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Expansion of Interstitial T
elomeric Sequences inYeastGraphical Abstract
Highlights
d Yeast interstitial telomeric sequences (ITSs) are prone to
expansions
d Post-replicative repair and homologous recombination
contribute to ITS instability
d These data can explain length polymorphism characteristic of
ITSs in humans
d The proposed model may have implications for alternative
telomere lengthening in cancer
Aksenova et al., 2015, Cell Reports 13, 1545–1551November 24, 2015 ª2015 The Authorshttp://dx.doi.org/10.1016/j.celrep.2015.10.023
Authors
Anna Y. Aksenova, Gil Han,
Alexander A. Shishkin, Kirill V. Volkov,
Sergei M. Mirkin
Correspondencesergei.mirkin@tufts.edu
In Brief
Telomeric DNA repeats within
chromosomes are called interstitial
telomeric sequences (ITSs). ITSs are
variable in length and are likely hotspots
of chromosomal rearrangements linked
to human disease. Aksenova et al.
demonstrate that yeast ITSs are prone to
expansions and propose a model of their
instability.
Cell Reports
Report
Expansion of Interstitial TelomericSequences in YeastAnna Y. Aksenova,1,3 Gil Han,1 Alexander A. Shishkin,2 Kirill V. Volkov,3 and Sergei M. Mirkin1,*1Department of Biology, Tufts University, Medford, MA 02155, USA2Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA3Department of Genetics, St. Petersburg State University, St. Petersburg 199034, Russia
*Correspondence: sergei.mirkin@tufts.edu
http://dx.doi.org/10.1016/j.celrep.2015.10.023This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
SUMMARY
Telomeric repeats located within chromosomesare called interstitial telomeric sequences (ITSs).They are polymorphic in length and are likely hot-spots for initiation of chromosomal rearrangementsthat have been linked to human disease. Usingour S. cerevisiae system to study repeat-mediatedgenome instability, we have previously shown thatyeast telomeric (Ytel) repeats induce various grosschromosomal rearrangements (GCR) when theirG-rich strands serve as the lagging strand templatefor replication (G orientation). Here, we show thatinterstitial Ytel repeats in the opposite C orientationprefer to expand rather than cause GCR. A tract ofeight Ytel repeats expands at a rate of 4 3 10�4 perreplication, ranking them among the most expan-sion-prone DNA microsatellites. A candidate-basedgenetic analysis implicates both post-replicationrepair and homologous recombination pathwaysin the expansion process. We propose a model forYtel repeat expansions and discuss its applica-tions for genome instability and alternative telomerelengthening (ALT).
INTRODUCTION
Repetitive DNA at the ends of chromosomes protects them
from degradation. Tandem (T2AG3)nd(C3TA2)n repeats span up
to 15 kb on human telomeres, serving as a platform for the bind-
ing of a multi-protein complex called shelterin, which guards
chromosomal ends from degradation and fusions (Palm and de
Lange, 2008). Budding yeast, S. cerevisiae, has much shorter
telomeres composed of heterogeneous (G1-3T)nd(AC1-3)n re-
peats bound by the CST (Cdc13/Stn1/Ten1) complex (Forste-
mann et al., 2000;Wang and Zakian, 1990;Wellinger and Zakian,
2012). The bulk of telomeric DNA is double stranded, while the
very tip of the 30 end of the G-rich strand remains single stranded
and is extended by telomerase prior to cell division (Forstemann
et al., 2000). The protruding G strand can fold into the G-quartet
structure (Williamson et al., 1989) or invade the telomeric dsDNA
forming the so-called t loop, shielding the 30 terminus from repair
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machinery and regulating telomerase access (Griffith et al.,
1999). Telomerase adds repeats to the G-strand DNA using its
core RNA as a template. The complementary C-strand DNA is
then filled in by DNA polymerase alpha (Bianchi and Shore,
2008; Giraud-Panis et al., 2010). The activity of telomerase is
limited in most somatic cells. In many cancer cells, in contrast,
telomerase is upregulated, thereby increasing their proliferation
potential. Approximately 10%–15% of cancer cells do not have
active telomerase (Bryan et al., 1995) but use a different mecha-
nism to counteract attrition of chromosome ends called alterna-
tive lengthening of telomeres (ALT). It is generally believed that
recombination-mediated copying of telomeric DNA is a mecha-
nism accounting for ALT (Lundblad, 2002).
Besides chromosomal ends, telomeric repeats are also pre-
sent in the internal parts of chromosomes (Azzalin et al., 1997;
Ruiz-Herrera et al., 2008; Weber et al., 1991; Wells et al.,
1990). These sequences are called interstitial telomeric se-
quences (ITS). Short ITSs (s-ITSs), containing between 2 and
25 copies of the repetitive tract, are found in multiple locations
in the human genome (Azzalin et al., 1997; Bolzan, 2012; Lin and
Yan, 2008; Mondello et al., 2000; Ruiz-Herrera et al., 2008;
Samassekou and Yan, 2011). They are believed to result from
the insertions of telomeric repeats during the repair of double-
stranded DNA breaks via non-homologous end-joining (NHEJ)
(Azzalin et al., 2001; Nergadze et al., 2004), possibly involving
telomerase (Nergadze et al., 2007). Like many other microsatel-
lites, s-ITSs are polymorphic in length (Hastie and Allshire,
1989); for instance, their significant length polymorphism has
been observed in gastric tumors (Mondello et al., 2000). Cyto-
genetic analysis has co-localized ITSs with spontaneous and
induced chromosome breakage sites in primates (Ruiz-Herrera
et al., 2005) and rodents (Musio et al., 1996). For example, ITSs
located at 2q14 on human chromosome 2 behave as a common
fragile site and require the shelterin component TRF1 for its
stabilization (Bosco and de Lange, 2012). Consequently, ITSs
may be hotspots for the initiation of chromosomal rearrange-
ments (Hastie and Allshire, 1989). Supporting this idea, inser-
tion of an 800-bp-long telomeric tract into an intron of the
APRT gene increased rearrangements of the reporter gene
in CHO cells (Kilburn et al., 2001). Several observations link
ITSs and chromosomal rearrangements observed in human
disease. They were detected at the junction sites of transloca-
tions in patients with Prader-Willi syndrome (Boutouil et al.,
1996; Qui et al., 1993; Vermeesch et al., 1997), neuroblastoma
orts 13, 1545–1551, November 24, 2015 ª2015 The Authors 1545
Figure 1. System Used for the Detection of
Ytel Repeat Expansions
(A) Cassette used to generate strains with internal
Ytel repeats and its location relative to the prox-
imal replication origin (ARS306). An �3 kb-long
cassette was constructed as described previously
(Aksenova et al., 2013). The cassette contains
flanking sequences from chromosome III (black),
flanking and coding sequences from URA3 (yellow
and red, respectively), intron sequences from the
ACT1 gene (blue), and TRP1 flanking and coding
sequences (pale green and dark green, respec-
tively). Telomeric repeats (8 or 15 copies) were
inserted into the indicated XhoI site (blunted) within
the intron of the URA3-Intron gene. Numbers
above the cassette indicate the position in the
cassette, and numbers below the line are SGD
coordinates for S288C reference genome.
(B) Phenotypes of strains carrying Ytel repeats in
the URA3-Intron reporter gene. Suspensions con-
taining approximately equal amounts of cells were
plated as drops on three different types ofmedium:
5-FOA-containing synthetic media, synthetic me-
dia without Uracil and complete YPD media.
(C) Expression of the URA3-Intron gene in strains
with insertions of telomeric repeats.
We examined expression of the URA3-Intron gene by RT-PCR in SMY751 (8 copies of the telomere repeat) and SMY750 (15 copies of telomere repeat). The
control strain SMY803 has no repeats in theURA3-Intron gene. The rows labeled URA3mRNA and URA3 pre-mRNA show the relative amounts of spliced mRNA
and URA3 pre-mRNA in these strains. The row marked URA3 30-RNA shows the total level of the URA3 transcript. The row labeled Actin indicates the RT-PCR
products for the control actin mRNA.
(Schleiermacher et al., 2005), and acute myeloid leukemia
(Cuthbert et al., 1999).
Altogether, these observations imply that ITS can trigger
genome instability, potentially leading to human disease. How-
ever, the mechanisms responsible for length polymorphism,
chromosomal fragility, and GCRs mediated by s-ITSs are poorly
understood. This prompted us to study genome instabilities
mediated by yeast telomeric (Ytel) repeats placed into an internal
chromosomal position. We found that the generic Ytel repeat
was quite unstable in a yeast model system. When its G-rich
strand served as the lagging strand template for replication,
Ytel repeats triggered gross chromosomal rearrangements
(GCRs) and mutagenesis at a distance (Aksenova et al., 2013).
Here, we analyzed the instability of Ytel repeats in the opposite
C-orientation. In striking contrast with the G-orientation, no
GCRs mediated by Ytel repeats were detected in the C-orienta-
tion. Instead, those repeats were highly prone to expansions.
Our genetic analysis revealed that two important pathways,
post-replication repair (PRR) and homologous recombination
(HR), contribute to Ytel repeat expansions.
RESULTS
Ytel Repeats Are Potent Inhibitors of Gene ExpressionYeast telomeric runs (Ytel) of varying lengths were cloned into an
intron of the artificially split URA3 gene (Shishkin et al., 2009),
and the cassette carrying the URA3-Intron gene was inserted
near ARS306 on chromosome III (Figure 1A). 8 or 15 Ytel
(CCCACACA)n repeats were placed into the intron of the split
URA3 gene such that the C-rich sequence corresponded to
1546 Cell Reports 13, 1545–1551, November 24, 2015 ª2015 The Au
the sense strand for transcription and, which is also the lagging
strand template for DNA replication (C orientation). As we previ-
ously described (Shishkin et al., 2009), large-scale expansions of
GAA repeats lengthening the intron over�1 kb inhibited splicing,
which resulted in gene inactivation and 5-FOA-resistance.
In Ytel’s case, however, the presence of just 15 copies of the
repeat in the C orientation resulted in the complete inactivation
of the URA3 gene (Figure 1B). This result was unexpected, since
the total length of the intron in this case corresponded to only
475 bp, which was considerably below the splicing threshold
(Shishkin et al., 2009; Yu and Gabriel, 1999). Also, yeast clones
having 15 Ytel repeats within the intron in the opposite G orien-
tation remained Ura+ (Aksenova et al., 2013).
The effect of Ytel repeats on gene expression could be caused
by the inhibition of transcription or splicing. Our RT-PCR analysis
of URA3 mRNA versus pre-mRNA is consistent with the latter
case (Figure 1C). The amount ofURA3mRNAwasmeasured us-
ing primers, one of which was complementary to the exon/exon
junction; thus, it could only anneal to spliced RNA. The amount
of pre-mRNA was determined by a primer set, in which one
primer could only anneal to the intron. We observed a significant
decrease in the amount of URA3mRNA as the length of the Ytel
repeat increases, while the amount of pre-mRNAwas unaffected
by the presence of Ytel repeats (Figure 1C).
Ytel Repeats in C Orientation Are Highly Unstable andProne to ExpansionsOur selective system is very useful for scoring various events
that result in gene inactivation, as they can be accounted
for on 5-FOA-containing media. Those events include repeat
thors
Figure 2. Expansions of the (CCCACACA)8Repeat
(A) PCR analysis of 5-FOAR colonies derived
from strain SMY751. Primers (1819F/1819R)
flanking the repeat (Figure S3) were used to
amplify genomic DNA. Numbers above the lines
indicate the exact number of units within the re-
petitive tract as determined by DNA sequencing.
‘‘Quick-load’’ 50-bp DNA ladders (NEB) are
shown.
(B) Distribution of different expansion events
observed in strain SMY751 carrying (CCCACACA)8repeat.
(C) Representative PCR analysis of Ura+ colonies
derived from strain SMY751. Primers (1819F/
1819R) flanking the repeat (see Figure S3) were
used to amplify genomic DNA. Numbers above the
lines indicate the exact number of units within the
repetitive tract as determined by DNA sequencing.
Ladders are the same as in (A).
(D) Distribution of contractions events observed in
strain SMY751.
(E) Rates of expansion and contraction in
strain SMY751. Expansions were scored on
5-FOA media, while contractions were scored
on Ura� media. Rates and 95% confidence
intervals (error bars) were calculated based on distribution of expanded clones in independent cultures using the Ma-Sandri-Sarkar maximum
likelihood estimator with a correction for sampling efficiency as described previously (Aksenova et al., 2013).
expansions, mutations in the body of the URA3 gene, chro-
mosomal aberrations or rearrangements, and epigenetic events
(Aksenova et al., 2013; Cherng et al., 2011; Shishkin et al., 2009).
Since a strain carrying 15 (CCCACACA)n repeats was 5-FOA
resistant to begin with, we could not use it in the expansion
studies. Thus, we analyzed the rate of expansions in a strain car-
rying eight (CCCACACA)n repeats. This strain has a growth
pattern bordering on Ura�/Ura+ phenotypes (Figure 1B). This
borderline phenotype was advantageous for our study, as an
addition or contraction of just a few Ytel repeats was sufficient
to convert this strain into unambiguous Ura� or Ura+ clones,
respectively, which effectively eliminated selective pressure in
favor of longer expansions or contractions.
The rate of 5-FOA resistance increased �14,000-fold in our
strain with 8 Ytel repeats compared to the wild-type strain car-
rying no repeats (Table S1). Analysis of these 5-FOA-resistant
clones revealed two types of events (Figure 2A). First, even
this short Ytel repeat was prone to expansions. The distribution
of clones with different numbers of added repeats fits with the
Poisson distribution for a random variable with a mean of �3
(Figure 2B). The rate of expansions for the Ytel8 repeats in the
C orientation was �4 3 10�4 per replication (Figure 2E).
Sequencing of 48 expanded repeats from 31 independent
clones showed that perfect, non-interrupted Ytel repeats were
added in every case. Expansions of the Ytel repeats in our sys-
tem were length dependent: shortening the (CCCACACA)n run
to six units resulted in an almost three orders of magnitude
decrease in the expansion rate (Figure S1).
Second, a significant portion of 5-FOA-resistant clones did not
have changes in the length of the Ytel tract or gross chromo-
somal rearrangements (Table S1). All 130 sequenced clones
from 36 independent cultures had the initial-size Ytel repeat
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and did not contain any mutations in the URA3 reporter. How
could one in �104 cells with the original number of Ytel repeats
grow on 5-FOA-containing media without acquiring additional
mutations in the URA3 cassette? Notably, the level of URA3
mRNA was already decreased by �10-fold in the strain with 8
Ytel repeats (Figure 1C). It was further reduced in most of the
5-FOR-r clones with unchanged repeat length (Figure S2). We
speculate, therefore, that an already low level of the URA3
mRNA in the original strain was further decreased owing to
some epigenetic event in a small fraction of cells, allowing
them to survive in the presence of 5-FOA.
We also studied contractions by plating the strain with the
Ytel8 repeat on synthetic media lacking uracil and analyzing
repeat lengths in the resultant Ura+ clones. The scale of contrac-
tions varied between individual clones, with deletions of four
to five repeats being most prevalent (Figures 2C and 2D).
Sequencing analysis of 23 clones, which carried various con-
tractions of the Ytel repeat, showed that the remaining parts of
the repeats contained no interruptions. The rate of contractions
for the Ytel8 repeats in C orientation was �43 10�5 per replica-
tion, i.e., an order of magnitude less than the rate of expansions
for the same repeat (Figure 2E).
The high rate of expansions of the 64-bp-long Ytel repeat
makes it one of the most expansion-prone repeats studied. A
comparable in size (GAC)25 run expands at a rate of �2 3 10�7
(Miret et al., 1998), while 75-bp-long (CGG)n and (CTG)n repeats
both expand at the rate of�13 10�5 (Pelletier et al., 2003). Also,
we observed the addition of up to eight Ytel octamers to the
(CCCACACA)8 repeat in C orientation, in effect doubling its
size. Thus, the C orientation of the Ytel repeat is highly prone
to expansions. The rate of repeat contractions was also high,
albeit significantly lower than that of the expansions. The bias
orts 13, 1545–1551, November 24, 2015 ª2015 The Authors 1547
Figure 3. Genetic Control of Ytel Repeat Expansions
Effect of different gene knockouts on the expansion of the (CCCACACA)8repeat. Rates of expansion and 95% confidence intervals (error bars) were
calculated based on distribution of expanded clones in independent cultures
using the Ma-Sandri-Sarkar maximum likelihood estimator with a correction
for sampling efficiency as described elsewhere (Aksenova et al., 2013). Dis-
tribution of expanded clones in 12–36 independent cultures was studied for
each strain. Length of the repetitive tract for each analyzed clonewas validated
by PCR. Numbers below the confidence intervals reflect fold decrease over the
wild-type, and numbers above the confidence interval reflect fold increase
over the wild-type. Red dashed contours designate the range of 10-fold dif-
ference with the wild-type.
for expansions for the Ytel repeat in the C orientation could be
explained by the fact that its structure-prone G-rich strand is in
the nascent lagging strand during DNA replication, which is
known to favor expansions over contractions (Kang et al., 1995).
Genetic Analysis of Expansions of Ytel RepeatsTo elucidate the mechanisms of expansions of Ytel repeats, we
used a candidate gene approach by comparing the rates of ex-
pansions for the (CCCACACA)8 repeat in individual knockouts of
genes involved in NHEJ, HR, and PRR pathways. We also stud-
ied the knockouts of genes encoding replication fork stabilizers,
RecQ- and UvrD-like DNA helicases, as well as telomere mainte-
nance proteins. The results of these analyses are summarized in
Figure 3.
Deletion of the RAD6 gene had the biggest effect on expan-
sions of the (CCCACACA)8 repeat (>200-fold reduction in the
expansion rate as compared to the wild-type strain). Rad6p is
an E2-ubiquitin-conjugating enzyme that interacts with several
E3 ubiquitin ligases. It plays a principal role in the PRR pathway
through PCNA ubiquitination at damaged or stalled replication
forks (Hedglin and Benkovic, 2015). It also regulates HR by ubiq-
uitinating histone H2B (Game and Chernikova, 2009; Robzyk
et al., 2000).
RAD5 and SRS2 constitute a branch of the RAD6 epistasis
group responsible for PRR. Rad5 was specifically implicated in
fork reversal (Klein, 2007). Srs2 DNA helicase is believed to
channel stalled replication forks into the PRR pathway (Marini
and Krejci, 2010; Putnam et al., 2010). We observed a 15- and
25-fold reduction of the Ytel repeat expansions in the rad5D
and srs2D strains, respectively (Figure 3).
Rad51 andRad52 drive the strand exchange reaction, which is
at the heart of HR (Krogh and Symington, 2004). Knocking out
1548 Cell Reports 13, 1545–1551, November 24, 2015 ª2015 The Au
the HR pathway led to a 30-fold reduction in expansions for
rad52D or a 10-fold reduction for rad51D strains.
Combining the srs2D and rad51D deletions together resulted
in a 236-fold decrease in the expansion rate of the Ytel repeat,
which is remarkably close to the effect of the individual RAD6
knockout (Figure 3B). This synergy demonstrates that the inter-
play of two pathways, HR and PRR, determines the rate of Ytel
repeat expansions.
Besides its role in the damage-avoidance pathway, the Srs2
helicase disrupts recombination intermediates and promote
synthesis-dependent strand annealing (SDSA) (Branzei and
Foiani, 2007; Macris and Sung, 2005). Two other DNA helicases,
Mph1 and Sgs1, are also known to dismantle D loops, thus,
promoting SDSA. Knocking out the SGS1 gene had no effect
on the expansions of Ytel repeats, while anMPH1 gene knockout
decreased their expansion rate by 11-fold. The fact that the
Mph1 helicase upholds expansions of the Ytel repeats impli-
cates SDSA in the process.
Tof1, Csm3, and Mrc1 form the so-called fork-stabilizing or
fork-pausing complex. It facilitates replication fork progression
through stable DNA structures formed on template DNA strands
(Voineagu et al., 2008, 2009) but commands replication forks to
pause at potent protein-DNA complexes (Calzada et al., 2005;
Mohanty et al., 2006). Fork stalling at protein-DNA complexes
specifically requires Tof1 and Csm3, but not Mrc1 (Hodgson
et al., 2007). We found that the expansion rate of Ytel repeats
is significantly reduced in tof1 and csm3, but notmrc1 knockouts
(Figure 3), implying that protein-mediated fork stalling is involved
in repeat expansions. In addition, we found a 26-fold stimulation
of Ytel expansions in a strain with the deletion of the SIR2 gene,
which is essential for chromatin silencing at telomeres.
We observed only a modest (4-fold) decrease in Ytel expan-
sions in the yku70D and yku80D strainswith impairedNHEJ, indi-
cating that NHEJ is not a major player in the process.
Finally, an est2 knockout and an rnh1-rnh202 double knockout
had only a slight effect on Ytel repeat expansions.
DISCUSSION
We found that interstitial telomeric repeats are intrinsically unsta-
ble in our yeast system.When theG-rich strand of the repeat was
in the lagging strand template (G orientation), gross chromo-
somal rearrangements frequently occurred (Aksenova et al.,
2013). In contrast, the same length Ytel repeat in the C orienta-
tion did not cause chromosomal rearrangements but was prone
to expansions. Internal Ytel repeats cause a potent replication
block in the G orientation but a much more subtle stall in the C
orientation (Anand et al., 2012). This orientation dependence in
fork stalling could explain the differences in GCR rates: modest
fork stalling in the C orientation is unlikely to result in double-
strand breaks and, consequently, GCR formation.
We previously proposed that a protein complex at Ytel repeats
is responsible for Ytel-mediated fork stalling (Anand et al., 2012),
since it depended on Tof1 protein that facilitates fork pausing at
protein-DNA complexes (Calzada et al., 2005; Mohanty et al.,
2006). Tof1 acts together with Mrc1 and Csm3 proteins, but
Mrc1 was not implicated in protein-mediated fork stalling (Hodg-
son et al., 2007).We found that the expansion rate of Ytel repeats
thors
Figure 4. Mechanisms Responsible for Ytel Repeat Instability in the
C Orientation
The replication fork progresses slowly through the repeat bound to Rap1 and
other proteins. Tof1 andCsm3 components of the replication pausing complex
sense the protein bulge at the repetitive tract and slow replication progression
through this region (black pause symbol). The replication slow down can lead
to repeat expansions via the PRR pathway or HR pathway.
was strongly reduced in TOF1 and CSM3 gene knockouts
but unaffected in the mrc1D strain (Figure 3). Thus, replication
stalling at the Ytel-protein complex is a likely trigger for repeat
expansions.
What could happen upon replication fork stalling at a Ytel
repeat? We show that Rad6 is crucial for Ytel expansions, which
implicates PRR in the process (Figure 4). This notion is addition-
ally supported by the data that a knockout of the Srs2 DNA
helicase, which channels stalled replication forks into the PRR
pathway (Marini and Krejci, 2010; Putnam et al., 2010), strongly
decreases Ytel expansions. Another important player in the
Rad6-dependent PRR pathway is Rad5, which promotes tem-
plate switching at stalled replication forks (Minca and Kowalski,
2010). Such template switching can occur through either the in-
vasion of the nascent leading strand into a sister chromatid or via
fork reversal (Klein, 2007; Sale, 2012). We observed a 15-fold
reduction in the expansion rate of the Ytel repeat when we
knocked out the RAD5 gene, implying that template switching
is involved (Figure 4, left panel). Extra repeats can be added
when the quasi-D loop is dismantled and the nascent leading
strand switches back to its original template fostering fork restart
(Figure 4, left panel), a process possibly facilitated by the Srs2
helicase (Marini and Krejci, 2010).
If the fork fails to restart via the PRR pathway, a repetitive gap
can be repaired by HR. Supporting this idea, we show that both
the RAD51 and RAD52 genes are important for the expansion of
Ytel repeats.We believe, therefore, that Ytel expansions can also
occur via the HR pathway (Figure 4, right panel). The D-loop for-
mation can be counteracted by several helicases (Srs2, Mph1,
and Sgs1; Dupaigne et al., 2008; Prakash et al., 2009) driving
the recombination process into SDSA (Ira et al., 2003; Mitchel
et al., 2013). We have showed that Srs2 and Mph1 helicases
play an important role in the process of Ytel repeat expansions,
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as their knockouts strongly reduced repeat expansion rates.
Thus, repeats can be added either during sister chromatid
exchange owing to out-of-register strand invasion, or during
SDSA, owing to out-of-register re-annealing of two repetitive
strands (Figure 4, right panel).
Overall, our genetic analysis demonstrates that the interplay of
two pathways, HR and PRR, contributes to expansions of Ytel
repeats in the C-orientation. Knocking out each of these path-
ways individually leads to a 10- to 30-fold reduction in the expan-
sion rates, while knocking out both of them together results in a
synergetic, 230-fold decrease in the expansion rate (Figure 3).
We also found a strong increase in the rate of Ytel expansions
in the sir2D strain (Figure 3). Rap1 protein is bound to interstitial
Ytel repeats (Aksenova et al., 2013; Anand et al., 2012); thus, we
expect sirtuins to be there as well (Moretti et al., 1994). Sirtuins
were shown to suppress HR at stalled replication forks (Bengurıa
et al., 2003), which can account for the stabilizing effect of sir-
tuins on Ytel repeats.
Based on our genetic analysis, the mechanisms of expansions
of Ytel repeats differ from that for other unstable repeats. HR pro-
motes expansions of Ytel repeats, in contrast to previous reports
showing that it prevents small-scale expansions of CAG repeats
(Sundararajan et al., 2010) or has no role in large-scale expan-
sions of GAA repeats (Shishkin et al., 2009). Similarly, PRR pro-
motes Ytel repeat expansions, while it was previously shown to
inhibit small-scale expansions of various trinucleotide repeats
(Bhattacharyya and Lahue, 2004; Daee et al., 2007; Kerrest
et al., 2009). We speculate that these differences might be due
todifferentmechanismsof fork stalling at unusualDNAstructures
formed by trinucleotide repeats versus nucleoprotein complexes
assembled at interstitial telomeric repeats.
Finally, our data hint to a possible overlap in themechanismsof
expansions of interstitial telomeric repeats and ALT, a telomere
maintenance pathway in cells lacking telomerase, including
manyhumancancers (Cesare andReddel, 2010). ALT is a recom-
bination-dependent process (Lundblad, 2002; Wellinger and Za-
kian, 2012), similar towhatweseehere for interstitial Ytel repeats.
Furthermore, Rad6was implicated in telomere formation in some
yeast ALT strains (Hu et al., 2013).
EXPERIMENTAL PROCEDURES
Yeast Strains
Isogenic haploid strains used in this study (Table S3) were derived from
SMY710 (Aksenova et al., 2013). All strains carried an artificially split URA3
gene within chromosome III, containing different numbers of telomeric repeats
within its intron (SMY803, 0 repeats; SMY751, 8 repeats; and SMY750, 15 re-
peats). Strains were constructed using the plasmids pISL-UR-Intron-A3-
TRP1-ISR, UIRL-Ytel18/19, and UIRL-Ytel11/13 (Table S2).
Various gene knockouts (Table S4) were made in the SMY751 strain using a
PCR-based method for gene disruption (Wach et al., 1994). Either the hygB
cassette (Goldstein and McCusker, 1999) or HIS3 cassette (Sikorski and
Hieter, 1989) amplified with primers containing 50 flanks of homology to the
target genes were used to make knockouts.
Plasmids
Plasmid pISL-UR-Intron-A3-TRP1-ISR was described previously (Aksenova
et al., 2013). Plasmids UIRL-Ytel18/19 and UIRL-Ytel11/13 were constructed
on the basis of pISL-UR-Intron-A3-TRP1-ISR (Table S2).
DNA fragments containing telomeric repeats were excised from pUC19-
YTEL18 and pUC19-YTEL11 (Aksenova et al., 2013) and inserted into the
orts 13, 1545–1551, November 24, 2015 ª2015 The Authors 1549
XhoI-digested and blunt-ended pISL-UR-Intron-A3-TRP1-ISR plasmid. The
sequence corresponding to the Ytel-containing URA3-Intron gene is shown
in Figure S3.
Measurements of Rates of Expansion, Contraction, and Gene
Inactivation
Rates were calculated using the Ma-Sandri-Sarkar maximum likelihood esti-
mator (MSS-MLE) method with correction for plating efficiency as described
earlier (Aksenova et al., 2013). See Supplemental Experimental Procedures
for more details.
Other Methods
Selection of 5-FOA-resistant colonies was performed on 0.1% 5-FOA media
and analyzed by colony PCR as described previously (Aksenova et al., 2013).
The entire coding sequence of the Ytel-containingURA3-Intron gene with its
promoter and 30 UTR was amplified from yeast genomic DNA and sequenced
at the University of Chicago Sequencing Facility or at the Research Resource
Center for Molecular and Cell Technologies (Research Park, St. Petersburg
State University, Russia).
RNA expression analysis was performed as described previously (Aksenova
et al., 2013).
SUPPLEMENTAL INFORMATION
Supplemental information includes Supplemental Experimental Procedures,
four figures, and four tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2015.10.023.
AUTHOR CONTRIBUTIONS
A.Y.A. designed and performed experiments, analyzed data, and wrote the
manuscript. G.H, A.A.S., and K.V.V. performed experiments. S.M.M. super-
vised the whole project, analyzed data, and wrote the manuscript.
ACKNOWLEDGMENTS
We thank Tom Petes for fruitful discussions, Jane Kim and Ryan McGinty for
critical reading of the manuscript, Durwood Marshall for statistical consulting,
and Alexey Masharsky and Elizaveta Gorodilova for support with sequencing.
This study was funded by NIH grants GM105473 and GM60987 to S.M.M and
RFBR grant #15-04-08658 to A.Y.A.
Received: January 29, 2015
Revised: August 7, 2015
Accepted: October 8, 2015
Published: November 12, 2015
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