Evidence that the S.cerevisiae Sgs1 protein
facilitates recombinational repair of telomeres
Mahrukh Azam1, Julia Y. Lee1, Veena Abraham1, Rebecca Chanoux2,
Kimberly A. Schoenly2and F. Brad Johnson1,2,*
1Department of Pathology and Laboratory Medicine and and2Cell and Molecular Biology
Graduate Program, University of Pennsylvania School of Medicine, Philadelphia,
Received as resubmission October 30, 2005; Revised December 22, 2005; Accepted January 4, 2006
RecQ DNA helicases, including yeast Sgs1p and
the human Werner and Bloom syndrome proteins,
participate in telomere biology, but the underlying
mechanisms are not fully understood. Here, we
explore the protein sequences and genetic inte-
ractors of Sgs1p that function to slow the senesc-
ence of telomerase (tlc1) mutants. We find that the
S-phase checkpointfunctionofSgs1p isdispensable
for preventing rapid senescence, but that Sgs1p
sequences required for homologous recombination,
including the helicase domain and topoisomerase
III interaction domain, are essential. sgs1 and rad52
mutations are epistatic during senescence, indicat-
ing that Sgs1p participates in a RAD52-dependent
recombinational pathway of telomere maintenance.
Several mutations that are synthetically lethal with
sgs1 mutation and which individually lead to genome
instability, including mus81, srs2, rrm3, slx1 and
top1, do not speed the senescence of tlc1 mutants,
indicating that the rapid senescence of sgs1 tlc1
mutants is not caused by generic genome instability.
However, mutations in SLX5 or SLX8, which encode
proteins that function together in a complex that is
required for viability in sgs1 mutants, do speed the
senescence of tlc1 mutants. These observations
further define roles for RecQ helicases and related
proteins in telomere maintenance.
Saccharomyces cerevisiae Sgs1p is a member of the
RecQ family of 30–50DNA helicases (1–4). Human RecQ
helicases include WRN, BLM and RTS, which are deficient
in the Werner, Bloom and Rothmund-Thomson syndromes,
respectively (5–7). These are recessive disorders characterized
bygenome instability, cancer predisposition and—particularly
in the case of Werner syndrome—by premature features of
aging (8–11). RecQ-family proteins maintain the genome
through several mechanisms (3,4,12). For example, Sgs1p
stabilizes stalled replication forks and facilitates the proper
resumption of DNA synthesis from stalled forks (13–19).
Sgs1p inhibits crossover events during homologous recomb-
ination (HR) (20), and certain phenotypes in sgs1 mutants can
be suppressed by deletion of genes in the RAD52 epistasis
group, indicating that Sgs1p normally prevents inappropriate
recombination or facilitates resolution of recombination
intermediates (21,22). Sgs1p, similar to WRN and BLM,
can unwind a variety of DNA structures that may be important
during replication or recombination, including replication
fork-like substrates, Holliday junctions and G-quadruplexes
(23–27). Sgs1p also functions in parallel with Rad24p to
ensure a complete S-phase checkpoint response to DNA dam-
age (28). Failure of these activities in sgs1 mutants leads to
aberrant recombination in repeated sequences, chromosome
loss, gross chromosomal rearrangements, unequal sister
chromatid exchange, sensitivity to methyl methanesulfonate
(MMS) and hydroxyurea (HU), as well as to defects in ribo-
somal DNA replication and to the premature cessation of
budding by yeast mother cells (1,13,15,29–32).
*To whom correspondence should be addressed. Tel: +1 215 573 5037; Fax: +1 215 573 6317; Email: firstname.lastname@example.org
Mahrukh Azam, Department of Chemistry, West Chester University, West Chester, PA, USA
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
? The Author 2006. Published by Oxford University Press. All rights reserved.
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Nucleic Acids Research, 2006, Vol. 34, No. 2
Sgs1p also participates in telomere function. Yeast
telomeres are maintained naturally in part through the actions
of telomerase, which is expressed constitutively (33,34).
Telomerase can be inactivated genetically, for example by
deleting TLC1, the gene encoding the RNA template compon-
ent of the enzyme (35). Telomeres shorten with cell division in
tlc1 mutants and this leads eventually to induction of a DNA
damage response and G2/M cell cycle arrest, a process termed
survivors use RAD52-dependent recombination mechanisms
to maintain their telomeres (39–41). There are two such sur-
vivormechanisms,type Iinvolvingthe amplificationprimarily
of subtelomeric Y0elements along with telomere repeat DNA
and type II involving primarily the amplification of the
telomere repeat DNA alone. Although deletion of SGS1
does not affect steady state telomere length in cells containing
telomerase, mutants lacking both SGS1 and telomerase sen-
esce more rapidly than mutants lacking telomerase alone, and
only type I survivors emerge in the absence of SGS1
(29,36,42,43). Thus, Sgs1p slows senescence and is required
for initiation of the type II survivor recombination mech-
anism. Recent findings also support a role for the mammalian
WRN and BLM proteins in telomere function. For example,
each protein interacts physically and functionally with
the telomere chromatin protein TRF2 (44,45), which itself
plays a critical role in maintaining telomeres in a protected
or ‘capped’ state (46). The capacity of purified TRF2 to
catalyze T-loop assembly is thought to be related to this cap-
ping function (47), and therefore demonstrations that the
WRN helicase and exonuclease activities process telomeric
T-loop structures in vitro support the idea that WRN and TRF2
cooperate to maintain telomeres (48,49). Moreover, Werner
mutant cells suffer from an elevated level of S-phase-
dependent telomere loss (50,51). Furthermore, BLM overex-
pression lengthens telomeres in cells that use recombination
to maintain their telomeres (52), and Wrn and Blm mutations
each synergize with telomerase (Terc) mutation to accelerate
telomere dysfunction and cause pathology in mice (53,54).
Our yeast model provides a relatively simple system
in which to probe mechanisms by which RecQ helicases
participate in telomere function.
The rapid senescence of tlc1 sgs1 mutants is due, at least
in part, to a synergistic interaction between the mutations
that renders the cells more prone to arrest at a given average
extent of telomere shortening (36). This observation might
be explained by a role for Sgs1p in processes that are parallel
to or downstream from telomere defects and thus affect
the sensitivity of cells to telomere dysfunction. One such pos-
sibility is that rapid senescence might result from an elevated
level of global genome instability caused by sgs1 mutation,
which could sum with telomere dysfunction to yield an overall
DNA damage signal that leads to premature cell cycle arrest.
A second such possibility is that rapid senescence might
instead be due to loss of SGS1-dependent intra-S-phase check-
point function, which may in turn preclude normal repair of
telomere damage and thus cause premature telomere dysfunc-
tion. Alternatively, the premature arrest of tlc1 sgs1 mutants at
longer mean telomere lengths might reflect a direct role for
Sgs1p in maintaining telomere integrity, perhaps via recom-
binational repair of shortened telomeres. Its requirement for
type II survivor formation is consistent with a role for Sgs1p
in recombination involving the G-rich telomere sequences
(36,42,43). One dysfunctional telomere is sufficient to cause
cell cycle arrest (55), and so only a rare telomere might require
recombinational repair, thus explaining the premature arrest
could be repaired by telomerase, when present, and obviate a
requirement for Sgs1p, thus explaining the normal telomere
lengths and absence of G2/M arrest in TLC1 sgs1 cells (36).
Here, we present evidence that neither loss of the SGS1-
dependent intra-S-phase checkpoint nor increased global
genome instability explains the rapid senescence of tlc1
sgs1 mutants. Our findings rather support a role for Sgs1p
in the recombinational repair of telomeres.
MATERIALS AND METHODS
All yeasts were grown at 30?C using standard yeast media
and techniques (56). All strains are described in Table 1 and
were isogenic derivatives of JKM111 (57). The sgs1 deletion
in YBJ133 is a null allele (58) but retains the first 481 nt of
the open reading frame (ORF) which might generate an
N-terminal Sgs1p fragment that, although inert itself, could
conceivably function in trans with other Sgs1p derivatives
(59). To avoid such potential trans complementation from
the various SGS1 derivatives that were examined, the entire
SGS1 ORF except for the start and stop codons was deleted
by PCR-mediated gene disruption to generate YMA75b.
rad52::LEU2 was generated with pSM20 (58). The top1,
mus81, slx1, slx5, slx8, est2, rrm3 and srs2 mutations were
Table 1. S.cerevisiae strains used in this study
YBJ133MATa/a, Dho Dhml::ADE1
Dhmr::ADE1 ade1 ura3-52,
leu2-3,112, lys5, TLC1/Dtlc1::kanMX,
YBJ133 TOP3/Dtop3:: HygB
YBJ133 SLX1/Dslx1:: LEU2
MATa/a, Dho Dhml::ADE1
Dhmr::ADE1 ade1 ura3-52,
leu2-3,112, lys5, TLC1/Dtlc1::kanMX
MATa/a, Dho Dhml::ADE1
Dhmr::ADE1 ade1 ura3-52,
leu2-3,112, lys5, SGS/Dsgs1::hisG-URA3,
Nucleic Acids Research, 2006, Vol. 34, No. 2 507
complete PCR-mediated ORF deletions, using pRS405, pUG6
(kanR) or pAG32 (hphMX4) as templates for marker gene
amplification (60). The top3 deletion extended from nucle-
otides 10 to 1666 of the ORF, to avoid disruption of the
overlapping ORFs YLR235C and YLR236C. Deletions were
confirmed by PCR amplification of fragments that crossed
both mutation breakpoints. Oligonucleotide sequences are
available on request.
The SGS1 deletion and point mutation derivatives (pJM526,
pJM511, pJM531, pJM512 and pRL1; kindly provided by
S. J. Brill) were cloned downstream a 0.15 kb SGS1 promoter
fragment in pRS405, as described previously (59). The
TOP3-SGS1-DN106 allele was generated by PCR amplifi-
cation of the TOP3 ORF from pRK500 (kindly provided
by J. Wang) with incorporation of NdeI and NcoI sites at
the 50and 30ends, respectively, and insertion of NdeI and
NcoI digested product into NdeI and partially-NcoI digested
pJM531; this yielded a fusion of the full TOP3 ORF to the
SGS1 ORF beginning at amino acid 107, in a fashion similar
to that described previously (61). For all SGS1 derivatives,
BstEII-digested plasmid DNA was transformed into yeast
cells, and proper integration of a single copy of each SGS1*
mutant at the LEU2 locus was confirmed by Southern blot
analysis of SacI-digested genomic DNA probed with pRS405.
Liquid senescence assays
To ensure that senescence comparisons were made between
strains that inherited similar telomeres, senescence assays
were performed on the haploid spore products of diploids
that were heterozygous for tlc1 and the various test mutations.
Liquid senescence assays were performed as described previ-
ously (36), starting with spore products from reference plates
(estimated population doubling of 30). At the start of each
day of growth, 2 · 106cells were inoculated into 5 ml of YPD
followed by growth for 22 h, and the process was repeated
until survivors emerged. Cells numbers were determined with
a Coulter counter, and population doubling calculated as
log2(final cell number/starting cell number). Each genotype
was represented by three to six independent spore products,
and the mean and standard errors of measurements are shown
Spot growth assays
Cells were grown overnight on YPD plates and were
suspended in sterile water at an OD600of 3.0. Cells were
spotted at this concentration and at four serial 10-fold dilutions
on YPAD plates and were grown at 30?C for 60 h.
Telomere Southern analysis
Southern analysis was performed essentially as described
previously (36). Briefly, purified genomic DNA was digested
with XhoI, separated by electrophoresis in 1% agarose gels,
transferred to Hybond-XL membranes, and probed and
washed at high stringency with a cloned 256 bp fragment
of S.cerevisiae telomere repeat DNA (BamHI–MluI fragment
of pJL8-50, Julia Y. Lee and F. Brad Johnson, manuscript in
preparation) or with a 784 bp Y0fragment distal to the XhoI
site (36). tlc1 rad51 type II survivor DNA was from (36).
The C-terminus of Sgs1p, required for intra-S-phase
checkpoint function, is not required to rescue
To begin to dissect how Sgs1p slows the rate of senescence
of telomerase (tlc1) mutants, we tested different deletion and
point mutation alleles of SGS1 (Figure 1) for their ability
to rescue the rapid senescence of tlc1 sgs1 mutants. The deriv-
ative alleles, designated with an asterisk, were placed down-
stream of the SGS1 promoter and integrated at the LEU2 locus
(59). Diploids heterozygous for tlc1 and sgs1 mutations and
an*allele were sporulated, and the rates of senescence of
haploid products lacking telomerase were determined using
liquid senescence assays. We first confirmed that wild type
SGS1 sequences integrated at LEU2 (denoted SGS1*) could
rescue an sgs1 mutation during senescence. tlc1 sgs1 mutants
carrying SGS1* senesced at a rate equivalent to tlc1 mutants
(Figure 2A), while a vector control lacking all SGS1 sequences
integrated at LEU2 failed to have any effect (Figure 2B).
Furthermore, tlc1 mutants carrying SGS1* senesced at the
same rate as tlc1 mutants, indicating that SGS1* did not have
effects beyond the rescue of the sgs1 mutation. Thus, integ-
rated SGS1* can function in the senescence assay in the same
fashion as the native SGS1 locus.
The C-terminal 200 amino acids of Sgs1p have been shown
to be essential for the intra-S-phase checkpoint activity of
the protein (28). We therefore tested the ability of SGS1-
DC200*, which encode an Sgs1p derivative lacking these
amino acids, to rescue rapid senescence. Similar to SGS1*,
SGS1-DC200* slowed the rate of senescence to that observed
in tlc1 mutants (Figure 2C). Therefore, the intra-S-phase
checkpoint function of SGS1 does not appear to be required
to prevent rapid senescence.
Sgs1p slows senescence through an
Because the checkpoint function of SGS1 appeared not to
be involved in slowing senescence, we considered the possib-
ility that the homologous (or homeologous, in the case of
the imperfect yeast telomere repeats) recombination (HR)
functionsofSGS1 mightinsteadplay arole.RAD52 isrequired
for nearly all HR reactions in yeast (62), and so we tested
for a possible epistatic relationship between sgs1 and rad52
are the helicase domain, the RQC domain (a C-terminal extension from the
helicase domain of shared homology among RecQ-family helicases, which is
involved in protein–protein interactions) and the HRCD domain (helicase/
RNase D C-terminal domain, which appears to be involved in DNA binding).
The locations of the N- and C-terminal deletion breakpoints used in this study
are indicated by thick vertical lines and labels above the protein, as is the
helicase-deficient alanine substitution mutant, K706A. Sequences containing
508 Nucleic Acids Research, 2006, Vol. 34, No. 2
mutations in the absence of telomerase. As reported previ-
ously, tlc1 rad52 mutants senesced rapidly (39,57), consistent
with a role for HR in slowing senescence, and at a rate
similar to tlc1 sgs1 mutants (Figure 3A); also, as expected,
no survivors of senescence were formed in the absence of
RAD52 function. Although tlc1 rad52 sgs1 mutants senesced
slightly faster than tlc1 rad52 or tlc1 sgs1 mutants, the
contribution of the sgs1 or rad52 mutation to the faster
senescence of the triple mutant was far less than the con-
tribution of either mutation to the rapid senescence in the
double mutants, and thus the rad52 and sgs1 mutations are
largely epistatic in our assay. This result was repeated twice
using independent sets of spore products and was also
observed when telomerase was inactivated via deletion
of EST2, rather than TLC1 (Figure 3B). SGS1 therefore
appears to function in a RAD52-dependent pathway during
senescence. Furthermore, the helicase activity of Sgs1p,
which is required for Sgs1p HR activities in other contexts
(31,59), was necessary to prevent rapid senescence because
the SGS1-hd* allele, which encodes a K706A mutant devoid
of helicase activity, failed to rescue the rapid senescence of
tlc1 sgs1 mutants (Figure 3C). Similarly, the SGS1-DC795*
allele, which encodes a protein lacking the helicase domain
and C-terminus of Sgs1p, did not rescue rapid senescence
(Figure 3D). These observations are consistent with a role
for SGS1 in HR during senescence.
To confirm that the failure of the SGS1-hd* and SGS1-
DC795* alleles to rescue rapid senescence was not simply
due to lack of their expression, we examined their activities
in cells lacking topoisomerase I (top1 mutants). Loss of SGS1
function causes a slight decrease in growth rate, as does loss of
TOP1 function, but an sgs1 top1 double mutant has a synthetic
growth defect (63). As reported previously (59), the SGS1-hd*
and SGS1-DC795* alleles partially and fully, respectively,
restored the growth rate of sgs1 top1 mutants to that of
top1 mutants (Figure 3E), confirming the activities of the
alleles. Furthermore, because Sgs1p helicase activity is critical
during senescence but less important for growth in top1
mutants, different functions of SGS1 are apparently involved
in each case, and this in turn implies that TOP1 function
may not play a role during senescence. Indeed, tlc1 top1
null mutants senesce at the same rate as tlc1 mutants
(Figure 3F). This is remarkable because, similar to sgs1 dele-
tion, top1 deletion causes genome instability, particularly in
the ribosomal DNA (64,65). Therefore, a decrease in genome
stability, at least in a generic sense, is not sufficient to speed
senescence. This point is explored more fully below.
Sgs1p cooperates with Top3p to slow senescence
A derivative of Sgs1p lacking the N-terminal 50 amino acids,
encoded by SGS1-DN50*, was tested and found to be devoid
of SGS1 function in the senescence assay (Figure 4A). The
deleted N-terminus includes residues that are essential for
the functional and physical interaction of the protein with
topoisomerase III (Top3p), a type I topoisomerase that co-
operates with Sgs1p during homologous recombination
(22,61,66–69). Indeed, this may be the only critical function
of the N-terminus of Sgs1p because when the TOP3 ORF is
fused to a fragment of the SGS1 ORF lacking the first 106
codons, the fusion product (Top3p-DN106Sgs1p) is able to
rescue the recombination defects and sensitivity of sgs1
mutants to MMS and HU (61). The failure of SGS1-DN50*
to rescue rapid senescence therefore implies a role for TOP3
during senescence. We investigated this possibility by first
Figure 2. Full-length and DC200 forms of Sgs1p rescue the fast senescence of
tlc1 sgs1 mutants. Full-length SGS1, SGS1-DC200 or control vector sequences
were integrated at the LEU2 locus of diploids heterozygous for sgs1 and tlc1
null mutations. tlc1 and tlc1 sgs1 spore products without or with the integrated
SGS1 alleles (indicated by an asterisk) were compared in liquid senescence
spore germination, and the density of cells after 22 h of growth, from cultures
inoculated with 4 · 105cells/ml are shown. For each time point, the mean and
standard errors for at least three independent spore products for each genotype
are indicated. For each experiment, filled diamonds indicate tlc1, filled circles
indicate tlc1 sgs1, open diamonds indicate tlc1 with an integrated*allele, and
by full-length integrated SGS1* (B) Lack of rescue by the pRS405 vector used
Sgs1p derivative lacking the C-terminal 200 amino acids.
Nucleic Acids Research, 2006, Vol. 34, No. 2 509
examining the rate of senescence of tlc1 top3 null mutants.
Haploid spores were derived from diploids with TLC1/tlc1
and TOP3/top3 mutations and tlc1 and tlc1 top3 products
compared with each other. top3 and tlc1 top3 mutant spores
germinated at very low frequency in our strain background.
However, we obtained one tlc1 top3 spore product, and this
senesced rapidly, ?20 generations after germination (data
not shown). Additional evidence for the importance of
TOP3 during senescence was provided by the ability of
the Top3p-DN106Sgs1p fusion, encoded by TOP3-SGS1-
DN106*, to rescue the rapid senescence of tlc1 sgs1 mutants
(Figure 4B). Because the fusion lacks all the residues from
the N-terminus of Sgs1p that are absent in Sgs1p-DN50, this
indicates that the inactivity of Sgs1p-DN50 was due to its
inability to bind productively to Top3p. We conclude that
Sgs1p and Top3p cooperate to slow the senescence of tlc1
Compared with most proteins that function with Sgs1p
in other settings, Sgs1p plays a more important role
A number of mutations, referred to as ‘slx’ mutations, have
been found to cause synthetic lethality (or extremely poor
growth) in combination with sgs1 null mutations (70–73).
Among these are null mutations in SLX1, SLX5, MUS81,
SRS2 and RRM3, as well as in TOP1 as described above.
Slx1p together with Slx4p forms an endonuclease complex
that cleaves 50flap structures and is required for normal
replication of the ribosomal DNA and for resistance to
MMS (13,70,74). Slx5p together with Slx8p forms a complex
of undetermined biochemical activity, although the complex
is known to promote resistance to HU (70). Mus81p com-
plexes with Mms4p to form an endonuclease that cleaves
30flap structures, D-loops and nicked Holliday junctions,
and the complex appears to help rescue stalled replication
forks (75–77). Srs2p is a 30–50DNA helicase that promotes
Rad51p-mediated strand invasion, and that is also required
for adaptation following DNA damage (20,78–80). Rrm3p
is a 50–30DNA helicase that prevents replication fork stalling
in non-nucleosomal chromatin regions, including telomeres
and ribosomal DNA sequences (81–83). Similar to SGS1,
each of the genes in this group is required for the maintenance
of DNA stability throughout the genome.
A mutation that is synthetically lethal with sgs1 may identi-
fy a gene that accomplishes an essential function through
a pathway that is distinct from and parallel to an SGS1-
dependent pathway. Thus, either SGS1 or the gene in question
can support a critical biochemical pathway at a level that
Figure 3. Evidence that Sgs1p functions via recombination to slow senescence. Senescence rates were measured in the same fashion as Figure 2. (A) rad52 and
sgs1 mutationsare epistaticduringsenescence.Haploid sporeproductswere derivedfrom diploidsheterozygous for tlc1, sgs1 andrad52mutations.Curvesfor tlc1
times with independently derived spore products (data not shown). (B) est2 and tlc1 mutations are equivalent in the context of sgs1 and rad52 mutations. The
experiment was the same as in (A), but with telomerase inactivation via est2 rather than tlc1 deletion (C) Sgs1p helicase activity is required to slow senescence.
Diploids heterozygous for tlc1 and sgs1 mutations and with an integrated SGS1-hd* allele, which encodes the helicase-defective K706A point mutant, were
sporulated and the senesce rates of tlc1 (filled diamonds), tlc1 sgs1 (filled circles), tlc1 SGS1-hd* (open diamonds) and tlc1 sgs1 SGS1-hd* haploid products were
measured. (D) Same as (C), except that the SGS1-DC795* allele, which encodes a derivative lacking the C-terminal 795 amino acids of Sgs1p was used instead of
SGS1-hd*. (E) SGS1-hd* and SGS1-DC795* encode active proteins. The ability of the*alleles to rescue the synthetic slow growth of sgs1 top1 mutants was tested
by generating top1 deletions in haploid cells with sgs1 deletion and the indicated*allele. Shown is growth of serial 10-fold dilutions of double mutants containing
integrated vector, SGS1*, SGS1-DC795* or SGS1-hd* alleles, as indicated. (F) top1 deletion does not speed senescence. Diploids heterozygous for top1 and tlc1
deletions were sporulated and senescence rates of tlc1 (filled diamonds) and tlc1 top1 (open diamonds) haploid spore products were measured.
510Nucleic Acids Research, 2006, Vol. 34, No. 2
is sufficient for viability, but loss of both genes is lethal.
Alternatively, the two genes may each contribute to the
efficiency of a single pathway, so that loss of both genes
slows the pathway to below a threshold needed for viability.
By either model, some of the synthetic lethal genes might
possibly function, like SGS1, to slow the senescence of tlc1
mutants. We therefore compared rates of senescence in tlc1
strains with tlc1 strains also containing deletions of SLX1,
SLX5, MUS81, SRS2 or RRM3 (Figure 5A, B, D–F). Remark-
ably, of all the mutations in this group only slx5 had an appre-
ciable effectontherateofsenescence,accelerating senescence
by 10 population doublings (PD) (Figure 5A–F). To confirm
the effect of the slx5 mutation, we examined the effect of
mutating SLX8, which encodes the partner of Slx5p. As expec-
ted, tlc1 slx8 mutants senesced more rapidly than tlc1 mutants,
by ?20 PD (Figure 5C). Although srs2 mutation did not speed
senescence appreciably, it did delay by 2–3 days the formation
of survivors and caused a concomitant depression of the min-
imum cell density prior to survivor formation (Figure 5E).
We conclude that, with the exception of SLX5/8, none of
the genes we examined plays a role as important as SGS1
toslowsenescence.Furthermore,asdetailed inthe Discussion,
the findings indicate that generalized genome instability is not
the cause of rapid senescence in tlc1 sgs1 mutants, but rather
that Sgs1p plays a more specific role at telomeres or in
response to telomere dysfunction.
In addition to speeding the senescence of tlc1 cells, sgs1
mutation increases telomere shortening per population doub-
ling and blocks the formation of type II survivors (36,42,43).
We therefore asked if slx8 mutation also had these other
effects. Weexamined Y0-containing telomere length by South-
ern analysis (Figure 6A). slx8 mutation did not affect steady-
state telomere length in TLC1+ cells. Comparison of telomere
lengths for four tlc1 mutants at days 1 and 4 of growth in liquid
(mean PD 49 versus 75) and four tlc1 slx8 mutants at 1 and 5
(mean PD 48 versus 71), yielded telomere shortening rates
of 2.3 ± 0.12 bp/PD for tlc1 and 4.0 ± 0.62 bp/PD for tlc1
slx8 cells (P ¼ 0.04), indicating that telomeres shorten faster
per population doubling in the absence of SLX8 function
(Figure 6A and data not shown; samples were from the cul-
tures shown in Figure 5C). We next examined survivor type by
Southern analysis of XhoI-digested genomic DNA. Type I
survivors are typified by amplification of subtelomeric Y0ele-
ments of 5.2 and 6.7 kb in size, the presence of ?1 kb Y0
fragments containing terminal telomere repeat DNA, and by
the relative absence of telomere repeat-containing fragments
between these two signals. Type II survivors appear as frag-
mentsof heterogeneous size that range insize from 1to >10 kb
and they usually have less amplification of Y0elements. In
general, type I survivors predominate early in survivor forma-
tion, but because of their faster growth rate type II survivors
eventually overtake liquid cultures (40). Three of four tlc1
cultures formed type II survivors by the 17th day after loss
oftelomerase(PD 155–170)andallfourweretype IIbyday22
(PD 200–215) (Figure 6B). In comparison, two of four tlc1
slx8cultureshadcleartype IIcharacter atday17,buttherewas
little further progression to type II by day 22, and one of the
cultures (Figure 6B, G) actually developed more type I char-
acter. Thus, SLX8 may facilitate the formation or growth of
type II survivors, but, in contrast to SGS1, is not absolutely
required for type II survivors to form.
We have investigated possible mechanisms by which the
Sgs1p helicase slows the senescence of tlc1 mutants. Our
findings are most compatible with a role for Sgs1p in HR-
mediated repair of telomeres, rather than a role for the Sgs1p-
dependent S-phase checkpoint or for a generalized role of
Sgs1p in genome stability. In support of an HR role are (i)
the epistatic relationship of sgs1 and rad52 mutations in
telomerase mutants, (ii) a requirement for Sgs1p helicase
activity and (iii) a role for Top3p. Helicase activity and
cooperation with Top3p have been shown to be essential
for all of the recombination functions of Sgs1p so far invest-
igated (22,59,61,69,84). Furthermore, Rad52p is a component
for Sgs1p in Rad52p-dependent HR (85). The higher rate of
recombination among shortened telomeres in Kluyveromyces
lactis (86), the direct observation of recombinant sequences
Figure 4. Sgs1p cooperates with Top3p to slow senescence. Strains were
generated and senescence experiments were performed in the same fashion
as in Figure 2. (A) SGS1-DN50*, which encodes a derivative lacking the
N-terminal 50 amino acids of Sgs1p, does not rescue the fast senescence of
circles), tlc1 SGS1-DN50*(open diamonds) and tlc1 sgs1 SGS1-DN50* (open
circles) haploid spore products are indicated. (B) TOP3-SGS1-DN106*, which
encodes Top3p fused to an Sgs1p derivative lacking the N-terminal 106 amino
acids, rescues rapid senescence. Senescence curves for tlc1 (filled diamonds),
tlc1 sgs1 (filled circles) and tlc1 sgs1 TOP3-SGS1-DN106* (open circles)
haploid spore products are indicated.
Nucleic Acids Research, 2006, Vol. 34, No. 2 511
at the termini of ?6% of telomeres in tlc1 mutants (87), and
the rapid senescence of tlc1 mutants also lacking RAD52,
RAD51, RAD54 or RAD57 (57) support the idea that HR
can contribute to telomere maintenance during senescence.
We note that a different interpretation of the relationship
between RAD52 and SGS1 incellssenescing due to telomerase
inactivation was reported previously (43), where an additive
effect of rad52 and sgs1 mutations on senescence was
observed. However, in the report telomerase was inactivated
in haploid strains already carrying the rad52 and/or sgs1 muta-
tions, and it is thus possible that differences in telomere integ-
rity in the different mutant backgrounds prior to telomerase
inactivation contributed to senescence rates. Our experimental
approacheliminatesthisconfoundbycomparing haploid spore
products derived from diploids that contain one wild-type
allele for each mutation being studied and, further, compares
cells with telomeres derived from the same diploid parent and
is thus less subject to random fluctuations in steady state
telomere length. We are therefore confident that SGS1 and
RAD52 function in a less-than-additive, i.e. epistatic, fashion
during the senescence of telomerase mutants.
A reasonable candidate for an HR-based mechanism of
telomere repair is break-induced replication (BIR) (88–90).
Such a BIR mechanism could, for example, involve invasion
of a critically shortened telomere end into a longer telomere,
which would then be used as a template for DNA synthesis
to extend the shortened telomere. A role for the Sgs1p–Top3p
complex in this context could be to facilitate progression
during DNA synthesis, and thus resolution at the telomere
terminus, of a Holliday junction formed by such an invasion
event. Particularly in a case where the telomere DNA was
not free to rotate, as could be the case in telomeric chromatin,
the helicase activity of Sgs1p could cooperate with the strand-
passage activity of Top3p to enable separation of the newly
synthesized strand from the template. This would be mechan-
istically similar to the branch migration and resolution of
double Holliday junctions shown to be catalyzed by the
BLM–TOP3a complex (91), and may also be related to the
stallingof DNA synthesis during HRthat occursin Drosophila
blm mutants (92). A BIR mechanism has been proposed pre-
of telomerase deletion (57), and it is possible that BIR operates
both during and after senescence. BIR is RAD51-independent
when initiated from chromosomal sites cleaved by the HO-
endonuclease, but proceeds by a RAD51-dependent mechan-
ism when initiated from a transformed chromosomal fragment
(88,90). Because tlc1 rad51 mutants senesce rapidly [(57) and
Q. Chen and F.B. Johnson, unpublished], Rad51p is also
involved in slowing senescence and so any such telomere
BIR mechanism would apparently be of the RAD51-dependent
type. Consistent with the chromosome fragment assay reflect-
ing a BIR process similar to that proposed to operate at
telomeres during senescence, RAD50 and RAD59 are dispens-
able forBIR inthis assay (despite being required forBIR inthe
HO-cleavage assay) and tlc1 rad50 or tlc1 rad59 mutants
senesce at normal rates (57,90,93). Furthermore, although
SGS1 is not required for BIR at an internal chromosomal
site cleaved by the HO endonuclease (94), it remains possible
Figure 5. Effectsofsgs1syntheticlethalmutationsonsenescence.Theindicatedsyntheticlethalmutationswereintroducedindividuallyintodiploidsheterozygous
indicate tlc1 plus the synthetic lethal mutation, and filled and open triangles indicate wild-type and the synthetic lethal mutant, respectively. Comparison of tlc1
and tlc1 with (A) slx1 deletion, (B) slx5 deletion, (C) slx8 deletion, (D) mus81 deletion, (E) srs2 deletion and (F) rrm3 deletion. Note the increased number of days
(data points) spent in the nadir in tlc1 srs2 mutants.
512 Nucleic Acids Research, 2006, Vol. 34, No. 2
that it is required for BIR from non-HO cleaved ends or there
could be particular requirements for Sgs1p in recombination
involving G-rich telomere repeats, for example the unwinding
We were surprised to find that so many different genes
involved in maintenance of genome stability had no sig-
nificant effect on senescence rates. These include five ‘slx’
genes (SLX1, MUS81, SRS2, RRM3 and TOP1) whose loss
of function causes synthetic sickness or lethality in combina-
tion with sgs1 mutation, and so which might be expected to
have some functions similar to Sgs1p. Indeed, one such pair
of genes, SLX5 and SLX8, do have effects on the senescence
rate (see below). Even though the slx genes have genetic
relationships to SGS1, they clearly affect different classes
of processes required for genome stability, based on their
different genetic interactions with other factors, biochemical
activities and mutant phenotypes. Thus, the five mutations
sample the importance during senescence of several distinct
functions required for genome maintenance. Our findings are
consistent with previous demonstrations that mutations in
RAD1 or RAD10, which are required for nucleotide excision
repair and the single-strand annealing pathway of double-
strand break repair, mutations in MSH2, which is required
for mismatch repair, the pol3-01 mutation, which leads to
an elevated rate of point mutations, and mutations in TEL1
or MEC1, which mediate DNA damage responses, do not
speed the senescence of telomerase mutants (38,95). Together
with our findings, these results indicate that induction of sev-
eral different types of genome instability is not sufficient to
speed senescence, and they further suggest that Sgs1p plays a
relatively specialized role in telomere function.
In contrast to the other sgs1-synthetic lethal mutations we
tested, slx5 and slx8 mutations sped the senescence of tlc1
mutants. The molecular function of the Slx5p/8p complex is
currently unknown, and so this finding does not yet provide
any detailed mechanistic insight. However, it is interesting
that the synthetic lethality of sgs1 slx5 mutants is not sup-
pressed by rad52 mutation, unlike the synthetic lethality of
sgs1 with several other mutations including mus81, srs2 and
rrm3 (17,18,21,22,76). This might reflect an involvement of
Slx5p in a pathway for which HR is an essential component;
the key role of HR in slowing senescence is consistent with an
HR-related role of Slx5p being important during senescence.
We also found that SLX8 is not required for the formation of
type II survivors of tlc1 deletion, although their appearance
appears to have been inhibited. This contrasts with an apparent
absolute requirement for SGS1 in type II survivor formation.
However, we note that once formed, type II survivors no
longer absolutely require the continued presence of SGS1
(36), and so the functional differences between SGS1 and
SLX8 in the type II mechanism may be subtle and reflect a
particularly important role for SGS1 during the initial stages
of the formation of type II survivors.
Although srs2 mutation did not speed the senescence of tlc1
mutants, it did substantially delay the appearance of survivors.
We do not yet understand the basis for this delay, but it might
be connected with the requirement for srs2 in the process of
adaptation. Adaptation enables cells that have arrested their
cell cycle in response to DNA damage to resume growth even
prior to repair of the offending lesion. srs2 mutants show
impaired adaptation after incurring an irreparable double
strand chromosomal break (78). Because critically shortened
telomeres are interpreted by the cell as a form of DNA damage
(37,38), the survivor mechanism might involve an adaptation-
like response to enable the resumption of growth. Further
experiments will be necessary to determine if other compon-
ents of the adaptation response are required for the formation
Recently, it was reported that telomere sequences that are
the products of lagging-strand DNA synthesis are selectively
lost in Werner mutant cells (51). This was attributed toa defect
in the removal of G-quadruplexes from the replication tem-
plate caused by the absence of WRN. Our findings are con-
sistent with this possibility. However, our data suggest a role
for Sgs1p in a HR-based pathway at telomeres, and because
replication forks stall naturally in telomere repeat DNA
(81,96), and because HR is critical for the restart of stalled
replication forks (97), we also suggest an alternative that
RecQ-family helicases might remove G-quadruplexes that
Figure 6. Telomere shortening and survivor formation in tlc1 slx8 cells.
(A) Y0-containing telomere lengths from wild type, slx8 and from two inde-
pendent cultures each of tlc1 and tlc1 slx8 mutants during senescence were
(E–F) cultures was examined by Southern analysis of XhoI-digested genomic
DNA visualized with a telomere repeat probe. Samples are from days 17
(PD 155–170) and 22 (PD 200–215) after sporulation, and samples were
obtained from the cultures shown in Figure 5C. Y0and Y0term indicate the
position of tandemly-repeated Y0elements, and the terminal Y0fragment,
respectively, that are most prominent in type I survivors. On the right, samples
are provided for comparison, and the position of DNA markers is indicated
with sizes in kilobases (kb).
Nucleic Acids Research, 2006, Vol. 34, No. 2513
would otherwise interfere with HR-based rescue of stalled
replication forks at telomeres.
Nina Luning-Prak and the members of the Johnson laboratory
for discussions and comments on the manuscript. This work
was supported by grants from American Federation for Aging
Research and the National Institute on Aging to F.B.J.
(5R01AG021521) and a National Research Service Award
to J.Y.L. is (F32AG22769). Funding to pay the Open Access
publication charges for this article was provided by the
University of Pennsylvania School of Medicine.
Conflict of interest statement. None declared.
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