d n a r e p a i r 6 ( 2 0 0 7 ) 1607–1617

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Mrc1 protects uncapped budding yeasttelomeres from exonuclease EXO1

Avgi Tsolou, David Lydall ∗

Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Henry Wellcome Laboratory forBiogerontology Research, Newcastle University, Newcastle upon Tyne NE4 6BE, UK

a r t i c l e i n f o

Article history:

Received 7 March 2007

Received in revised form

18 May 2007

Accepted 22 May 2007

Published on line 6 July 2007

Keywords:

a b s t r a c t

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork

machinery and is necessary for checkpoint activation after replication stress. In this study,

we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1

contributes to the vitality of both cdc13-1 and yku70� telomere capping mutants. Cells with

telomere capping defects containing MRC1 or mrc1AQ, a checkpoint defective allele, exhibit

similar growth, suggesting growth defects of cdc13-1 mrc1� are not due to checkpoint defects.

This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping.

Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded

DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding

Mrc1

Telomere

ssDNA

CDC13

YKU70

E

a nuclease that contributes to single stranded DNA accumulation after telomere uncapping,

improves growth of cdc13-1 mrc1� strains and decreases ssDNA production. Our observations

show that Mrc1, a core component of the replication fork, plays an important role in telomere

capping, protecting from nucleases and checkpoint pathways.

1

TeibdcSDwcg

ta

which include – among other components – mediators. Medi-

1d

XO1

. Introduction

elomeres are specialized DNA–protein complexes at thend of eukaryotic chromosomes. Proper telomere structures essential for chromosome integrity and genome stabilityecause telomeres protect natural chromosome ends fromegradation and end-to-end fusion and because they ensureomplete genome replication. Telomeres differ from Doubletrand Breaks (DSBs) in that they normally fail to activateNA repair or DNA damage checkpoint pathways. If thatas the case, then they would undergo recombination and

hromosomal fusions and dicentric chromosomes would beenerated [1–4].

Many proteins associate with telomeric DNA. These pro-eins ensure that telomeres behave differently from DSB endsnd help maintain chromosomal stability. Some telomeric

∗ Corresponding author. Tel.: +44 191 256 3449; fax: +44 191 256 3445.E-mail address: d.a.lydall@ncl.ac.uk (D. Lydall).

568-7864 © 2007 Elsevier B.V.oi:10.1016/j.dnarep.2007.05.010

Open access under CC BY license.

© 2007 Elsevier B.V.

proteins bind specifically to dsDNA and others show higheraffinity to ssDNA. In budding yeast, there are numerous pro-teins with affinity for telomeric dsDNA, such as Rap1, Sir2,Sir3, Sir4, Rif1 and Yku70/Yku80 [5]. The budding yeast telom-eric ssDNA ends are thought to be protected by three essentialproteins, Cdc13, Stn1 and Ten1 [6–11].

If telomeres become uncapped, they activate a DNA dam-age response pathway leading to cell cycle arrest [12–14].Moreover, recently it has been suggested that telomeres trig-ger a transient DNA damage response in each S phase inorder to complete DNA replication and cap chromosome ends[15]. DNA damage response pathways are complex networks

Open access under CC BY license.

ators facilitate the transmission of the DNA damage signalfrom sensors to downstream effectors; activation of the lat-ter affecting cell cycle progression [16]. Mrc1 (Mediator of

( 2 0

1608 d n a r e p a i r 6

Replication Checkpoint 1) appears to take the mediator role inRad53 activation under replication stress [17]. However, paral-lel pathways exist because in mrc1� mutants Rad53 activationoccurs through Rad9 (another mediator protein), presum-ably because the accumulation of “DNA damage” rather than“replication defects” in mrc1� mutants leads to activation ofRad9 and thereby activation of Rad53 [17].

Mrc1 also appears to be directly involved in DNA replica-tion and, because of this, mrc1� cells display a slow S phase[18]. Mrc1 is an active component of the replication machin-ery, loaded onto DNA shortly after replication initiates, andmoving with other components of replication forks [19–21]. Inthe presence of hydroxyurea, a type of replication stress, Mrc1appears to form a stable replication-pausing complex prevent-ing the uncoupling of the replication machinery from DNAsynthesis [20–22]. According to this model, Mrc1 mediatesactivation of Rad53 under conditions of replication stress sothat subsequent DNA repair events occur and cell replicationresumes normal function [22]. However, recent experimentssuggest that the role of Mrc1 at stalled replication forks ismore than activating Rad53, since mrc1AQ cells, defective inRad53 activation, are not defective in replication fork initi-ation or progression [18,20,21]. mrc1AQ is a mutant allele inwhich SQ/TQ residues have been substituted with AQ, result-ing in its inability to mediate phosphorylation and activationof Rad53 [18].

Although Mrc1 is involved in the DNA replication check-point, it has been shown that it is not required for theDNA damage checkpoint, since cdc13-1 mrc1� double mutantsarrest in G2 at non-permissive temperatures [17]. It has beenreported that activation of Rad53 in response to telomereshortening still occurs in the absence of Rad9 and that Mrc1is responsible for this activation in telomerase-deficient cells,in which telomeres continually shorten until they activatea checkpoint [23]. Surprisingly, though, tlc1� mrc1� dou-ble mutants arrest cell division, suggesting that Mrc1 is notrequired for cell cycle arrest in telomerase negative cells. Incontrast, after cdc13-1 induced telomere uncapping, Rad53activation is entirely Rad9-dependent and Mrc1-independent[23].

Here we investigated the role of Mrc1 at uncapped telom-eres, using the temperature sensitive cdc13-1 and yku70�

mutations to uncap telomeres. Our experiments indicate thatMrc1 protects telomeres from the DNA damage response andthat the role of Mrc1 in DNA replication forks, rather than incheckpoint activation, is important for protection of telom-eres.

2. Materials and methods

2.1. Yeast strains and plasmids

All strains in the W303 background are RAD5 and they containan ade2-1 mutation (Supplemental Table 1); therefore yeastextract/peptone/dextrose (YEPD) was supplemented with ade-

nine at 50 mg/l. Strains 3393–3402 are in the S288C background(Supplemental Table 1) and they were generated by mating asingle gene deletion mutant array [24] with a cdc13-1 querystrain [25]. To construct strains, standard genetic procedures

0 7 ) 1607–1617

of transformation and tetrad analysis were used [26]. pMRC1and pmrc1AQ, also carrying the URA3 gene were a gift fromSteven Elledge [17,18].

MRC1 was disrupted in two different ways. Firstly, the MRC1ORF was substituted with KanMX6, with a PCR based method[27]. Primers 5′-tcgttattcgcttttgaacttatcaccaaatattttagtgCGGA-TCCCCGGGTTAATTAA-3′ (#878) and 5′-ctggagttcaatcaacttc-ttcggaaaagataaaaaaccaGAATTCGAGCTCGTTTAAAC-3′ (#881),which contain homology to upstream and downstreamsequences of MRC1 (bases in lowercase), were used toamplify a 1559 bp KanMX6 sequence (pFA6a-kanMX6; [27]).The PCR product was transformed into yeast and candi-date colonies were selected for G418 resistance. Integrationof the KanMX6 marker into the MRC1 locus was con-firmed by PCR, using two sets of primers: (i) forward5′-CCAAGAACAGACAAACAACTAAGGA-3′ (#876) with reverseprimer 5′-TCAGCATCCATGTTGGAATT-3′ (#81) and (ii) for-ward 5′-CCATCCTATGGAACTGCCTC-3′(#82) with reverse 5′-CCTAGACTCGGGTGCCATCT-3′ (#880). Disruption of MRC1 wasalso confirmed by Southern blot (data not shown). Alterna-tively, MRC1 was substituted with URA3 using a restrictionenzyme digest approach. First, pMRC1 was digested with XhoIand a 5008 bp fragment containing the full MRC1 gene wascloned into XhoI digested pIC19H vector (2.7 kb) to createpAT1065. A correct clone was identified by restriction digests.pAT1065 was digested with SpeI to remove a 2.31 kb DNA frag-ment containing the bulk of MRC1, which was replaced witha 1.3 kb BamHI URA3 gene fragment from pDL349 (pBSB + KScontaining a BamHI fragment containing the URA3 gene)by blunt cloning following treatment with DNA polymeraseI Large (Klenow) fragment (New England Biolabs). Positiveclones were selected by restriction enzyme digests to iden-tify the disruption of the bulk of MRC1 with URA3 (pAT1066).Disruption of MRC1 was also confirmed by Southern blot (datanot shown). To disrupt MRC1, pAT1066 was digested with XhoIprior to transformation of yeast.

2.2. Spot tests

Single colonies were inoculated into 2 ml YEPDextrose (YEPD)and incubated overnight, with aeration, at 23 ◦C. The follow-ing day, 200 �l of each culture was inoculated into 2 ml of freshYEPD and returned to 23 ◦C. Cells were grown for three morehours, and cell numbers were determined in a haemocytome-ter. The cells were then centrifuged (13,000 rpm for 10 s in amicrocentrifuge), washed twice with sterile water and resus-pended in water to a final concentration of 1.5 × 107 cells/ml.A five-fold dilution series of each of the cultures was pre-pared using sterile water in a 96 well plate and 3–5 �l spottedonto plates using a 48-prong replica plating device. Plates wereincubated at various temperatures for 2–3 days before beingphotographed. For spot tests with strains containing pMRC1,pmrc1AQ or pRS416 the steps were as described above, butstrains were grown on selective medium (-URA). All strainsshown as if on a single agar plate were grown on the sameplate, although in some cases their positions were moved

using Adobe Photoshop and Adobe Illustrator CS. Unless oth-erwise stated, at least two different strains of the samegenotype were spot tested and representative examples areshown.

0 0 7

2

Hwt

2

PT(mmWb[(ba1c

2

SfDocTdI

2

cSI8hTgamtp

2

cgatf1cer

d n a r e p a i r 6 ( 2

.3. Yeast transformation

igh efficiency transformations needed for gene disruptionsere performed according to Gietz et al. [28]. For plasmid

ransformations a more rapid method was used [29].

.4. Western blots

rotein extracts were prepared by glass bead breakage inCA, essentially as previously described [30,31]. Bio-Rad gels

7.5% Tris–HCl), Schleicher and Schuell Protan Nitrocelluloseembranes and the Pierce Supersignal West Pico Chemilu-inescent Substrate detection kit were used in a standardestern blot procedure. Rabbit anti-Rad53 polyclonal anti-

ody (AbDL50, 1:1000 dilution, a gift from Dan Durocher32]) was a primary antibody used with an anti-rabbit-HRPAbDL7, 1:10,000 dilution, Dako P0448) as a secondary anti-ody used. Mouse anti-tubulin (TAT-1, AbDL42, 1:2000 dilution,

gift from Keith Gull [33] and anti-mouse-HRP (AbDL6,:10,000 dilution, Dako P0447) were used for tubulin loadingontrols.

.5. Telomere length measurement by Southern blot

trains were grown to saturation in liquid YEPD at 23 ◦C. DNArom each strain was subjected to XhoI cut. The digestedNA was loaded on a 0.8% agarose gel, run at low voltagevernight, transferred to a Magna nylon membrane and UVross-linked. The membrane was then hybridised with a Y′-G probe [34]. A non-radioactive detection kit was used for theetection of the hybridisation (Amersham, Arlington Heights,

L).

.6. Synchronous cultures

dc13-1 cdc15-2 bar1� strains with additional mutations (seeupplemental Table 1) were grown in YEPD at 23 ◦C overnight.n the morning, cells were adjusted to a concentration of× 106 buds/ml in 250 ml. Cultures were grown for three moreours, then arrested with 20 nM �-factor for a further 2.5 h.he cultures were then released from G1 arrest by centrifu-ation and washed twice in YEPD and placed at 36 ◦C, 40 minfter the culture was first centrifuged. Cell cycle position wasonitored as previously described [13]. DNA was prepared and

he fraction of single stranded DNA (ssDNA) was measured asreviously described [35].

.7. Asynchronous cultures

dc13-1 strains with additional mutations indicated wererown in YEPD at 23 ◦C overnight. In the morning, cells weredjusted to a concentration of 1 × 107 cells/ml and tempera-ure was raised to 27.3 ◦C. Every 90 min samples were takenor cell cycle position and cell density was re-adjusted to

× 107 cells/ml. Cell numbers were determined with a haemo-ytometer. Samples for Western blots were collected fromxponentially growing cultures 2 h after the temperature wasaised from 23 ◦C to 36 ◦C.

) 1607–1617 1609

2.8. Cell cycle position determination

A 1 ml sample of culture was centrifuged for 8–10 s at highspeed, the supernatant was aspirated, and cells were fixedat 70% ethanol overnight. The fixed cells were washedtwice in water before being resuspended in 4,6-diamidino-2-phenylindole (DAPI, 0.2 �g/ml). Cell cycle distribution wasmonitored by DAPI staining of nuclei and fluorescencemicroscopy. For DAPI staining, 100 cells for each sample werecounted and classified as: (1) unbudded, single DAPI-stainedbody; (2) small budded, single DAPI-stained body, with the bud<50% of the diameter of the mother cell; (3) medial nucleardivision, single DAPI-stained body, with bud >50% diameterof mother cell, the cdc13-1 arrest point; (4) late nuclear divi-sion, two buds, and two DAPI-stained bodies, the cdc15-2 arrestpoint [36].

3. Results

3.1. Mrc1 contributes to the vitality of cdc13-1 andyku70� mutants

Since Mrc1 plays a role in the checkpoint response to stalledreplication, we wondered if it also plays a role at uncappedtelomeres. The temperature sensitive cdc13-1 mutation causesa defect in Cdc13, a telomere binding protein, and cells con-taining this mutation accumulate large amounts of ssDNA attelomeres at non-permissive temperatures [12,37,38]. Interest-ingly, deletion of checkpoint proteins, like Chk1, Mec1, Mec3,Rad9, Rad17, Rad24 and Rad53 improves growth of cdc13-1strains at semi-permissive temperatures [39–41]. This is pre-sumably because checkpoint pathways inhibit cell divisionby responding to low levels of ssDNA that accumulates attelomeres at semi-permissive temperatures. Deletion of othercheckpoint proteins, like the MRX complex, which appears toplay a role in telomere capping, worsens the growth of cdc13-1 strains [42]. Therefore, we wanted to investigate whetherMrc1 plays a role at uncapped telomeres and, if so, whether itbehaved like Rad9 or MRX. Fig. 1A shows that deletion of MRC1dramatically reduces the growth of cdc13-1 mutant strains at25 ◦C. The effect of Mrc1 is not as profound as that of theMRX complex, as cdc13-1 mre11� and cdc13-1 rad50� displaymore severe growth defects than cdc13-1 mrc1� even at 23 ◦C(Supplementary Fig. 1). Thus, Mrc1, like MRX, but unlike themajority of checkpoint proteins, contributes to the vitality ofcdc13-1 strains.

We next wanted to investigate whether analogous growthdefects of cdc13-1 mrc1� strains occur in yku70� strains.Yku70 is a telomere capping protein which is also involvedin dsDNA damage repair and in Non-Homologous End Joining(reviewed in Ref. [43]). Deletion of YKU70 results in a tempera-ture sensitive phenotype at 37 ◦C, due to telomere uncapping,which activates a Chk1-dependent cell cycle arrest [36]. Fig. 1Bdemonstrates that deletion of MRC1 results in a severe growth

defect of yku70� mutant strains at 35 ◦C and 36 ◦C. Thus, Mrc1contributes to the vitality of yku70� strains. We conclude thatMrc1 contributes to the vitality of two cell types defective intelomere capping.

1610 d n a r e p a i r 6 ( 2 0 0 7 ) 1607–1617

Fig. 1 – Mrc1 contributes to the vitality of cdc13-1 and yku70� mutants. Small aliquots of five-fold dilution series of thestrains indicated, and growing at 23 ◦C, were transferred to plates and incubated at the temperatures shown for 3 days

catedu70�

before being photographed. The relevant genotypes are indi(A) Growth of W303 cdc13-1 mutants. (B) Growth of W303 yk

Mrc1, Tof1 and Csm3 are three proteins that play similar,although distinct roles in DNA replication [19–21,44]. There-fore, we wished to address whether Tof1 and Csm3, like Mrc1,contributed to the vitality of cdc13-1 mutants. Spot test analy-sis showed that although deletion of TOF1 or CSM3 also conferssome growth defects on cdc13-1 mutants, deletion of MRC1has a stronger phenotype (Fig. 1C). Therefore, we decidedto focus on understanding the role of MRC1 at uncappedtelomeres.

3.2. Growth defects of cdc13-1 mrc1� cells are not dueto checkpoint defects

In budding yeast, two independent roles have been previouslyreported for Mrc1. One role implicates Mrc1 as a media-tor of checkpoint activation under replication stress and theother role is as part of the replication machinery [17–21].Therefore, we investigated whether the heightened temper-ature sensitivity phenotype of cdc13-1 mrc1� mutant strainsis a result of a replication defect, a checkpoint defect orboth.

cdc13-1 mrc1� mutants were complemented with eitherwild type pMRC1, pmrc1AQ or an empty vector (pRS416) andstrains were grown at various temperatures. Fig. 2 shows thatat 26.2 ◦C, complementation of mrc1� cdc13-1 mutant strains,with either pMRC1 or pmrc1AQ allele improves growth com-pared to the empty vector control (compare rows 1–3). Thus,we conclude that the checkpoint role of Mrc1 is not important

for the vitality of cdc13-1 strains.

At higher temperature we noticed an increased growth ofcdc13-1 mrc1� cells carrying the mrc1AQ allele, compared topMRC1. However, this phenotype was observed even in the

on the left, and strain numbers are shown in parentheses.mutants. (C) Growth of S288C cdc13-1 mutants.

presence of the wild type MRC1 allele, suggesting that thiseffect is due to some type of dominant effect of mrc1AQ (Fig. 2,compare rows 2 and 11).

3.3. Exo1 inhibits growth of cdc13-1 mrc1� andyku70� mrc1� mutants

EXO1 encodes a nuclease known to contribute to ssDNA pro-duction at uncapped telomeres of cdc13-1 and yku70� strains[36,39]. If Exo1-dependent ssDNA production at uncappedtelomeres is responsible for the poor growth of cdc13-1 mrc1�

and yku70� mrc1� mutants, then removing Exo1 should sup-press their poor growth. Fig. 3A demonstrates that at 25 ◦C,cdc13-1 mrc1� exo1� triple mutants exhibit better growththan cdc13-1 mrc1� strains, showing that Exo1 contributes tothe growth defects observed in cdc13-1 mrc1� strains. Impor-tantly, deleting EXO1 also reverses the growth defect of mrc1�

yku70� mutants (Fig. 3B). These data suggest that Mrc1 pro-tects uncapped telomeres from Exo1.

3.4. Effects of checkpoint mutations on cdc13-1 mrc1�

and yku70� mrc1� growth

To understand if checkpoint pathways are activated in mrc1�

strains after telomere uncapping, we wanted to combinecdc13-1 mrc1� and yku70� mrc1� strains with checkpointmutations. A genetic screen has revealed that mrc1� is syn-thetically lethal with rad9�, rad17� or rad24� checkpoint

mutations [24]. Consistent with these results we were unableto recover viable offspring carrying mrc1� in combinations ofany of these checkpoint genes (data not shown). However, wewere able to combine mrc1� with rad53� and chk1�, encod-

d n a r e p a i r 6 ( 2 0 0 7 ) 1607–1617 1611

Fig. 2 – The checkpoint role of Mrc1 is not important for the vitality of cdc13-1 strains. Small aliquots of five-fold dilutionseries of the strains indicated, and growing at 23 ◦C, were transferred to plates and incubated at the temperatures shown for3 re inp

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Fi

days before being photographed. The relevant genotypes aarentheses. Row numbers are designated on the right.

ng two downstream checkpoint kinases (analogues of humanhk2 and Chk1, respectively).

When cdc13-1 mutants grow at non-permissive temper-tures Rad53 and Chk1 dependent parallel pathways arectivated [36,45,46]. We deleted RAD53 or CHK1 and exam-ned the effects in cdc13-1 mrc1� mutants. Deletion of RAD53equires simultaneous deletion of SML1 to obtain viablepores. Sml1 is a small protein that inhibits the activityf ribonucleotide reductase (RNR) which catalyzes the rate-

imiting step of de novo dNTP synthesis [47]. Normally Sml1s degraded in a Rad53-dependent manner during S phase48]. We found that removal of RAD53 and SML1 improvedhe growth of cdc13-1 mrc1� strains (Fig. 4A). However, dele-

ion of SML1 alone (cdc13-1 mrc1� sml1�) also rescued therowth defects associated with MRC1 deletion (Fig. 4A). Thus,e were unable to observe any strong role for Rad53, inaintaining vitality of cdc13-1 mrc1� mutants, other than in

ig. 3 – Exo1 contributes to the poor growth of cdc13-1 mrc1� andn Fig. 1. The relevant genotypes are indicated on the left, and st

dicated on the left, and strain numbers are shown in

degrading Sml1. We suggest that the reason that deleting Sml1improves the growth of cdc13-1 mrc1� strains is that increasedribonucleotide reductase activity may stabilise the replicationforks.

Removal of Chk1, like removal of Rad53, does not rescuegrowth of cdc13-1 mrc1� strains, indicating that Chk1 does notinhibit growth of these mutants (Fig. 4B). Therefore, we find noevidence that inactivating DNA damage checkpoint pathwaysimproves growth of cdc13-1 mrc1� mutants.

A CHK1 deletion strongly rescues growth of yku70�

mrc1� mutants at restrictive temperatures (Fig. 4C) sim-ilarly to its effect in yku70� mutants [36]. Thus, mrc1�

yku70� uncapped telomeres qualitatively behave like yku70�

uncapped mutants. The effects of chk1� in yku70� mrc1�

and cdc13-1 mrc1� strains are consistent with earlier findingsshowing that the Chk1-dependent pathway is more importantin yku70� mutants [36,45].

yku70� mrc1� strains. Colonies were plated as describedrain numbers are shown in parentheses.

1612 d n a r e p a i r 6 ( 2 0 0 7 ) 1607–1617

Fig. 4 – Differential suppression of cdc13-1 mrc1� and yku70� mrc1� growth defects by checkpoint mutations. Colonies werecate

plated as described in Fig. 1. The relevant genotypes are indi

3.5. Mrc1 is not required for the cell cycle arrest aftercdc13-1 uncapping

To directly test whether Mrc1 plays a checkpoint role incdc13-1 strains, cdc13-1 strains with additional mutationswere first grown at the semi-permissive temperature 27.3 ◦C.Strains defective in telomere capping (cdc13-1) arrest at medialnuclear division before entry to anaphase after 3 h at 27.3 ◦C(Fig. 5A). As expected, when the checkpoint is compromisedin cdc13-1 chk1�, cdc13-1 rad9� or cdc13-1 rad53� cells, noaccumulation at medial nuclear division is observed over 9 h(Fig. 5A). In contrast, cdc13-1 and cdc13-1 mrc1� strains rapidlyaccumulate at medial nuclear division and within 3 h morethan 90% of cells are arrested at this point. Consistent withthese conclusions cell numbers stopped increasing in cdc13-1 and cdc13-1 mrc1� strains, but continued to increase in theother strains (Fig. 5B). We conclude that Mrc1 is not requiredfor the checkpoint response to cdc13-1 dependent telomereuncapping.

Rad53 and Chk1 are components of parallel checkpointpathways that respond to cdc13-1 induced telomere uncapping[45,46]. It appears that the Rad53 pathway is more important

for arrest of cdc13-1 mrc1� mutants because 80% of cdc13-1mrc1� chk1� cells have arrested at medial nuclear divisionby 7.5 h, whereas there is no arrest of cdc13-1 mrc1� rad53�

cells.

d on the left, and strain numbers are shown in parentheses.

To test the role of Mrc1 in checkpoint control in a sin-gle cell cycle we combined mrc1� with cdc13-1 cdc15-2 andbar1 mutations. Over many years we and others have usedthese mutations to determine the effects of checkpoint pro-teins in responding to telomere uncapping [13,39,40,45]. Bar1encodes a protease that degrades the mating pheromone �-factor. Cells bearing the bar1 mutation can efficiently arrest inG1 phase of the cell cycle with low levels of �-factor. Cdc15 isnecessary for mitotic exit. At 36 ◦C, cdc13-1 cdc15-2 bar1 con-trol strains, released from alpha factor arrest, accumulate atmedial nuclear division due to cdc13-1-dependent telomereuncapping. However, if cells have escaped the G2/M check-point, like cdc13-1 rad9� cdc15-2 bar1 strains, they arrest at latenuclear division due to cdc15-2 and they are unable to proceedto the next cycle. The cdc15-2 dependent cell cycle arrest helpsin two ways, it ensures that DNA damage checkpoint defectsare easily quantified because cells with checkpoint defectsaccumulate at a later stage of the cell cycle and that DNA dam-age caused by cdc13-1 is not amplified during new rounds ofDNA replication.

cdc13-1 cdc15-2 bar1 strains with additional mutations werearrested with �-factor, then released from G1 and transferred

to a non-permissive temperature to induce telomere uncap-ping and the cell cycle position was monitored. Fig. 5C showsthat in contrast to cdc13-1 rad9� strains cdc13-1 and cdc13-1mrc1� strains arrest at medial nuclear division with similar

d n a r e p a i r 6 ( 2 0 0 7 ) 1607–1617 1613

Fig. 5 – Mrc1 does not contribute to checkpoint activation after cdc13-1 dependent telomere uncapping. (A and B) 7 cdc13-1strains, whose genotypes are shown in e, were switched from 23 ◦C to 27.3 ◦C and their cell cycle position and growth weremonitored, as described in Section 2. (C and D) cdc15-2 bar1 strains with additional mutations [DLY2646 (cdc13-1 mrc1�),DLY3071 (cdc13-1 mrc1� exo1�), DLY1468 (cdc13-1), DLY1470 (cdc13-1 rad9�)] were synchronised with �-factor, released fromG1 to non-permissive temperature (36 ◦C) to induce telomere uncapping and cell cycle position was measured, after cellswere fixed in 70% ethanol and stained with DAPI. (E) Genotypes of strains used for the asynchronous cultures demonstratedin (a and b) are shown. (F) Western blot demonstrating Rad53 phosphorylation in various cdc13-1 strains with the additionalmutations indicated [DLY1108 (cdc13-1 RAD+), DLY1256 (cdc13-1 rad9�), DLY2532 (cdc13-1 mrc1�), DLY2533 (cdc13-1 mrc1�)].Cultures were grown overnight at 23 ◦C, diluted in the morning and divided in two. While still growing exponentially, thetemperature was raised to 36 ◦C in one of the aliquots and samples were taken 2 h later and processed for Western blots.A

kapmbtmhm

attuRp

nti-Tubulin was used as a loading control.

inetics at 36 ◦C, supporting the idea that Mrc1 does not playrole in checkpoint activation after cdc13-1 telomere uncap-

ing. Interestingly, cdc13-1 mrc1� exo1� remain arrested atedial nuclear division (Fig. 5C and D) which contrasts to the

ehaviour of cdc13-1 exo1� that begin to escape arrest duringhe 4-h period in analogous experiments [39]. This difference

ost likely reflects the fact that cdc13-1 mrc1� exo1� mutantsave more severe telomere capping defect than cdc13-1 exo1�

utants and therefore activate a stronger checkpoint signal.In response to both replication stress and DNA damage,

ctivation of the checkpoint machinery induces phosphoryla-ion and activation of Rad53 kinase. Therefore, we addressed

he role of Mrc1 in Rad53 phosphorylation after telomerencapping. cdc13-1 strains were exposed to 36 ◦C for 2 h andad53 phosphorylation was measured by Western blot. Rad53hosphorylation is observed in cdc13-1 mrc1� strains but not

in cdc13-1 rad9� mutants (Fig. 5F), confirming a previousstudy [23]. We conclude that activation of Rad53 after cdc13-1 dependent uncapping at non-permissive temperatures isRad9-dependent but Mrc1-independent.

3.6. Mrc1 contributes to telomere length regulation

If Mrc1 plays a protective role at telomeres, this predicts thatstrains lacking Mrc1 may have short telomeres. Fig. 6A showsthat absence of Mrc1 results in shorter telomeres, compared tothe wild type. However, the telomere length defects of mrc1�

mutants are not as severe as in rad50� or yku70� mutants, and

this may help explain why cdc13-1 mrc1� cells grow better thancdc13-1 rad50� cells (Supplementary Fig. 1). Our experiment isconsistent with replication proteins having an important rolein telomere length regulation [49–52].

1614 d n a r e p a i r 6 ( 2 0 0 7 ) 1607–1617

Fig. 6 – Mrc1 protects telomeres from shortening and inhibits ssDNA generation at uncapped telomeres. (A) Southern blotwhere the telomere length of various strains was examined. DNA was extracted from strains grown at 23 ◦C in liquid YEPD.Strains used were DLY640 (wild type), DLY641 (wild type), DLY2512 (mrc1�), DLY2709 (mrc1�), DLY1366 (yku70�), DLY2584(yku70�) and DLY1091 (rad50�). (B) Schematic figure to show the PDA1 locus on chromosome V. (C) ssDNA measurements inthe single copy locus of cdc15-2 bar1 strains with the additional mutations indicated; DLY2646 (cdc13-1 mrc1�), DLY3071(cdc13-1 mrc1� exo1�), DLY1468 (cdc13-1), DLY1470 (cdc13-1 rad9�). Samples were taken, during synchronous cultures, atthe indicated time points after cultures were released from G1 arrest and transferred to non-permissive temperatures(36 ◦C). DNA preparations were assessed by quantitative amplification of ssDNA (QAOS) [37] to measure ssDNA on the TGand AC strand at PDA1 locus. The error bars represent the standard error of the mean calculated from three independent

measurements of each sample.

3.7. Mrc1 protects telomeres from extended ssDNAaccumulation

All our data suggest an important role of Mrc1 in telomerecapping but no role in cell cycle arrest. To directly test therole of Mrc1 in telomere capping, we measured ssDNA accu-mulation on the 3′ TG strand, at PDA1, a single copy locusapproximately 30 kb away from the right end of chromosome Vin cdc13-1 strains (Fig. 6B). cdc13-1 strains were synchronised,as in Fig. 5C and D in order to follow the effects of Mrc1 on

ssDNA accumulation at non-permissive temperatures withina single cell cycle. We find that cdc13-1 mrc1� mutants, likecdc13-1 rad9� strains, accumulate more 3′ TG ssDNA at PDA1,30 kb from the uncapped telomere, compared to cdc13-1 strains

(Fig. 6C) [39]. This shows that Mrc1, like Rad9, protects cdc13-1mutants from ssDNA production. Consistent with our conclu-sion, increased ssDNA accumulation, closer to the telomere,in telomere repeats, was recently reported in cdc13-1 mrc1�

and yku70� mrc1� mutants using both in gel and dot blotanalyses [53]. Importantly, ssDNA production is reduced incdc13-1 mrc1� exo1� strains in comparison to cdc13-1 mrc1�

strains showing that Mrc1 protects uncapped telomeres fromExo1-dependent nuclease action. This ssDNA data is consis-tent with our finding that Exo1 contributes to the poor growth

of cdc13-1 mrc1� and yku70� mrc1� mutants (Fig. 3). Takentogether, we conclude that Mrc1 inhibits accumulation ofExo1-dependent ssDNA accumulation after telomere uncap-ping and, by this criterion, contributes to telomere capping.

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. Discussion

ur experiments demonstrate that Mrc1 contributes to theitality of budding yeast cells with uncapped telomeres.herefore, Mrc1 behaves differently from many other knownheckpoint proteins such as Chk1, Mec1, Rad9, Rad17, Rad24r Rad53, deletion of which improves the growth of cdc13-

mutants at semi-permissive temperatures [39,40]. Theffect of Mrc1 is more similar to that of the MRX complex,nother checkpoint complex with roles in telomere capping36,42,54,55]. It seems that it is the role of Mrc1 at the replica-ion fork that contributes to the vitality of telomere capping

utants, rather than its role in checkpoint activation. Ourndings are in accordance with recent work that demon-trated a protective role of Mrc1 in cells with cdc13-1 or yku70�

ncapped telomeres or in telomerase deficient cells [53]. Addi-ionally, our work demonstrates that the growth defects ofdc13-1 mrc1� and yku70� mrc1� mutants and enhancedsDNA levels of cdc13-1 mrc1� strains are suppressed when theuclease encoded by EXO1 is deleted. Therefore, Mrc1 protectsncapped telomeres from Exo1.

Mrc1 is recruited to the replication machinery as DNA repli-ation initiates and is required for normal rates of replicationork progression [17,18,22]. The biochemical role of Mrc1 ineplication fork progression is unclear which makes it diffi-ult to know its precise role in telomere capping. Mrc1 is alsoart of a replication-pausing complex formed when replica-ion is arrested by the S phase poison hydroxyurea (HU), andequired for replication fork restart after HU. However, thisestart role for Mrc1 is not universal, since Mrc1 plays no rolen replication restart after cells are treated with the alkylatinggent MMS [22].

It is interesting that there is evidence from budding yeast,ssion yeast and human cells that telomeric sequences con-ain DNA regions that slow or stall replication forks [15,56,57].rom this it seems plausible that telomeric DNA may be moreependent on proteins like Mrc1, which contribute to forktability and restart, than other chromosomal regions. Thats because the replication fork struggles to reach the end ofhe chromosome in mrc1� mutants where a telomere cappingefect is observed.

Numerous studies on budding yeast mutants with DNAeplication defects have demonstrated interactions betweenNA replication and telomere structure. For example both

dc17/pol1 and cdc44/rfc1 (large subunit of replication factor) mutants affect telomere length [49]. Here we show thatudding yeast mrc1� mutants have short telomeres. In S.erevisiae, cdc17/pol1 mutants, encoding temperature sensitiveNA polymerase �, exhibit very long telomeres, high levelsf telomeric ssDNA and elevated recombination at telomeres

50,51]. Interestingly, the B subunit of DNA polymerase � phys-cally interacts with Stn1, which in turn interacts with Cdc1310,52]. This shows there is a very direct interaction betweenudding yeast telomere capping proteins and the replica-ion fork machinery, and suggests that telomere capping is

ntimately linked with DNA replication. In this regard it is,erhaps, relevant that the 5′–3′ exonuclease Exo1 is involved inenerating single stranded DNA at uncapped telomeres [36,39]nd in processing stalled replication forks [58] and highlights

) 1607–1617 1615

the similarities between uncapped telomeres and stalled repli-cation forks.

cdc13-1 cells maintain a functional telomere cap (low lev-els of telomeric ssDNA), when released from G1 arrest intothe S phase poison hydroxyurea. HU stalls replication forksand stops late origins of replication from firing. However,if the same cdc13-1 cells are permitted to complete DNAreplication, by removing the S phase poison HU, telomereuncapping occurs and high levels of ssDNA are observed [59].Therefore, Cdc13-dependent telomere capping may dependon a coordinated interaction between the chromosome end,Cdc13/Stn1/Ten1 and the DNA replication fork. Further stud-ies examining the interactions between telomeric DNA, thetelomere cap and the replication fork will be necessary to bet-ter understand these interactions.

Acknowledgements

This work was supported by the Wellcome Trust (Grantnumbers 054371 and 075294) and a Newcastle University stu-dentship awarded to Avgi Tsolou. We thank Steven Elledge forMRC1 plasmids, Steve Addinall for S288C cdc13-1 strains, DanDurocher for rabbit anti-Rad53 polyclonal antibody and KeithGull for the TAT-1 antibody. We are also grateful to Thomas vonZglinicki, Steve Foster, Misha Zubko, Isabelle Morin, AmandaGreenall, Laura Maringele, Vasso Sapountzi, for comments onthe manuscript and Alan Leake for help making media andsolutions.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.dnarep.2007.05.010.

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