌬strain is propagated in liquid,
yku80⌬est2⌬survivors generated by serial single-colony
isolation were examined, and all exhibited telomeric only type II survivors are eventually recovered, due to the selective advantage of this class relative to type I restriction fragment profiles characteristic of type II
sur-vivors, similar to that shown in Figure 4A. In contrast, survivors (TengandZakian1999). However, even when
yku80⌬est2⌬exo1⌬strains were grown in liquid culture 2 of 5 survivors isolated in a similar manner from an
est2⌬ strain were of the type I class, indicating type I until each culture was overgrown with survivors, type I survivors could be identified (Figure 4B, lane 9). survivors could be readily recovered from anest2⌬strain
when Ku proficient. To determine whether thisEXO1-dependent bias was
specific only to strains that were defective for both te-The absence of type I survivors suggested that the
severe telomere uncapping defect displayed by the lomerase and YKU80, the consequences of an exo1⌬
mutation on the type of survivors that emerged from an
yku80⌬ est2⌬strain influenced the type of
recombina-tion events—and hence the types of survivors that could est2⌬strain following serial liquid culture were assessed. Although these conditions should favor the outgrowth be recovered—when telomeres become precipitously
short in this double-mutant strain. To ask whetherEXO1 of type II survivors, loss ofEXO1 shifted the telomere profile exhibited by anest2⌬strain from the exclusively action influenced this process, survivors from yku80⌬
est2⌬exo1⌬strains were similarly isolated. Although this type II pattern to a predominantly type I pattern (Figure 4C). Eighteen of 18est2⌬survivors displayed the hetero-triple-mutant strain was characterized by a prolonged
period of microcolony formation and poor plating effi- geneous, long telomeres characteristic of type II survi-vors, whereas 13 of 16est2⌬exo1⌬survivors exhibited a ciency, eventually discrete small colonies could be
recov-ered and analyzed for telomere structure. In sharp con- type I pattern, with Y⬘amplification and a short terminal G-rich telomeric tract. Therefore, just as was observed trast to the type II telomere profile exhibited by every
survivor recovered fromyku80⌬est2⌬strains, both type for a yku80⌬est2⌬strain, Exo1 influences the form of telomere recombination utilized for survival in telo-I and type telo-Itelo-I types of survivors could be isolated from
yku80⌬est2⌬exo1⌬strains (Figure 4B). Theyku80⌬est2⌬ merase-deficient mutants, resulting in an increased rela-tive frequency of type I survivors in its absence.
DISCUSSION the combined effects of the loss of a mechanism to
elongate telomeres and the inability to protect telo-Exo1 influences the balance between elongation and
meres from shortening activities. In fact, anexo1⌬ muta-shortening activities at telomeres:Telomere length
ho-tion can extend the propagaho-tion ofyku80⌬ est2⌬ and meostasis is a genetically regulated process that
main-yku80⌬ est1⌬ strains, consistent with the premise that tains chromosome termini within a carefully controlled
shortening due to nuclease action has been partially length range. Careful analysis of individual telomeres,
relieved. however, has revealed that the length of telomeric ends
At a mechanistic level, how does increased action of can vary, even in telomerase-proficient cells, resulting
Exo1 at telomeres lead to telomere shortening, at least in a certain degree of length heterogeneity (Shampay
in Ku-deficient strains? Molecular studies indicate that
et al.1984;Lansdorpet al.1996). This clonal variation
the substrate that is susceptible to the 5⬘ to 3⬘ Exo1 in the length of individual telomeres is thought to be
enzyme is the C strand of the telomere (Maringele the consequence of a balance between lengthening and
andLydall2002), whereas telomerase acts on the telo-shortening processes that can occur during each round
meric G strand. One simple model to explain a telomere of replication (Blackburn2001).
length alteration induced by Exo1 action rests on the In wild-type cells, telomerase is the primary activity
idea that Exo1-dependent changes in the length of the responsible for elongating telomeres, whereas
incom-C strand will be passed on to subsequent progeny, fol-plete replication and potential nuclease-mediated
deg-lowing replication. This model does not easily explain, radation have been proposed to contribute to telomere
however, why loss of Exo1 activity does not cause a notable shortening. In yku70⌬ and yku80⌬ cells that express
effect on telomere length, even though there is a repro-telomerase, the balance between shortening and
length-ducible decrease in G-strand single-strandedness in an ening activities is shifted, such that telomeres are
main-exo1⌬ strain. One possibility is that any increase in tained at a much shorter mean length. Part of this
telo-mere length resulting from an Exo1 deficiency may be mere length decline is due to loss of an interaction
only transient inYKU80cells, due tocis-inhibition of telo-between Ku and a 48-nucleotide stem-loop of the yeast
merase action on these slightly elongated telomeres in telomerase RNA. This interaction facilitates
telomerase-subsequent cell divisions (Marcandet al.1997, 1999). mediated telomere elongation, by contributing to either
In contrast, telomere length regulation via telomerase telomerase recruitment or activation (Peterson et al. has clearly been compromised in Ku-defective strains,
2001;Stellwagenet al.2003). However, loss of the Ku
and hence reduced resection by Exo1 would result in heterodimer also results in an end protection defect a detectable change in telomere length.
that is manifested by increased single-stranded regions Exo1 mediates telomerase-independent prolifera-at chromosome termini (Gravel et al. 1998; Polot- tion:The above observations indicate that, in cells that niankaet al.1998). The action of various nucleases on express telomerase, Exo1 action opposes telomere elon-these partially exposed termini might be expected to gation. In contrast, Exo1 appears to directly contribute contribute to telomere shortening in yku70⌬ and to telomere maintenance when telomeres are
main-yku80⌬strains. tained by recombination. The effect of Exo1 on telo-Consistent with such a prediction, this work demon- merase-independent pathways for telomere mainte-strates that the severe telomere length defect displayed nance is twofold. First, in a strain that is defective for by Ku-deficient cells is partially rescued in an exo1⌬ both telomerase and the Ku heterodimer, Exo1
pro-background. In parallel, the end protection defect of a motes the formation of telomerase-independent
survi-yku80⌬strain is also substantially relieved by an exo1⌬ vors. Second, in telomerase-defective strains that also
mutation, as evidenced by the reduction in the extent lack Exo1, there is a shift in the type of recombination-of the terminal G-strand overhang in a yku80⌬ exo1⌬ dependent survivors that are recovered: whereas liquid
strain (this work and Maringele and Lydall 2002). propagation of est2⌬ strains yields only survivors with Even in Ku-proficient cells, there is a very slight, but the characteristic type II telomeric pattern of rearrange-reproducible, effect of the loss ofEXO1activity on the ments, both type I and type II patterns can be observed extent of the G-strand single-strandedness (Figure 1B). in est2⌬exo1⌬survivors.
These observations indicate that one role of the Ku Telomere maintenance in the absence of telomerase heterodimer is to protect termini from the unregulated has been proposed to employ break-induced replication action of the Exo1 nuclease. Whether Ku similarly pro- (BIR; reviewed inKrauset al.2001). Two similar genetic tects chromosome ends from other nucleases is cur- pathways have been described for
telomerase-indepen-rently under investigation. dent telomere maintenance and BIR, one that isRAD50/
Award (to A.B.), National Institutes of Health grant K08 HD01231
strand invasion. In the case of telomerase-independent
(to A.B.), and National Institutes of Health grant R01 AG16626 (to
telomere maintenance, the recombination processes
ap-V.L.).
pear to involve different regions of homology. For type I recombination, BIR is likely initiated either between terminal and TG1-3 repeats present between
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