Expansion of Interstitial Telomeric Sequences in Yeast

Report

Expansion of Interstitial Telomeric Sequences in Yeast Graphical Abstract

Authors Anna Y. Aksenova, Gil Han, Alexander A. Shishkin, Kirill V. Volkov, Sergei M. Mirkin

Correspondence [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.

Highlights d

Yeast interstitial telomeric sequences (ITSs) are prone to expansions

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Post-replicative repair and homologous recombination contribute to ITS instability

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These data can explain length polymorphism characteristic of ITSs in humans

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The proposed model may have implications for alternative telomere lengthening in cancer

Aksenova et al., 2015, Cell Reports 13, 1545–1551 November 24, 2015 ª2015 The Authors http://dx.doi.org/10.1016/j.celrep.2015.10.023

Cell Reports

Report Expansion of Interstitial Telomeric Sequences in Yeast Anna 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, USA Institute of MIT and Harvard, Cambridge, MA 02142, USA 3Department of Genetics, St. Petersburg State University, St. Petersburg 199034, Russia *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2015.10.023 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2Broad

SUMMARY

Telomeric repeats located within chromosomes are called interstitial telomeric sequences (ITSs). They are polymorphic in length and are likely hotspots for initiation of chromosomal rearrangements that have been linked to human disease. Using our S. cerevisiae system to study repeat-mediated genome instability, we have previously shown that yeast telomeric (Ytel) repeats induce various gross chromosomal rearrangements (GCR) when their G-rich strands serve as the lagging strand template for replication (G orientation). Here, we show that interstitial Ytel repeats in the opposite C orientation prefer to expand rather than cause GCR. A tract of eight Ytel repeats expands at a rate of 4 3 104 per replication, ranking them among the most expansion-prone DNA microsatellites. A candidate-based genetic analysis implicates both post-replication repair and homologous recombination pathways in the expansion process. We propose a model for Ytel repeat expansions and discuss its applications for genome instability and alternative telomere lengthening (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 binding 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 repeats bound by the CST (Cdc13/Stn1/Ten1) complex (Fo¨rstemann 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 (Fo¨rstemann 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

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 mechanism to counteract attrition of chromosome ends called alternative lengthening of telomeres (ALT). It is generally believed that recombination-mediated copying of telomeric DNA is a mechanism accounting for ALT (Lundblad, 2002). Besides chromosomal ends, telomeric repeats are also present 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 sequences (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; Bolzań, 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 doublestranded 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 microsatellites, 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). Cytogenetic 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 rearrangements (Hastie and Allshire, 1989). Supporting this idea, insertion 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 translocations in patients with Prader-Willi syndrome (Boutouil et al., 1996; Qui et al., 1993; Vermeesch et al., 1997), neuroblastoma

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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 proximal 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, respectively). 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 containing approximately equal amounts of cells were plated as drops on three different types of medium: 5-FOA-containing synthetic media, synthetic media 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 the URA3-Intron gene. The rows labeled URA3 mRNA 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. However, 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-orientation. 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 Expression Yeast 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

the sense strand for transcription and, which is also the lagging strand template for DNA replication (C orientation). As we previously 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 orientation 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 of URA3 mRNA was measured using 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 URA3 mRNA as the length of the Ytel repeat increases, while the amount of pre-mRNA was unaffected by the presence of Ytel repeats (Figure 1C). Ytel Repeats in C Orientation Are Highly Unstable and Prone to Expansions Our 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

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Figure 2. Expansions of the (CCCACACA)8 Repeat (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 repetitive 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)8 repeat. (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, chromosomal 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 carrying 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 carrying 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 3 104 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 system 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 chromosomal rearrangements (Table S1). All 130 sequenced clones from 36 independent cultures had the initial-size Ytel repeat

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 contractions 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 contractions 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 4 3 105 per replication, 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 3 107 (Miret et al., 1998), while 75-bp-long (CGG)n and (CTG)n repeats both expand at the rate of 1 3 105 (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


Figure 3. Genetic Control of Ytel Repeat Expansions Effect of different gene knockouts on the expansion of the (CCCACACA)8 repeat. 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). Distribution of expanded clones in 12–36 independent cultures was studied for each strain. Length of the repetitive tract for each analyzed clone was 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 difference 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 Repeats To elucidate the mechanisms of expansions of Ytel repeats, we used a candidate gene approach by comparing the rates of expansions for the (CCCACACA)8 repeat in individual knockouts of genes involved in NHEJ, HR, and PRR pathways. We also studied the knockouts of genes encoding replication fork stabilizers, RecQ- and UvrD-like DNA helicases, as well as telomere maintenance proteins. The results of these analyses are summarized in Figure 3. Deletion of the RAD6 gene had the biggest effect on expansions 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 ubiquitinating 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 and Rad52 drive the strand exchange reaction, which is at the heart of HR (Krogh and Symington, 2004). Knocking out

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 interplay 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 an MPH1 gene knockout decreased their expansion rate by 11-fold. The fact that the Mph1 helicase upholds expansions of the Ytel repeats implicates 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 not mrc1 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 expansions in the yku70D and yku80D strains with impaired NHEJ, indicating 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 unstable in our yeast system. When the G-rich strand of the repeat was in the lagging strand template (G orientation), gross chromosomal rearrangements frequently occurred (Aksenova et al., 2013). In contrast, the same length Ytel repeat in the C orientation 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 doublestrand 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 (Hodgson et al., 2007). We found that the expansion rate of Ytel repeats


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 and Csm3 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 additionally 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 template switching at stalled replication forks (Minca and Kowalski, 2010). Such template switching can occur through either the invasion 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 formation 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,

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 pathways individually leads to a 10- to 30-fold reduction in the expansion 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 (Bengurıá et al., 2003), which can account for the stabilizing effect of sirtuins on Ytel repeats. Based on our genetic analysis, the mechanisms of expansions of Ytel repeats differ from that for other unstable repeats. HR promotes 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 expansions of GAA repeats (Shishkin et al., 2009). Similarly, PRR promotes 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 to different mechanisms of fork stalling at unusual DNA structures formed by trinucleotide repeats versus nucleoprotein complexes assembled at interstitial telomeric repeats. Finally, our data hint to a possible overlap in the mechanisms of expansions of interstitial telomeric repeats and ALT, a telomere maintenance pathway in cells lacking telomerase, including many human cancers (Cesare and Reddel, 2010). ALT is a recombination-dependent process (Lundblad, 2002; Wellinger and Zakian, 2012), similar to what we see here for interstitial Ytel repeats. Furthermore, Rad6 was 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 repeats). Strains were constructed using the plasmids pISL-UR-Intron-A3TRP1-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 pUC19YTEL18 and pUC19-YTEL11 (Aksenova et al., 2013) and inserted into the


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 estimator (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-containing URA3-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. supervised 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 REFERENCES Aksenova, A.Y., Greenwell, P.W., Dominska, M., Shishkin, A.A., Kim, J.C., Petes, T.D., and Mirkin, S.M. (2013). Genome rearrangements caused by interstitial telomeric sequences in yeast. Proc. Natl. Acad. Sci. USA 110, 19866–19871. Anand, R.P., Shah, K.A., Niu, H., Sung, P., Mirkin, S.M., and Freudenreich, C.H. (2012). Overcoming natural replication barriers: differential helicase requirements. Nucleic Acids Res. 40, 1091–1105.

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