Expansion of Interstitial Telomeric Sequences in Yeastase.tufts.edu/biology/labs/mirkin/documents/2015expansionTelomeri… · Ytel Repeats in C Orientation Are Highly Unstable and

Report

Expansion of Interstitial T

Graphical 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

[email protected]

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: [email protected]

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

Cell Rep

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

Cell Rep

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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