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Molecular and Cellular Biology, March 2001, p. 1819-1827, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1819-1827.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Two Survivor Pathways That Allow Growth in the
Absence of Telomerase Are Generated by Distinct Telomere
Recombination Events
Qijun
Chen,
Arne
Ijpma, and
Carol W.
Greider*
Department of Molecular Biology and Genetics,
Graduate Program in Cell and Molecular Medicine, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
Received 25 July 2000/Returned for modification 20 September
2000/Accepted 30 November 2000
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ABSTRACT |
Yeast cells can survive in the absence of telomerase RNA,
TLC1, by recombination-mediated telomere elongation. Two
types of survivors, type I and type II, can be distinguished by their
characteristic telomere patterns. RAD52 is essential for
the generation of both types of survivors. Deletion of both
RAD50 and RAD51 produces a phenotype similar to
that produced by deletion of RAD52. Here we examined the
effects of the RAD50 and the RAD51 epistasis
groups as well as the RAD52 homologue, RAD59,
on the types of survivors generated in the absence of telomerase.
rad59 mutations completely abolished the ability to
generate type II survivors, while rad50 mutations decreased
the growth viability of type II survivors but did not completely
eliminate their appearance. Mutations in RAD51, RAD54, and
RAD57 had the converse affect: they eliminated the ability
of cells to generate type I survivors in a tlc1 strain. The
triple mutant, tlc1 rad51 rad59, was not able to generate survivors. Thus either type I or type II recombination pathways can
allow cells to survive in the absence of telomerase; however, elimination of both pathways in a telomerase mutant leads to the inability to elongate telomeres and ultimately cell death.
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INTRODUCTION |
Telomere integrity is essential for
stable chromosome transmission and for cell viability. In
Saccharomyces cerevisiae, telomeres contain a characteristic
pattern of repeated sequences. The most terminal telomere sequences
consist of an irregular repeat of TG1-3 sequences
(25). Just internal to the TG1-3 repeats,
there are more complex telomere-associated repeats, termed Y' elements
and X elements. All yeast telomeres have one X element, whereas Y'
elements are found only at some telomeres (3). Y' elements
are often found in multiple copies, and in some cases there are
TG1-3 repeats between adjacent Y' elements
(3). Two classes of Y' elements have been defined, Y'-long
and Y'-short, which are 6.7 and 5.2 kb in length, respectively
(17, 18).
Telomere function depends on a number of specific proteins that bind to
the terminal TG1-3 repeats (reviewed in reference 6). In wild-type yeast cells, the overall length of the
TG1-3 sequence is maintained at an equilibrium of around
300 bp, by activities that remove and those that add TG1-3
telomeric repeat sequences. Telomere repeats are added by the enzyme
telomerase (8), which contains an essential RNA component
(TLC1) (27) and a catalytic protein component
(EST2) (16). In the absence of telomerase,
telomeres shorten progressively due to incomplete end replication and
possibly also specific nuclease activity, and after a lag of about 60 cell divisions, the growth potential of the culture decreases
(19, 21, 27). This suggests that after a certain number of
TG1-3 repeats have been lost from yeast chromosomes,
telomere function is lost.
Although telomerase is the major pathway for telomere elongation, in
the absence of telomerase, recombination can efficiently elongate
telomeres and lead to the survival of a population of cells
(19). In a tlc1 culture, there is a progressive
decline in the fraction of growing cells to less than 1%, followed by the selection for a small population of cells that maintains telomeres via a recombination-mediated pathway (19). If the major
recombination gene, RAD52, is deleted, these "survivor"
cells are not generated. Thus, telomere elongation in the survivors is
thought to occur via a recombinational mechanism (19).
Break-induced replication (BIR) is a gene conversion mechanism that can
allow telomere healing. A centromere-proximal double-stranded break can
be healed by the broken end invading a homologous region on another
chromosome (20, 23). A replication fork can be established, and the entire chromosome arm can be copied, resulting in
the duplication of a large portion of the chromosome arm
(12). The mechanism proposed for the generation of
survivors in the absence of telomerase is similar to the mechanism
proposed for BIR (5, 19, 29).
Two apparently distinct types of telomere elongation events can occur
via recombination in the absence of telomerase (19, 29).
The two types of survivors can be distinguished on a Southern blot by
the characteristic telomere bands that appear. Type I survivors show
amplification of the telomere-associated Y' elements and have very
short TG1-3 repeat tracts on the ends. Type II survivors
show a variable pattern of long tracts of TG1-3 repeats
and only modest Y' amplification (29). In a
tlc1 mutant, there is a distribution of the two survivor
types. The exact percentage of each type is strain dependent (19,
29). Type II survivors grow faster than type I survivors. Thus,
if a continuous liquid culture is grown, only type II will be seen at
the end of a long growth period. To determine the distribution of
survivor types, single colonies from plates where there was no growth
competition must be assayed (29).
Two independent pathways defined by RAD50 and
RAD51 are both capable of generating survivors
(15). The RAD51 pathway involves RAD51,
RAD54, and RAD57 (15). These genes are
known to interact with each other genetically, and the protein products
interact physically (11). The RAD50 pathway
involves RAD50, XRS2, and MRE11. The protein
products from these genes interact to form a complex that participates
in both homologous recombination and DNA end-joining pathways (reviewed
in reference 10). Deletion of either RAD50 or
RAD51 alone does not abolish the ability of yeast cells to
generate survivors. However, deletion of RAD50 and
RAD51 prevents the generation of survivors in the absence of
telomerase (15).
To explore these two recombination pathways in more detail, we examined
the role of RAD59 in the generation of survivors. RAD59 was identified as a gene that when mutated decreased
recombination in a rad51 background (1).
RAD59 has homology to RAD52, and overexpression
of RAD52 will rescue a RAD59 mutant but not vise versa. Deletion of RAD59 has little effect on its own in
most recombination assays; however, in a rad51 background,
rad59 significantly decreases spontaneous intrachromosomal
recombination (1). This synergistic effect of the two
mutations suggests that rad59 plays a role in a
recombination pathway separate from the rad51 pathway. In
other assays, however, such as HO cleavage-induced recombination, there
is little synergy between the rad59 and rad51
mutations (28).
To investigate the role of RAD59 and the RAD50
and RAD51 epistasis groups in telomere maintenance, we
combined mutations in the telomerase RNA, TLC1, with single
and double mutations in these pathways. The tlc1 rad50 and
tlc1 rad59 mutants generated predominantly type I survivors,
while the tlc1 rad51, tlc1 rad54, and tlc1 rad57
mutants generated only type II survivors. This suggests that the two
genetic pathways, which we previously defined, can be distinguished by
their different physical effects at telomeres. Both RAD50
and RAD59 play a role in a RAD51-independent
survivor pathway. Recent experiments that demonstrate a
RAD51-independent BIR pathway involving RAD59 and
the RAD50-MRE11-XRS2 complex suggested that BIR may be the
mechanism that allows the generation of survivors in the absence of
telomerase (L. Signon, A. Malkova, M. Naylor, H. Klein, and J. E. Haber, unpublished data).
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MATERIALS AND METHODS |
Yeast strains and plasmid constructs.
All yeast strains used
in this study are summarized in Table 1.
The isogenic strains with mutations in several RAD genes and
TLC1 gene have been described previously (22).
Strains JHUY564 and JHUY563
(tlc1::URA3/TLC1
rad50::hisG'/RAD50
rad59::kanMX4/RAD59 and
tlc1::URA3/TLC1
rad51::LEU2/RAD51
rad59::kanMX4/RAD59, respectively) were
constructed by PCR-mediated gene disruption (2) of the RAD59 gene in strains CSHY92 and CSHY91, respectively. The
rad59::kanMX4 deletion fragment was PCR
amplified from pRS400 (2) with oligonucleotides AY23F
(5'AGTTTAGCACATGCTTTGGACCATTAAAGGGTTACGTAGAGATTGTACTGAGAGTGCAC 3')
and AY24R
(5'ATCAACGATACTGTTGATAAAGGTAAGTCGATACCTGTTCTGTGCGGTATTTCACACCG 3'). The yeast strains CSHY91 and CSHY92 were transformed
using the lithium acetate method (24). The
rad59 deletions were confirmed by Southern blot analysis.
The genomic DNA was digested with BstEII and probed with a
DNA sequence produced by PCR using the oligonucleotides AY25F (5'
CATACCATTCCAAGGTAA 3') and AY26R (5' TAGCAGGCGACGAAGAAT 3')
by random hexamer primed DNA synthesis with the Klenow fragment in the presence of 32P-labeled dATP and dGTP
(7). Deletion of RAD59, TLC1, and
RAD50 in all diploid strains was confirmed by Southern blot
analysis. The pRAD59 plasmid was constructed by cloning the 1.2-kb
ScaI-BglII fragment of the RAD59 gene
(1) by PCR amplification with oligonucleotides QC22F
(5'CGGGATCCCGGCATCACCCATAATTG) and QC23R
(5'GCTCTAGAGCCCATGCCTTCGTTACC). The resulting fragment was
cloned into the vector pRS315 (26) to make pRAD59. The
full 1.2-kb ScaI-BglII DNA fragment was sequenced to verify its accuracy. To generate the appropriate haploid strains for
the cell density assay, triple- or double-mutant diploid cells were
sporulated, tetrads were dissected, and spore clones were tested for
the appropriate markers or by Southern blot analysis.
Cell viability assay.
Cells of the appropriate genotype were
picked up from a fresh dissecting plate and grown in yeast
extract-peptone-dextrose (YPD) medium to saturation (1 × 108 to 2 × 108 cells/ml). Every 24 h the
cell density was measured by counting cells in a hemocytometer, and
then the culture was diluted with fresh YPD liquid medium to a density
of 105 cells/ml (15, 27). This cycle was
repeated for 10 to 16 days. At various time points during growth, cells
were plated to examine for possible contamination and some samples were
collected and frozen for telomere length analysis.
Single-colony streak assay.
Cells of the appropriate
genotype were picked from a fresh dissecting plate and streaked onto a
YPD plate. After incubation for 48 h at 30°C, single colonies
were picked and restreaked on a YPD plate. This restreaking was
repeated four times to allow loss of viability and appearance of
survivors. Generation of survivors typically occurred after 3 to 4 days
at 30°C. Single colonies from streak 5 were grown in YPD medium
overnight, and cells were pelleted for the telomere length assay.
Telomere Southern blots.
Southern blot analysis was
performed for examination of telomere length. Yeast genomic DNA was
isolated, and ~4 µg of DNA was digested with XhoI and
separated on a 1% agarose gel. DNA was then transferred to a HybondN+
(Amersham, Piscataway, N.J.) membrane and UV cross-linked. The membrane
was then hybridized with a random primed telomeric poly(d[GT/CA])
(Pharmacia, Piscataway, N.J.), and hybridization was detected with an
ECL direct nucleotide labeling kit (Amersham).
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RESULTS |
RAD59 is required to generate survivors in a
rad51 background.
RAD59 defines a
RAD51-independent recombination pathway (1). To
examine how RAD59 affects the two previously defined
telomere maintenance pathways involving RAD51 and
RAD50, we compared the viabilities and telomere lengths of a
set of isogenic mutants, including the tlc1, rad59, tlc1 rad59,
tlc1 rad51 rad59, and tlc1 rad50 rad59 mutants. The
growth of these cells and the ability to generate survivors were
compared to growth and survivor generation of the wild-type, tlc1
rad52, and tlc1 rad50 rad51 cells. Cell viability was
measured by diluting liquid cultures to 105 cells/ml,
allowing them to grow for 24 h, and then measuring the cell
density. While wild-type cells reach a density of 108
cells/ml in 24 h, mutant cells that have a loss of viability or a
decreased growth rate will reach lower cell densities (15, 27). The ability to generate survivors was defined as the
ability of the culture to recover after reaching a minimum growth rate after about 6 days in culture. The tlc1 rad59 double mutant
showed a decline in its growth rate similar to that of the single
tlc1 mutant, and survivors were generated. In contrast, the
tlc1 rad51 rad59 triple mutant showed an accelerated decline
in growth similar to the accelerated rate of decline seen in tlc1
rad52 (Fig. 1) (15,
19). As with the tlc1 rad52 and tlc1 rad50
rad51 mutants, no survivors were generated in the tlc1 rad51
rad59 mutant culture. These results indicate that RAD59
plays a role in generation of survivors and that RAD59 and
RAD51 are in separate pathways for the generation of
survivors in the absence of telomerase.
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FIG. 1.
RAD59 is required to generate survivors in a
rad51 background. Cells were grown to saturation in YPD
medium and then diluted to 105 cells/ml every 24 h with
fresh YPD medium. Cells were counted every 24 h with a
hemocytometer. The curves shown are the average of results for four
independent clones from each genotype.
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To determine whether the accelerated decline in the growth rate for the
tlc1 rad51 rad59 mutant resulted from a more rapid
telomere
shortening, we examined telomere lengths in three mutants,
tlc1,
tlc1 rad52, and
tlc1 rad51 rad59. There was no evidence
for a more rapid telomere shortening in the
tlc1 rad51 rad59
or
tlc1 rad52 mutants than in the
tlc1 mutant in
the first 3 days
of culturing (Fig.
2).
Thus the accelerated decline in the growth
rate of
tlc1 rad51
rad59 mutants is not due to a faster decrease
in telomere length.
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FIG. 2.
The tlc1 rad51 rad59 mutant does not show a
higher rate of telomere shortening. tlc1, tlc1
rad52, and tlc1 rad51 rad59 cells were grown for five
successive days in liquid culture, and telomere length was determined
on Southern blots for the first 3 days. The tlc1 rad52 and
tlc1 rad51 rad59 mutants reached a minimum growth rate at
day 4. Solid arrows indicate X telomeres and the open arrow indicates
Y' telomeres in the wild-type strain.
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In contrast to results for the
tlc1 rad51 rad59 triple
mutant, the
tlc1 rad50 rad59 triple mutant did generate
survivors (Fig.
1). However, the recovery for the
tlc1 rad50
rad59 triple mutant
was slower than that for
tlc1. This
slow recovery is similar to
the slow recovery of
tlc1 rad50
mutants (
15) and may be due
to the low growth rate of
rad50 mutants (
14). The ability of
the
tlc1 rad50 rad59 mutants to generate survivors as do the
tlc1 rad50 and
tlc1 rad59 mutants suggests that
RAD50 and
RAD59 are
in the same pathway for
telomere maintenance in the absence of
telomerase. The failure of
tlc1 rad51 rad59 to generate survivors
supports the
conclusion that
RAD59 and
RAD51 are in different
pathways.
tlc1 rad59 mutants generate only type I survivors.
In the absence of telomerase, at least two distinct types of survivors
are generated (19, 29). Type I and type II survivors can
be distinguished by the pattern of telomere restriction fragments on
Southern blots. The telomere bands in type II survivors have long
tracts of telomeric TG1-3 sequence and some amplification of Y' elements (Fig. 3) (29). In these survivors, the
multiple distinct telomere bands between 1 and 6 kb represent
individual chromosomes with different lengths of TG1-3
repeats. The major XhoI band near 1 kb, representing
telomeres containing Y' elements, is no longer prominent, since these
short telomeres are all elongated. In contrast, type I survivors have a
short XhoI band just below 1 kb, lack telomere bands between
1 and 6 kb, and show strong amplification of the 5.2- and 6.7-kb Y'
elements. Since type II survivors have a growth advantage over type I
survivors, type II survivors are expected to predominate when cells are
grown in liquid culture (29). Consistent with this, the
Southern analysis of tlc1 survivors grown in liquid culture
showed type II survivors. In contrast, the liquid cultures of
tlc1 rad59 mutants showed only type I survivors (Fig.
3). Thus the absence of RAD59
inhibits type II survivor formation.
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FIG. 3.
tlc1 rad59 double mutants generate only type
I survivors. Southern blots of tlc1 and tlc1
rad59 mutants are shown. The numbers at the top of the lane
represent the number of days that cells were grown in liquid culture.
The letter S at the top of the lanes represents survivor cells that
were streaked out at least two times after survivors were generated.
The numbers on the side indicate the DNA molecular size markers in
kilobases. The survivor type is indicated below each lane as type I or
type II. Solid arrows indicate X telomeres and open arrows indicate Y'
telomeres in the wild-type strain. (A) tlc1 mutants were
grown in liquid culture, and telomeres were measured on Southern blots
at days 1 and 4, before the generation of survivors (lanes 1 and 2). At
days 9, 10, and 15, type II survivors were apparent (lanes 3 to 5).
Wild-type telomeres are shown for comparison in lane 6. (B) The
telomere patterns of tlc1 rad59 double mutants are shown
before the generation of survivors at days 1, 4, and 9 (lanes 2 to 4)
and after type I survivors were generated at days 13, 17, and 21 (lanes
5 to 7). (C) The single-colony assay was used for tlc1 rad59
cells. Wild-type cells (lane 1), a presurvivor colony at the second
streak-out (lane 2), and four independent type I tlc1 rad59
survivor colonies are shown.
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Growth in liquid culture for many population doublings provides a
strong selection for the fastest-growing cells. Thus we
used a
single-colony assay to determine the distribution of survivor
types in
mutant strains. We plated for single colonies and assayed
for type I or
type II telomere length patterns on Southern blots.
The
tlc1
and
tlc1 rad59 cells were each streaked five successive
times on YPD plates until survivors were generated (approximately
125 cell divisions). Single colonies were then inoculated into
liquid
culture for overnight growth, and genomic DNA was isolated.
Of 39 single
tlc1 colonies assayed, 24 (62%) showed a type I
pattern
and 15 (38%) showed a type II telomere pattern (Table
2 and Fig.
5A). This ratio of type I to
type II is similar to that found
in other strain backgrounds (
19,
29). In contrast, of the
22
tlc1 rad59 single
colonies analyzed, all 22 showed type I survivors
(Table
2 and Fig.
3C). This result further supports the conclusion
that
RAD59
is required for the formation of type II survivors.
tlc1 rad50 mutants have a reduced percentage of type II
survivors.
The specific requirement for RAD59 for the
generation of type II survivors suggested that the two different
survivor pathways defined by the RAD50 and RAD51
epistasis groups might independently affect the two survivor types. We
thus assayed whether deletion of genes in these groups would also
affect the distribution of survivor types. In a liquid culture assay,
the cell density of the tlc1 rad50 double mutant declined
continuously until survivors were generated, and then the cell density
increased (data not shown) (15). Telomere length in the
tlc1 rad50 double mutant shortened progressively, and in two
of three independent experiments only type I survivors were found. In
the third experiment, type II survivors were detected at day 10, but
they were no longer present at days 15, 19, and 23 (Fig.
4). The cell growth rate from day 10 to
23 was similar to that of wild-type cells (data not shown). This
suggested that type II survivors were initially formed but that they
were not maintained during continued growth.
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FIG. 4.
Type II survivors that are generated in tlc1
rad50 cells are not maintained during continuous culturing.
tlc1 cells were cultured for 15 days, and telomeres were
examined at days 1, 4, 9, 10, and 15 (lanes 1 to 5). Type II survivors
were detected at days 9, 10, and 15 (lanes 3 to 5). tlc1
rad50 cells were grown for 23 days, and telomeres were examined at
days 1, 4, 10, 15, 19, and 23 (lanes 8 to 12). Type II survivors were
detected at day 10 but not at days 15, 19, and 23. The survivor type is
indicated below each lane as type I or type II.
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The instability of type II survivors in
tlc1 rad50 cells was
also seen in single-colony assays. In the first streak after
survivor
generation, 2 of 20 individual
tlc1 rad50 survivor colonies
assayed showed the type II pattern (Table
2). However, after
streaking
an additional three times, only type I survivors were
detected (data
not shown). This is consistent with the appearance
of type II survivors
in
tlc1 rad50 mutants seen in earlier work
(
15)
when cells were assayed soon after survivor generation.
These data
suggest that type II survivors are generated at a low
frequency in
tlc1 rad50 mutants but cannot be maintained during
subsequent growth. Thus
RAD50 facilitates the formation and
maintenance
of type II
survivors.
tlc1 rad51, tlc1 rad54, and tlc1 rad57
mutants generate only type II survivors.
RAD51 defines
a pathway that includes RAD54 and RAD57 and is
separate from the RAD50 pathway that is able to maintain
telomere length in the absence of telomerase (15). We used
Southern analysis to determine the type of survivors that are formed in
tlc1 rad51, tlc1 rad54, and tlc1 rad57
double-mutant cells. In liquid culture, all survivors showed a type II
pattern (15) (data not shown). Since type II survivors
have a growth advantage and will take over a liquid culture, we assayed
single colonies to examine the distribution of survivor types.
tlc1 rad51, tlc1 rad54, and tlc1 rad57 double
mutants were streaked five times on YPD plates to allow the generation
of survivors, and single colonies were examined by Southern blot
analysis. Each of the double mutants tlc1 rad51, tlc1 rad54,
and tlc1 rad57 generated only type II survivor clones (Table
2 and Fig. 5B, C, and D). This differs
significantly from the tlc1 single-mutant survivors in the
same genetic background, where only 38% of the survivors were type II
(Table 2 and Fig. 5A). Thus RAD51, RAD54, and
RAD57 are required for the formation of type I
survivors in the absence of telomerase.
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FIG. 5.
tlc1 rad51 mutants generate only type II
survivors in the single-colony assay. tlc1 and tlc1
rad51 cells were streaked out repeatedly until survivors were
generated (see Material and Methods). DNA samples from either
presenescence cells (streak 2) or survivor cells (S) were used for
telomere length pattern analysis. (A) For tlc1, both type I
(lanes 3 to 5) and type II (lanes 2 and 6) survivors were detected. (B)
For tlc1 rad51, of four independent survivors, all were type
II (lanes 3 to 6). (C) For tlc1 rad54, of four independent
survivors, all were type II (lanes 3 to 6). (D) For tlc1
rad57, of four independent survivors, all were type II (lanes 3 to
6). The survivor type is indicated below each lane. Solid arrows
indicate X telomeres and open arrows indicate Y' telomeres in the
wild-type strain.
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Reintroduction of the RAD59 gene into late tlc1
rad59 survivors does not reestablish a wild-type distribution of
type I and type II survivors.
To examine the role that
RAD59 might play in the generation of type II survivors, we
tested whether reintroduction of the wild-type gene at three different
points during survivor generation would rescue the ability to generate
type II survivors (Fig. 6A). A plasmid
containing RAD59 was transformed into a diploid heterozygous for tlc1 and rad59. The diploid was sporulated,
and haploid spore clones were selected. tlc1 rad59 spore
clones without the pRAD59 plasmid generated only type I survivors in
liquid culture (Fig. 6B). However, tlc1 rad59 spore clones
with the pRAD59 plasmid generated type II survivors, showing that the
plasmid can complement the loss of rad59 from the genome
(Fig. 6B) (see Materials and Methods). As an independent control, we
tested the methyl methanesulfonate (MMS) sensitivity of the spore
clones. The cells without pRAD59 were sensitive to MMS, but the cells
with pRAD59 were not (data not shown). We next tested whether
transformation of the pRAD59 plasmid into tlc1 rad59 cells
just before or just after survivors were generated would affect the
distribution of survivor types. Transformation into a culture that had
undergone approximately 60 cell divisions (before survivors were
generated) restored the ability to generate type II survivors in 10 of
the 18 transformants examined. The remaining 8 transformants showed the
type I pattern (Fig. 6C and data not shown). Transformation of a
control vector plasmid generated only type I survivors, indicating that
the transformation itself did not restore type II survivors.
Strikingly, transformation of the pRAD59 plasmid into cells that were
initially grown for 130 cell divisions (after survivors were generated)
did not restore the appearance of type II survivors (Fig. 6D).
Telomeres were examined for five independent transformants at days 1, 2, 3, and 4 during growth of the culture, and an additional seven
transformants were assayed at day 4 only. None of these transformants
showed type II survivors (Fig. 6d and data not shown). Thus the
restoration of RAD59 function after the establishment of
type I survivors does not allow conversion to type II despite the type
II survivor growth advantage.
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FIG. 6.
Reintroduction of the RAD59 gene into
late-generation tlc1 rad59 survivor cultures does not
reestablish a wild-type distribution of survivor types. (A) The pRAD59
plasmid was transformed into tlc1 rad59 cells at various
times during the generation of survivor cells as indicated. (B)
tlc1 rad59 spore clones that did not contain the pRAD59
plasmid (lanes 2 to 6) and those that did (lanes 7 to 11) were analyzed
after 12 days of growth in liquid culture. Two additional spore clones
containing the pRAD59 plasmid were also analyzed at day 12. (C) pRAD59
was transformed into tlc1 rad59 cells at generation ~60,
and independent transformants were examined for their survivor type
distribution by Southern blot analysis. Two independent transformants
containing pRAD59 (lanes 2 to 4 and 5 to 7) were examined at days 1, 5, and 8. Control cells transformed with the pRS315 vector only are shown
in lane 8. Additional independent transformants containing the pRAD59
plasmid were assayed at day 8 (lanes 9 to 13) or day 11 (lane 14). (D)
pRAD59 or the vector pRS315 was transformed into tlc1 rad59
cells at generation ~130 (see Material and Methods), and independent
transformants were cultured for 4 days to test survivor types. Two
transformants containing pRAD59 (lanes 2 to 5 and 6 to 9) and one with
the pRS315 vector (lanes 10 to 13) were assayed for telomere pattern.
Additional independent colonies containing pRAD59 (lanes 15 to 18) or
vector (lane 19) at day 4 were assayed. Numbers on the side are
molecular size markers in kilobases. Lanes marked wt contain wild-type
DNA as a control. Plas, plasmid.
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DISCUSSION |
Two types of survivors are generated via recombination-mediated
telomere elongation in the absence of telomerase. Specific genes
mediate the pathways that allow generation of the survivors; the
RAD51, RAD54, and RAD57 genes are involved in the
generation of type I survivors, and both RAD59 and
RAD50 mediate the generation of type II survivors (Fig.
7). When both survivor generation
pathways are eliminated, as in the tlc1 rad50 rad51 mutants
or the tlc1 rad51 rad59 mutants, no survivors are generated.
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FIG. 7.
Two types of survivors are generated by two distinct
genetic pathways. A telomere containing two tandem Y' elements (large
gray boxes) separated by TG1-3 repeats (small white boxes)
is represented at the top. Telomere shortening occurs in the absence of
telomerase. Survivors can be generated via two different mechanisms.
(A) The RAD51-, RAD54-, and
RAD57-dependent pathway generates type I survivors. Telomere
shortening continues into the Y' element, exposing single-stranded 3'
overhangs. The single-stranded DNA invades a homologous region in the
Y' element on some other telomere, and BIR allows duplication of the
intact telomere onto the telomere which had lost the TG1-3
repeats. The telomere that is copied is shown in light gray for
clarity, to reveal the 3' end elongation of the invading strand. This
kind of recombination event could also occur at X sequences on
telomeres that do not contain Y' elements (see the text). (B) Telomere
shortening results in recombination before all of the telomere repeats
are lost from the ends. RAD50 and RAD59 allow
recombination in the irregular TG1-3 repeats to occur
efficiently. Alternatively, the telomeric TG1-3 tracts
self-prime DNA replication and allow extension of the telomere
sequences with a rolling-circle-type mechanism.
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RAD51, sequence identity-dependent strand annealing,
and type I survivors.
Type I survivors involve the amplification
of the telomere-adjacent Y' elements and require the presence of
RAD51. These survivors may arise via continued resecting of
the telomere in the absence of telomerase until the Y' elements are at
the molecular terminus. Recombination is then initiated within the Y'
elements. If the recombination occurs with an internal Y' element on a
chromosome that has tandem Y' elements, amplification of Y's will occur
(Fig. 7A). Alternatively, on chromosomes that have only X elements, resectioning into the X sequences may allow recombination with other X
elements. This could lead to all chromosomes picking up Y's, since many
chromosomes have both X and Y' elements. This would explain the
apparent disappearance of the X-containing bands between 1 and 6 kb in
the type I survivors. The requirements for RAD51 for type I
survivors may be due to the fact that recombination in the Y's requires
a high level of sequence identity. RAD51 mediates strand
invasion during recombination, and it is very sensitive to the
mismatches in the homologous region (4). Y' elements are
very well conserved in sequence, showing only 1% sequence divergence
(17), while TG1-3 telomere repeats are much more irregular in sequence. Thus, RAD51 may be able to
mediate the recombination between Y's but not be able to mediate the
recombination that occurs between the less identical TG1-3
tracts. In the absence of RAD51, the recombination events
between Y' elements cannot be carried out.
RAD59 and the establishment of type II survivors.
RAD59 is involved in a RAD51-independent mitotic
recombination pathway mediating recombination between intrachromosomal
inverted repeats (1). Consistent with this, we found that
RAD59 plays a role in a RAD51-independent pathway
in the generation of tlc1 survivors. In the absence of
RAD59, all survivors were type I, indicating that
RAD59 promotes the generation of type II survivors. These
survivors likely arise via a recombination mechanism that is initiated
within the TG1-3 repeats themselves. This recombination event could be initiated through either an interchromosomal invasion and copying of the TG1-3 repeats or perhaps
intrachromosomal copying of a telomere that is looped back on itself
(Fig. 7B). There is evidence that mammalian telomeres form t-loops, in
which the telomere sequences are looped back on themselves and the 3' G-rich overhang is base paired with a duplex region of repeats forming
a D-loop (9). A D-loop resembles half of a replication fork and may be able to prime DNA polymerization. Perhaps such a
structure would become deregulated upon telomere shortening and serve
as the substrate for the generation of type II survivors (Fig. 7B).
RAD50 is required for maintenance of type II
survivors.
rad50 mutants can initially generate type II
survivors at a low rate, but these survivors are not maintained over
time (Table 2 and Fig. 3) (15). Recently it was found that
Rad50 protein is bound to human telomeres, and it was proposed that
Rad50 is involved in the establishment of the t-loop structure
(30). The initial appearance and subsequent loss of type
II survivors in tlc1 rad50 mutants is consistent with
rad50 playing a role in the efficient establishment of
t-loops. If t-loops form in rad50 cells but at a lower rate
than usual, those cells that were able to establish t-loops could
initially generate type II survivors via priming from the 3' end in the
t-loop. However, in the absence of telomerase, there is strong pressure
to maintain telomere length since many cells in the culture stop
dividing when telomeres become short; perhaps the impaired t-loop
formation in rad50 mutants cannot keep up. Thus the type I
survivors in the culture are able to take over and become the dominant
survivor type.
Break-induced replication and survivors arise through a similar
mechanism.
The mechanisms outlined in Fig. 7 by which survivors
are generated in the absence of telomerase may be similar to BIR. BIR is one mechanism by which a double strand break can be healed. If there
is sequence homology on one side of a break and not on the other, the
homologous region can invade, pair with, and copy an entire chromosome
arm (20). The mechanisms proposed for the generation of
survivors (5) (reviewed in references 13, 19, and
29) are very similar to that proposed for BIR. Recent
experiments indicate that the genetic requirements for BIR are similar
to those for survivor generation. There is a
RAD51-independent BIR pathway involving RAD59 and
the RAD50-MRE11-XRS2 complex (Signon et al., unpublished
data). rad51 or rad50 single mutants do not eliminate BIR, but rad51 rad50 double mutants dramatically
reduce BIR. Similarly, rad59 single mutants have little
effect on BIR, yet rad51 rad59 double mutants reduce BIR
significantly. These genetic requirements could reflect the two
mechanisms outlined in Fig. 7 for the two genetic pathways that allow
generation of survivors.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the NIH, GM43080, to
C.W.G.
We thank Anna Malkova for testing MMS sensitivity. We thank Siyuan Le,
Jennifer Hackett, and Kay Keyer-Opperman for critical reading of the
manuscript. We thank James Haber for sharing data before publication
and for helpful discussions.
| |
ADDENDUM IN PROOF |
While this paper was under review, Teng et al. published a paper
that also showed a requirement for RAD50 for type II survivors (S. Teng, J. Chang, B. McCowan, and V. A. Zakian, Mol. Cell
6:947-952, 2000).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Genetics, Johns Hopkins University School of
Medicine, 617 Hunterian, 725 N. Wolfe St., Baltimore, MD 21205. Phone:
(410) 614-6506. Fax: (410) 614-2987. E-mail:
cgreider{at}jhmi.edu.
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Molecular and Cellular Biology, March 2001, p. 1819-1827, Vol. 21, No. 5
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.5.1819-1827.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
