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Molecular and Cellular Biology, October 2001, p. 6559-6573, Vol. 21, No. 19
Department of Biochemistry, Tulane University
Health Sciences Center, New Orleans, Louisiana 70112
Received 12 February 2001/Returned for modification 12 March
2001/Accepted 22 June 2001
We have previously identified a process in the yeast
Saccharomyces cerevisiae that results in the contraction
of elongated telomeres to wild-type length within a few generations. We
have termed this process telomeric rapid deletion (TRD). In
this study, we use a combination of physical and genetic assays to
investigate the mechanism of TRD. First, to distinguish among several
recombinational and nucleolytic pathways, we developed a novel physical
assay in which HaeIII restriction sites are positioned
within the telomeric tract. Specific telomeres were
subsequently tested for HaeIII site movement between
telomeres and for HaeIII site retention during TRD.
Second, genetic analyses have demonstrated that mutations in
RAD50 and MRE11 inhibit TRD. TRD,
however, is independent of the Rap1p C-terminal domain, a central
regulator of telomere size control. Our results provide evidence that
TRD is an intrachromatid deletion process in which sequences near the
extreme terminus invade end-distal sequences and excise the intervening
sequences. We propose that the Mre11p-Rad50p-Xrs2p complex prepares the
invading telomeric overhang for strand invasion, possibly
through end processing or through alterations in chromatin structure.
The length of the simple sequence
tracts present at the telomere is tightly regulated by coordinated
mechanisms of telomere lengthening and shortening, leading to a
steady-state equilibrium (20, 63, 66, 73). In the yeast
Saccharomyces cerevisiae, individual telomeres consist
of irregular TG1-3 sequences, with each complete
telomeric tract maintained within 50 bp of a
genetically determined mean size (63, 73). In yeast, as in
most eukaryotes, the enzyme responsible for telomere lengthening is
telomerase, a ribonucleoprotein reverse transcriptase
consisting of a catalytic protein subunit, encoded by the
EST2 gene (36, 37, 41), and an RNA species,
encoded by the TLC1 gene (64). The RNA
component of telomerase contains the template for the
addition of telomeric repeats onto G-rich
single-stranded substrates (20, 21, 64). In cells
lacking telomerase, recombination among
subtelomeric or telomeric sequences can also
result in telomere lengthening (69, 70).
The mechanisms that counteract the possibly unlimited lengthening by
telomerase are less well understood. Recent studies have provided evidence for several non-mutually exclusive mechanisms for
maintaining a genetically set equilibrium of sizes. First, the major
telomere binding protein, Rap1p, associating with the telomeric tract sequence motif
GGTGTGTGGGTGT (14), is directly involved in achieving telomere length homeostasis. Rap1p activity is mediated in part through recruitment of positive and negative regulators of telomere addition to the Rap1p C-terminal 165 amino acids (10, 25, 30, 52, 77). The number of bound Rap1p C
termini, or of factors associating with the C termini, is counted through an as-yet-unknown mechanism, until an optimal stable length is
reached (44, 61). According to this model, shorter
telomeres are elongated via telomerase to wild-type
sizes, while overelongated telomeres gradually lose
telomeric repeats until the wild-type size is regained. In
this way, a homeostasis between addition and loss of
telomeric sequences could be established (43, 44, 72). Second, Rap1p interacts directly or indirectly with
telomerase to form a cap against the uncontrolled addition
of telomeric tracts (29, 65). Third, the
telomeric single-strand binding protein Cdc13p recruits
multiple complexes to the telomeric 3' single-stranded overhang, where they act to maintain a balance between telomere elongation and loss at the extreme terminus (9, 17, 18, 40, 56,
60).
In addition to these mechanisms, we have proposed that the rapid
truncation of overelongated telomeres, termed telomeric
rapid deletion (TRD), negatively regulates telomere length
(35). This mechanism is distinct from the slow attrition
of telomeric sequences observed after increases in
telomere tract size (43). TRD is observed in wild-type
RAP1 strains containing a subset of telomeres that
range in size from 400 to 3,000 bp greater than that of wild type, with the vast majority (>80%) of deletion events at an
individual telomere reducing telomeric tracts to
wild-type size (termed complete deletions). For the remaining events
(<20%), deletions lead to intermediate sizes (termed incomplete
deletions). TRD occurs at the relatively high rate of 3 × 10 Loss of the major recombination protein, RAD52, diminishes
the TRD rate, implicating recombination in TRD (35). In
addition, hpr1 cells, which display an elevated rate of
recombination between direct repeats (1), also show an
increase in TRD, raising the possibility that TRD may be an
intrachromatid recombination event. However, no definitive mechanistic
conclusions could be drawn from these initial genetic studies.
The frequency of complete rapid deletion events at an individual
telomere depends upon the lengths of other telomeres in the cell (35). Cells that contain an increased number of
wild-type length telomeres have a corresponding increase in
complete deletions. Based on these results, we have proposed two
components of TRD: a recombinational process between imperfect
(homeologous) repeats and a "yardstick" that measures telomere
lengths relative to one another.
The yeast and human Ku heterodimer, involved in
nonhomologous end joining, also associate with telomeres
in vivo and in vitro (3, 19, 32, 42). Interestingly, loss
of either of the subunits of the yeast Ku heterodimer (yKu70 or yKu80)
confers a large increase in the TRD rate (58), in
nucleolytic degradation (58), and in the length of
terminal 3' single-stranded overhangs (19, 58). The
yku70 and yku80 alleles also display a global decrease in telomere tract size (6, 59). These data
suggest that the yKu heterodimer is part of a telomeric cap
that guards against potentially deleterious processes such as
promiscuous recombination and end degradation.
yKu acts together with a trimeric complex consisting of Mre11p, Rad50p,
and Xrs2p (the MRX complex) in the nonhomologous end-joining pathway
(8, 23, 42, 76). However, the role of the
Mre11p-Rad50p-Xrs2p (MRX) complex is far more complicated and is
additionally required for mitotic homologous recombination, induced by
ionizing radiation (47), double-strand break formation
(7, 27), and meiotic recombination (23). At
telomeres, each MRX component plays a positive role in telomere
addition (6, 50), which is mediated through the
telomerase pathway (23, 34, 50). Further
indicating a role of the MRX complex at telomeres, human Rad50,
Mre11, and Nbs1 (the presumed Xrs2p homologue) associate with the
telomere in vivo (78). However, the role of
the MRX complex in TRD remains unknown.
We investigate here the mechanism of telomere rapid deletion.
Through a combination of physical and genetic assays, we provide evidence that TRD is an intrachromatid recombination process, with
sequences near or at the extreme terminus invading distal tract
sequences. Our studies also indicate that the MRX complex plays a
critical role in the TRD process.
Plasmids.
Plasmid AD3ARUGT-IV [pVIIL
URA3(
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.19.6559-6573.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Intrachromatid Excision of Telomeric DNA as a
Mechanism for Telomere Size Control in Saccharomyces
cerevisiae
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
3 events/cell division/telomere
(35).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
)tel] contains VIIL sequences, a "flipped"
URA3 gene transcribing toward subtelomeric
sequences [designated by ()], and an 80-bp
poly(TG1-3) seed sequence (64). pVR
AD3ARUGT [pVR URA3()] was derived by ligating the 2.8-kb HindIII fragment of pVR URA3()tel
(16) into the unique HindIII site of pVIIL
URA3()tel. pVR URA3() in which
the URA3 sequences are transcribed toward
subtelomeric sequences was identified by Southern analysis.
pADADE2 was used for labeling of VIIL telomeres with the
URA3/ADE2 marker, as previously described (16).
Yeast media and strains. Rich (YPD [yeast extract-peptone-dextrose]), synthetic complete (SC), and SC omission media were prepared by standard methods. YPAD is identical to YPD except for supplementation with 30 mg of adenine/ml.
Haploid yeast strains used in this study are listed in Table 1. All strains were isogenic to the W303 background, with the exception of strains derived from DVL227-6c and TVL345. DVL227-6c is isogenic to the TVL345 background. W303-based strains carry a weak rad5 allele that does not influence most classes of recombination (46). All strains containing subtelomeric genes that are transcribed in the telomere-proximal to distal direction are termed the "()" derivative. Transformations that give rise to marked telomeres were
performed as described previously (16). Each fragment used for telomere marking contained subtelomeric sequences,
a selectable marker, and 80 bp of telomeric seed sequence.
|
)-marked VIIL telomeres with
HaeIII sites (the MUH strains) were constructed as follows:
The yeast strain YDS/RAP1 was transformed with the 2.5-kb
EcoRI/XhoI fragment from pVIIL
URA3(), giving rise to YDS/RAP1/VIIL
URA3() containing the URA3()-marked
VIIL telomeres. A plasmid shuffle was subsequently used to replace
the wild-type RAP1 on pD130 with the rap1-17
allele on pRS313/rap1-17, yielding YDS/1-17/VIIL
URA3(). The marked telomeres range in size from
400 bp to 3,000 bp greater than the wild-type length (300 bp).
YDS/1-17/VIIL URA3() was transformed with the
centromeric plasmid pRS314/TLC1-1 in the presence of genomic
TLC1 and maintained for 20 to 60 generations before loss of
the plasmid. The resulting strains used in this study were MUH4, MUH5,
MUH6, and MUH7, containing HaeIII sites at 1,430 bp
(HaeIII 1430), 1,880 bp (HaeIII 1880), 1,930 bp
(HaeIII 1930), and 2,930 bp (HaeIII 2930) from
the subtelomeric junction, respectively. Wild-type
RAP1 strains containing HaeIII sites were
generated after a plasmid shuffle of an MUH strain carrying a
centromeric rap1-17 allele with the plasmid pD130 (Fig. 1A, right panel). This
gave rise to MUH15 that contains a HaeIII site 640 bp
(HaeIII 640) from the subtelomeric junction.
|
,
Restriction fragments derived from the telomeric copy of
ADE2; x, restriction fragments derived from the internal
ADE2 locus. Size markers (in kilobases) are shown on the
left. The positions of the Progenitor, the TRD product, and the
NdeI/HaeIII fragment carrying the
HaeIII 250 (H250) site are also indicated. M refers to
the molecular mass marker. (C, right panel) As a control, DNA was
isolated from CZY1/RAP1 and digested with NdeI or with
both NdeI and HaeIII. Note that digestion
of CZY1/RAP1 generates an internal 1.45-kb
NdeI/HaeIII fragment that overlaps with
the telomeric fragment. For presentation purposes, the
autoradiogram is a composite of two portions of a single gel, with the
junction borders indicated by the vertical line on top of the gel.
Similar confirmations of HaeIII incorporation were
carried out in MBH11-8b and MBH93-6d, carrying the
HaeIII 150- and the HaeIII 750-marked
telomeres, respectively.
) was transformed with the 3.6-kb
SalI/NotI fragment of pADADE2, thereby replacing
the URA3()-marked VIIL telomere with a
URA3/ADE2-marked VIIL telomere
(16). The transformation in this case was conducted in the
presence of pRS314/TLC1-1 and a genomic copy of
TLC1, so that HaeIII sites could be incorporated
during telomere lengthening in rap1-17 strains (Fig. 1A,
left panel). After loss of the pRS314/TLC1-1 plasmid as
described above, three strains were identified.
First, MBH1 contains a HaeIII site at 150 bp from the
subtelomeric junction (HaeIII 150). MBH11 is a
wild-type RAP1 strain that was derived from a cross between
MBH1 and W303a. Second, MBH2 contains an HaeIII site at 250 bp from the subtelomeric junction (HaeIII 250).
MBH21 and MBH22 are wild-type RAP1 strains following the
first and second backcrosses of MBH2 with W303, respectively. Third,
MBH9 has an HaeIII site 750 bp (HaeIII 750) from
the subtelomeric junction. The cross between MBH9 and W303a
formed the diploid MBH91. MBH91, after sporulation, generated the
wild-type RAP1 spore colony MBH91-3. Two additional
backcrosses (MBH92 and MBH93) were performed, and wild-type spore
products (MBH92-1a and MBH93-6d) were identified. The sizes of the
marked telomeric tracts within each progenitor diploid were
determined by Southern analysis. MBH9/ura3, containing the
ura3/ADE2 HaeIII 750 marked VIIL telomere, was selected after growth of MBH9 on 5-fluoroorotic acid (5-FOA). 5-FOA
allows growth of Ura3, but not
Ura3+, cells. MEH9 was the result of
transformation of the 14-kb SphI fragment of pUC140,
containing the est1::URA3 null allele, into MBH9. The
presence of the disrupted allele was confirmed by Southern analysis.
The positions of HaeIII sites within the
telomeric tract of elongated URA3/ADE2 marked
VIIL telomeres were determined after digestion of DNA with both
NdeI, which cleaves within the ADE2 gene, and
HaeIII. An identical approach was used to identify strains carrying HaeIII sites in elongated URA3()-
marked VIIL telomeres, except that DNA was digested with both
ApaI, which cleaves uniquely in the URA3 gene,
and HaeIII. The presence or absence of multiple sites was
determined by partial digestion of genomic DNA. Multiple sites collapse
to the HaeIII site closest to the subtelomeric or telomeric junction after complete digestion.
HaeIII sites were present at their original position in
>99% of mitotically grown cells. Strains containing these elongated
progenitor telomeres were used for subsequent site transfer and
retention assays.
YP17-24c carries an elongated URA3/ADE2 HaeIII
250-marked VIIL telomere and a wild-type length
URA3() marked VR telomere. To generate YP17,
MBH21-20a was backcrossed with W303 four times to eliminate other
elongated and HaeIII-marked telomeres, thereby forming
the diploid YP15. The spore colony YP15-2d was subsequently crossed
with WUF1 to form YP17. WUF1 was constructed by transformation of W303a
with the 3.8-kb EcoRI/SalI fragment of pVR
URA3().
DJ204-10b carries an elongated ura3/ADE2 HaeIII
750-marked VIIL telomere and a wild-type length
URA3()-marked VR telomere. A cross between
DJ309-1c and WUF1 generated DJ204. To generate DJ309,
MBH9/ura3 was backcrossed with W303 four times. Therefore, the VIIL marked telomere is likely to be the sole elongated and HaeIII-marked telomere in DJ204-10b and YP17-24c.
To construct a strain carrying a disruption of the RAD50
gene, the 6.0-kb EcoRI/BglII fragment of pNKY83,
carrying the rad50 disruption allele, was transformed into
YP20, giving rise to YPR50. YP20 was derived from a cross between
W303
and BL27-11a, carrying 50% elongated telomeres including
the ura3/ADE2-marked VIIL telomere. Sporulation of YPR50 gave rise to the
rad50::URA3 spore colonies YPR50-3b, -3d,
-14b, -15c, -15d, -16a, and -16c.
To construct a telomere-marked strain carrying a rad50
null allele in a second genetic background, DVL227-6c was crossed to MBR52-5b, isogenic to the W303 background, and the resulting diploid MBM1 was sporulated. The progeny after sporulation of MBM1 contained rad50 (MBM1-3d) carrying the elongated
URA3/ADE2-marked VIIL telomere. YP18 was formed by an
independent cross between DVL227-6c and MBR52-5b that gave rise to the
rad50 spore colonies YP18-12b and YP18-13a.
To construct a strain carrying a disruption of MRE11, the
2.1-kb PvuII/AseI fragment of pHO27 was
transformed into YP20, yielding YPM11. Sporulation of YPM11 gave rise
to the spore colonies YPM11-3d, -6a, -6b, and -9c containing the
mre11::URA3 disruption allele. Sporulation of MBX91 led
to the generation of the xrs2 spore colonies MBX91-13c and
MBX91-16c containing the elongated URA3/ADE2-750 HaeIII-marked VIIL telomere. MBX91 is the result of a
cross between the nonisogenic strain TVL345 and MBH9.
TRD assays.
Rapid deletion was measured by a change in the
size of the telomeric fragment relative to the elongated
progenitor. To this end, the progenitor and truncated forms of
URA3/ADE2 or URA3 VIIL()-marked VIIL
telomeres were digested with NdeI and ApaI,
respectively, and subjected to Southern analysis.
the rate of loss.) In each strain, the median of
the distribution of values is presented. The use of YPAD media is
critical, since elongated ADE2 marked telomeres have low
levels of ADE2 expression relative to wild-type-length marked telomeres (54). As a consequence, growth in
media containing limiting levels of adenine, such as YPD, selects for
cells containing deleted telomeres.
Site retention assay.
The site retention assay measures the
presence or absence of the HaeIII site in the marked VIIL
telomeres after a screen for cells containing partial deletion
events (i.e., a subset of incomplete deletions that are smaller than
the distance between the HaeIII site and the extreme
terminus). In the case of the URA3()
HaeIII-marked VIIL telomeres, ApaI and
ApaI/HaeIII double digests were used to determine
telomere length, the distance between the HaeIII site
and the extreme terminus, and the retention or loss of the HaeIII site after Southern blot hybridization with
URA3 sequences as a probe. A similar protocol was used with
strains containing URA3 (or ura3)/ADE2
HaeIII-marked VIIL telomeres, except that the
telomeric length was determined by digestion with
NdeI. NdeI/HaeIII double digests were
used to measure the distance from the HaeIII site to the
extreme terminus and the degree of HaeIII site retention after Southern blots were probed with ADE2 sequences.
Site transfer assay.
The site transfer assay measures the
movement of a HaeIII site in elongated URA3/ADE2
HaeIII 250- and ura3/ADE2 HaeIII
750-marked VIIL telomeres to a heterologous wild-type-length
URA3()-marked VR telomere lacking
HaeIII sites in strains YP17-24c and DJ204-10b, respectively.
) VR-marked telomere by digestion with
ApaI and HaeIII. For batch analysis, we
confirmed that a 
10:1 ratio between a telomeric and
unique species could be readily detected using PhosphorImager analysis.
In the absence of site transfer, only the URA3/ADE2
HaeIII 250- and ura3/ADE2 HaeIII
750-marked VIIL telomeres should contain the HaeIII
site. If site transfer were obligatorily associated with rapid
deletion, we would expect a probability of transfer to be 3% for any
interacting telomeres, assuming one HaeIII
site/telomere and random telomere pairing patterns among the 32 telomeres in a haploid cell. This value may be greater if other
telomeres recombine preferentially (see Discussion). This
expectation is valid for events occurring in G1,
DNA replication, and G2. If TRD occurs in
G1, then a single transfer event during rapid
deletion will give rise to two identical products. Similarly, the
presence of a second independent TRD event occurring during DNA
replication or in G2 is highly unlikely. Given
this expected value, we estimated that a sample size of 260 independent
cells was required for statistical significance as determined by
chi-square analysis.
Site acquisition assay.
Conversely, we determined whether
the VIIL telomere is capable of acquiring HaeIII sites
from heterologous telomeres. To this end, we first crossed AJL
437-1d and MBH9 to give rise to the strain AJL528. The diploid was
sporulated, and two spore colonies, AJL 528-1b, and AJL 528-2b, were
isolated. These strains carry 
50% elongated telomeres with a
wild-type length ura3/ADE2 VIIL telomere. A total of 221 independent subclones were selected and subsequently subcultured in
batches of 10 to 11 as described above.
33%
after 20 generations; see Table 4) would be equivalent to the combined
value for the association of individual heterologous telomeres
with the unique marked VIIL telomere. This value therefore reflects
the expected rate of site acquisition during TRD if both the marked
VIIL telomere and its interacting partner are capable of deletion
and if the TRD rates on the marked VIIL telomere and heterologous
telomeres were on average comparable. The latter possibility has
been verified by the observed rapid deletions at the left arm of
chromosome III, a chromosome XI telomere, and the right arm of
chromosome V (30, 31). Therefore, the expected percentage
of site acquisition is equal to: (% TRD) (% HaeIII-marked
elongated telomeres) × 102. Among the
50% of telomeres that are elongated in the strains, we estimate
that 50% contain a HaeIII site. Therefore, the percentage of site acquisition should equal 8.25%. For the sample size of 221 used in these experiments, we would therefore expect 18 events over
a period of 20 generations of growth.
Statistical analysis. Chi-square analysis was used for comparisons between expected and observed values in site transfer, retention, and acquisition assays. The rank sum (Mann-Whitney) test was employed to compare the degree of overlap between the distributions of wild-type and mutant cell values.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
A physical system to study telomeric dynamics.
We
have previously proposed that TRD is the consequence of
intrachromosomal recombination. However, further
tests of this hypothesis have been limited by the absence of
physical markers in telomeric simple sequences. To overcome
this obstacle, we introduced HaeIII restriction
enzyme sites at defined positions within URA3()-marked and URA3/ADE2-marked telomeres on the left arm of
chromosome VII (VIIL [35]). This was accomplished
by the use of an allele of TLC1, TLC1-1, that acts as a
template for the HaeIII sequence (64).
Transient expression of TLC1-1 was performed in cells containing both the VIIL marked telomere and the rap1-17
mutation, which confers overelongated telomeres (Fig. 1A; see
Materials and Methods). HaeIII sites were kept to a minimum
by the coexpression of wild-type and mutant telomerase
RNAs. Based on HaeIII digestion of genomic
DNA, we estimate that 50% of the telomeres incorporated one or
more HaeIII sites (data not shown).
)-marked VIIL
telomeres with one or more mitotically stable HaeIII sites (see Materials and Methods). The HaeIII sites at
the URA3/ADE2-marked VIIL telomere were 150, 250, and
750 bp from the subtelomeric junction (HaeIII
150, HaeIII 250, and HaeIII 750, respectively; Fig. 1B and C). HaeIII 250 and HaeIII 750 sites
were present as the sole HaeIII site within the marked
telomere, while the HaeIII 150 site consisted of
three HaeIII sites located at 150, 250, and 400 bp from
the subtelomeric junction (data not shown). At URA3()-marked VIIL telomeres,
HaeIII sites were identified at distances of 640, 1,430, 1,880, 1,930, and 2,900 bp from the subtelomeric junction
(data not shown). After replacement of the rap1-17 allele with wild-type RAP1, both sets of strains were used to
distinguish among potential mechanisms of TRD. The presence of the
HaeIII sites does not alter the ability of the elongated
progenitor telomeres to delete to 300 bp, the wild-type
telomeric tract size in these strains (Fig. 1C). Since
these telomeres lack the conserved elements found adjacent to most
telomeres, our assays measure TRD only in the absence of
subtelomeric recombination.
TRD is an end-mediated process.
To test whether TRD is
mediated through intrachromatid recombination, as postulated previously
(35), or through other intra- or interchromosomal
pathways, we designed an assay that explores the relationship between
the degree of deletion and the retention of the HaeIII site.
This site retention assay takes advantage of incomplete TRD events that
do not delete to wild-type sizes (see Materials and Methods; Fig.
2A). A subset of incomplete deletions, termed partial deletions in this study, form the basis of the site
retention assay. Partial deletions are operationally defined here as
incomplete deletions in which the size of the deletion is smaller than
the distance from the HaeIII site to the extreme terminus
(Fig. 2B, subclones 7, 8, 9, and 12). The behavior of larger incomplete
deletions (Fig. 2B, subclones 11 and 16) can be explained by a plethora
of mechanisms and are therefore not included in this analysis. Partial
deletion products are capable of deletion to wild-type
telomeric tract size in a manner indistinguishable from the
progenitor (Fig. 2B, right panel). Hence, partial deletions are likely
to be mechanistically related to complete deletions in the wild-type
TRD pathway (see Discussion).
|
|
)
HaeIII-marked and URA3/ADE2
HaeIII-marked VIIL telomeres. Strikingly, none of
these deletions lost the HaeIII site. In all cases a
terminal deletion pattern was observed (Fig. 2B, left; Table
3). This pattern was observed regardless
of whether the HaeIII site was present in end-distal or
end-proximal regions. The terminal deletion pattern is consistent with
two possible end-mediated processes: (i) invasion of
telomeric ends into a heterologous telomere, forming a
nonreciprocal translocation (5, 24), or (ii)
intrachromatid deletion proceeding from the extreme terminus (Table 2).
|
Absence of interchromosomal exchange in TRD.
To distinguish
between these possibilities, we designed a "site transfer" assay to
measure the movement of a HaeIII site between telomeres
during rapid deletion. Site transfer measures the ability of the VR
URA3()-marked telomere that lacks a
HaeIII site telomere to acquire a HaeIII site
from a heterologous HaeIII-marked VIIL telomere during
rapid deletion (Fig. 3A; see Materials
and Methods). In most cells, the marked VIIL telomere was the only
elongated and HaeIII-marked telomere. To carry out site
transfer, we first identified cells carrying a VIIL telomere which
had undergone a deletion to the wild-type size (Fig. 3B, right panel).
Transfer of HaeIII sites in these cells was subsequently
measured by the ability of the VR-marked telomere to be cleaved by
HaeIII. Site transfer would be expected to occur in a
nonreciprocal translocation, but not in an intrachromatid deletion
(Fig. 3A).
|
)-marked VR telomere (Fig. 3B, left; data not
shown). Among 260 samples for each VIIL telomere, we did not
observe a single HaeIII site transfer from the elongated
VIIL telomere to the VR-marked telomere. The
measured value for both the distal (0 of 260) and proximal (0 of
260) HaeIII sites is significantly different from the
expected value (8.7 of 260; 
2 = 6.9;
P < 0.01) if deletion and transfer are linked and if
the VIIL telomere can randomly recombine with the VR-marked
telomere (see Discussion).
For the pooled site transfer results from both set of strains, the
expectation of 17.4 site transfers out of 520 samples is clearly
different from the experimental results (0 of 520)
(2 = 16; P < 0.001). The
latter result indicates that even a VIIL-VR recombination frequency
threefold lower than the expected value would be statistically
significant. Our results suggest that site transfer between
end-proximal or end-distal HaeIII sites to the VR
telomere is not frequently associated with rapid deletion. These
data are not consistent with any interchromosomal mechanism, including
nonreciprocal translocation, which involves exchange between the
telomeres of heterologous chromosomes.
As a reciprocal assay, we determined the frequency of site acquisition
from all HaeIII-marked elongated telomeres to a
naive wild-type-length ura3/ADE2-marked VIIL telomere,
lacking HaeIII sites. If TRD is due to interchromosomal
exchange, then the TRD rate is equal to the sum of the rates of the
productive associations with heterologous telomeres. Given that TRD
was observed in 33% of cells after 20 generations of growth (Table
4) and that 25% of telomeres in
these strains were both elongated and HaeIII marked, we
would expect a site acquisition frequency of 8.25% (see Materials and
Methods). We would therefore expect 18 acquisition events out of the
221 samples that were tested. Strikingly, we never observed a single
site acquisition event (2 = 16.7;
P < 0.001). Similar results were obtained after
analysis of HaeIII site transfer from heterologous
telomeres to an elongated marked telomere (data not shown).
These data corroborate the site transfer results and suggest the
absence of interchromosomal exchange on TRD.
|
The Rap1p C terminus does not influence TRD. The 165-amino-acid Rap1p C terminus acts as a negative regulator of telomere addition. The rap1-17 mutant that lacks this domain displays multiple and frequent cycles of rapid loss and overelongation of telomeric tract sequences (30). However, it remains unknown whether this rapid loss of telomeric sequences proceeds through the wild-type mechanism for TRD. We therefore sought to determine the influence of the Rap1p C-terminal domain on TRD by examining the behavior of TRD in rap1-17 strains (30, 35).
One of the hallmarks of TRD in wild-type cells is a terminal deletion pattern of HaeIII site retention among partial deletion products. To test the possibility of a distinct TRD pathway in rap1-17 cells, we conducted the site retention assay in cells carrying either a URA3()- or a
URA3/ADE2-marked VIIL telomere with HaeIII
sites present at distinct telomeric positions (Table 3). Of
35 partial deletions analyzed in rap1-17 cells, 10 lost HaeIII sites, indicating an apparent complex pattern of site
retention (Table 3), a result significantly different from that for the wild type (2 = 22; P < 0.001).
These observations can be explained in two ways. First, TRD in
rap1-17 cells may operate either via a pathway that is
mechanistically distinct from that of the wild type or via a mixture of
wild-type and alternative pathways. Second, the telomeres of
rap1-17 cells may delete to positions beyond the
HaeIII site proximal to subtelomeric sequences.
Since the formation of the elongated telomeres in
rap1-17 cells is dependent on telomerase
activity (data not shown), such telomeres could re-elongate after
TRD to positions end proximal to the position of the original
HaeIII site but lacking the HaeIII site.
To address the latter hypothesis, we examined the effects of the
elimination of telomerase activity on TRD in
rap1-17 cells that also carried a mutation in one of the
telomerase holoenzyme components, Est1p. Est1p is a
single-stranded telomeric DNA-binding protein that acts
with Cdc13p as an essential component in telomerase loading
(12, 39). The est1 mutation alone in a
wild-type RAP1 background has no effect on rapid deletion
(35). As expected for a loss in telomerase
activity, all elongated telomeres were stabilized at distinct sizes
in rap1-17 est1::URA3 double mutants (data not shown).
If the deletion-re-elongation hypothesis were correct, we would
predict a recovery of the wild-type terminal deletion pattern of site
retention in double-mutant cells.
To test this possibility, we conducted the site retention assay in
rap1-17est1::URA3 cells carrying a URA3/ADE2
HaeIII 750-marked VIIL telomere. Strikingly, we observed
that all 19 partial TRD events examined retained the
HaeIII site (Table 3). This is a significantly higher
frequency of HaeIII retention than observed in
rap1-17 cells (2 = 4.7;
P < 0.025). Hence, the apparent complex deletion
pattern in rap1-17 cells is the probable consequence of
HaeIII site loss and subsequent re-elongation by
telomerase. These data strongly suggest that the C terminus
of Rap1p does not influence the mechanism of TRD and
thereby unlinks TRD from the process of Rap1p counting (44, 61).
Rad50p and Mre11p are positive regulators of TRD. The MRX complex participates in a multiplicity of homologous mitotic and meiotic recombinational pathways, nonhomologous end joining, and telomere size control (6, 7, 8, 23, 27, 28, 42, 47, 50, 76). At least one component of this complex, Rad50p, is known to influence the abrupt elongation of telomeres by telomere/telomere recombination in cells that lack telomerase (type II recombination [69]).
To test whether TRD falls within this pathway for recombination between telomeric repeats, we first tested the possibility of Rad50p involvement in TRD. Relative TRD rates were measured by a decrease in the retention of the undeleted form of URA3/ADE2-marked VIIL telomeres after liquid subculturing of both rad50 and wild-type cells (Table 4; Fig. 4B, left panel; see Materials and Methods). Strikingly, the rad50 mutant strongly reduced the frequency of TRD. The retention of the undeleted form increased from 67% in wild-type strains to 97% in isogenic rad50 cells after 20 generations of growth (P < 0.01; Table 4, background 1 strains). These data suggest that Rad50p is a positive regulator of rapid deletion. Similar results were observed in a second genetic background (Table 4, background 2 strains).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this investigation, we present the first mechanistic insights into TRD, a process that is likely to be a major participant in telomere size control. Our studies have been based on the development of a physical assay for TRD, in which HaeIII sites are introduced into the telomeric tract. The presence of telomeric HaeIII sites does not interfere with telomeric functions including silencing, size control, and TRD (Fig. 1C; also data not shown).
Two physical criteria were used to investigate TRD. The first criterion was the relationship between the loss of sequences and the retention of the HaeIII site. The site retention assay relies on the characteristics of partial TRD events, i.e., incomplete deletions that are smaller than the distance from the HaeIII site to the extreme terminus. Partial deletion follows the same pathway as complete deletions. The evidence for this is twofold: (i) partial deletion species subsequently delete to wild-type-length telomere size (Fig. 2B, right panel), and (ii) complete TRD occurs more frequently as the percentage of wild-type-length telomeres increases (35). Thus, the size of the deletion is dependent on the size of the majority of telomeres rather than being an obligatory intermediate step in TRD. Hence, partial TRD is likely to be the consequence of sizing relative to aberrantly elongated telomeres.
The second criterion is the movement of HaeIII sites between two marked telomeres during rapid deletion. We found no evidence for site transfer in our studies. For statistical evaluation of our data, we made the simplifying assumption that the VIIL telomere associates with VR and other telomeres at similar rates. One important difference, however, is the presence of subtelomeric elements in natural, but not artificially marked, telomeres. Previous studies have revealed the presence of selected groups of telomeres that exhibit preferentially high rates of interchromosomal recombination between conserved Y' subtelomeric elements (38). Subtelomeric regions that share the greatest degree of homology have the highest rates of recombination, suggesting that the degree of homology dictates the probability of subtelomeric recombination (38). If these preferentially interacting telomeres are functionally sequestered from the marked telomeres, site transfers between the VR and VIIL telomeres may occur at a higher frequency than expected. Hence, the actual significance of the difference between observed and expected values may be greater than we reported. Therefore, associations based on subtelomeric homology are unlikely to interfere with the ability to observe HaeIII site transfer. The lack of ura3/ADE2-marked VIIL telomere site acquisition from heterologous HaeIII-marked elongated telomeres further argues that interchromosomal exchange is not likely to be responsible for TRD.
The presence of a terminal pattern of site retention and the absence of site transfer between nonhomologous telomeres, lacking subtelomeric homology, effectively rule out interchromosomal recombination. These include those processes involving crossing over (see, e.g., references 5 and 24), double-strand break repair (68) and break-induced replication (53). The data are also inconsistent with intrachromosomal events such as sister chromatid exchange (57), single-strand annealing (26, 53), and other mechanisms of internal deletion (Table 2). Rather, the data support a mechanism of intrachromatid loss of telomeric sequences initiated at the extreme terminus. An exonucleolytic activity is an unlikely explanation of our data, given that this activity would be expected to generate heterodispersed telomeric fragments that decrease in size during growth, rather than the discrete TRD products that we observe. In addition, our physical data cannot be explained by endonuclease cleavage, unless cleavage occurs in the absence of DNA repair. We note that any recombinational basis for TRD must involve a homeologous interaction between telomeric sequences, given the limited sequence homology present in telomeric tracts.
TRD is independent of the C terminus of Rap1p. This finding strongly suggests that TRD is also independent of components of telomeric chromatin, including both negative (Rif1p and Rif2p) and positive (Sir3p and Sir4p) regulators of telomere size (10, 25, 52, 77). Additionally, TRD is not linked to the overall structure of subtelomeric regions, since loss of the C-terminal tail and Sir proteins results in an open chromatin state in subtelomeric regions (15, 31).
TRD is likely to be a mechanism that acts in concert with other elements of the telomere sizing system to achieve and maintain homeostasis. In particular, the Rap1p counting mechanism (44, 61), size regulation through Rap1p-telomerase interactions (29, 65), or Cdc13-mediated homeostatic mechanisms (9, 17, 18, 40, 56, 60) present at the terminal 3' overhang may maintain the size of the TRD product. It is noteworthy that the Rap1p C terminus is essential for the Rap1p counting mechanism, but dispensable for TRD, suggesting that the two processes are mechanistically distinct.
Participation of the MRX complex in TRD. The strong inhibitory effect of rad50 and mre11 on TRD, coupled with the diminution of TRD in rad52 cells (35; data not shown) supports a recombinational model for this process. However, the relationship between Rad50p stimulation and Rad52p stimulation of TRD is unclear, and the presence of Rad50p-dependent Rad52p-independent deletions remains a distinct possibility.
Rad50p, Mre11p, and Xrs2p are associated in a complex that is required for mitotic homologous recombination, induced by ionizing radiation (47), double-strand break formation (7, 27), nonhomologous end joining (8, 23, 42, 76), and meiotic recombination (23). Rad50p also plays an important, but nonessential, role in single-strand annealing (26). The precise function of Rad50p remains unknown, although it has been suggested to act through the preparation of DNA single strands for recombination (67) or through regulation of chromatin condensation prior to recombination, as observed in meiotic cells (51, 53). Alternatively, the participation of Rad50p in TRD may reflect a specific form of end processing required prior to intramolecular strand invasion. While Rad50p is important for TRD, it does not appear to be essential, since TRD events can be observed under growth conditions that select for the presence of shortened telomeres (54; data not shown; see Materials and Methods). A second component of the MRX complex, Mre11p, also has a central role in TRD. The exonucleolytic activities of Mre11p may play a role in the processing of ends for strand invasion during mitotic and meiotic recombination (13, 23, 71). However, while null mutations in MRE11 display short telomere tract sizes (6, 50), selective elimination of the Mre11p nuclease activities has no effect on wild-type-length telomeres (48). These data therefore indicate that the function of Mre11p in telomeric size control does not require the nucleolytic activity of Mre11p. Although the relationship of the role of Mre11p in telomere size control and TRD is not known, these data raise the possibility that Mre11p may play a different, possibly structural, role in TRD. The third component of the MRX complex, Xrs2p, may participate in TRD to a lesser extent, if at all. The MRX function in telomere size control is dependent on the presence of the ATM homologue Tel1p (62). However, in rapid deletion studies, we have found that a tel1 null allele confers a wide range of deletion frequencies (data not shown). Therefore, the role of Tel1p in the MRX pathway for TRD remains an open question. At least two distinct recombinational rescue pathways are present after inactivation of telomerase: Rad50p-independent recombination between subtelomeric Y' repeats (type I) and Rad50p-dependent recombination between the telomeric simple sequences (type II [69, 70]). The exclusive involvement of Rad50p in the generation of type II recombination is consistent with the participation of telomere-telomere recombination in TRD and may suggest that the generation of type II survivors and TRD are mechanistically related.TRD and homeologous recombination.
Unlike most homeologous
recombination, TRD is not constrained by mismatch repair enzymes
(35, 53). Mutations in MSH2, MSH3,
or PMS1 have no effect on the rate of TRD (35).
This apparent discrepancy has several possible explanations. First, the
effect of mismatch repair enzymes on homeologous sequences is dependent on the degree of homology (11). While mismatches of
10% are strongly repressed by Msh2p and Msh3p, DNA sequences
having lower levels of homology are independent of mismatch repair.
Second, cells may have multiple pathways for homeologous recombination, as suggested by the presence of a homeologous recombination
pathway independent of mismatch-repair in pms1 cells
(4). Third, the chromatin structure at telomeres may
influence the telomeric homology requirements or the role
of mismatch repair enzymes. For example, among telomeric
sequences are multiple copies of the highly conserved 13-bp binding
site for Rap1p (14, 74), which, in the context of
telomeric chromatin, may play a unique role in
telomere-telomere pairing. If Rap1p is involved, however,
it must be independent of the Rap1p C terminus, since the
rap1-17 mutation does not influence TRD. Similarly, a
specific structure formed at telomeres, such as a t-loop
(22) or a G-quadraplex structure (75),
may act in cis to promote TRD. Distinguishing among
these possibilities is a major goal for the future.
A model for telomeric recombination.
The
genetic and physical studies presented here suggest that TRD is a
3' end-mediated intrachromatid recombinational process (Fig.
5). Specifically, the components of
the MRX complex may directly or indirectly prepare the 3'
telomeric terminus for strand invasion. The
recombinationally proficient 3' extreme terminus would subsequently
invade distal telomeric DNA, leading to an excision of the
intervening sequences through branch migration, D-loop degradation, and
resolution of the Holliday junction (Fig. 5). This may result in the
transient formation of linear or circular degradation products,
depending upon the precise nature of resolution, which would be quickly
lost from the population. One intriguing feature of this model is that
multiple cycles of both semiconservative replication primed from the
invading strand and subsequent reinvasion could give rise to amplified
telomeric DNA. This interrelationship raises the
possibility that TRD and type II events may be mechanistically related.
Interestingly, the proposed TRD intermediate (Fig. 5, step 2) is
virtually identical in structure to the stable human t-loops involved
in telomere cap function (22). The function and
evolutionary relationship between TRD and t-loops, however, remains
unknown.
|
400 bp longer than the wild-type length are proficient for TRD, well within the range of telomeric sizes found in
different laboratory yeast strains (73). Smaller changes
in telomere size cannot be ascribed to TRD, since it is not
possible, under our assay conditions, to determine whether such
decreases in size are due to slow attrition or rapid deletion of
telomeric sequences. Second, the efficiency and precision
of TRD events suggest that any overelongated telomere is inherently
susceptible to rapid deletion to wild-type size. Third, the presence of
rapid deletion of telomeric tracts in a multiplicity of
organisms, including Tetrahymena spp. (33),
trypanosomes (55), and human cells (45, 49),
suggests that the rapid deletion of telomeric tracts is a
conserved process through evolution.
A further mechanistic understanding of TRD will provide paradigms for
the control of telomere size in yeast and higher systems. The
characterization of TRD and other size control regulatory systems in
yeast and higher eukaryotes will ultimately lead to an ability to
understand and to regulate the factors involved in telomere size control.
| |
ACKNOWLEDGMENTS |
|---|
M.B. and Y.P. contributed equally to this study.
We thank Charles Zhang for his excellent technical contribution to this study. We also thank E. B. Hoffman, M. Clancy, T. de Lange, D. Gottschling, M. A. Osley, T. D. Petes, M. Wellinger, and members of our laboratory for critical comments during the course of these studies and D. Gottschling, E. Louis, V. Lundblad, T. D. Petes, M. Resnick, and L. Symington for providing strains and plasmids.
This study was supported by NIH grant GM56526 and matching funds from the Tulane Cancer Center. Initial studies were funded by the National Science Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Tulane University Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112. Phone: (504) 584-3688. Fax: (504) 584-2739. E-mail: alustig{at}mailhost.tcs.tulane.edu.
Dedicated to E. B. Hoffman.
Present address: Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, 020106, Warsaw, Poland.
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Materials and Methods
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Discussion
References
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Molecular and Cellular Biology, October 2001, p. 6559-6573, Vol. 21, No. 19
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.19.6559-6573.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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