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DNA Repair 10 (2011) 536–544

Contents lists available at ScienceDirect

DNA Repair

journa l homepage: www.e lsev ier .com/ locate /dnarepai r

ubtelomeric regions in mammalian cells are deficient in DNA double-strandreak repair

ouglas Millera, Gloria E. Reynoldsa, Ricardo Mejiaa, Jeremy M. Starkb, John P. Murnanea,∗

Department of Radiation Oncology, University of California San Francisco, 2340 Sutter Street, San Francisco, CA 94143-1331, United StatesDepartment of Radiation Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, United States

r t i c l e i n f o

rticle history:eceived 22 November 2010eceived in revised form 2 March 2011ccepted 3 March 2011vailable online 3 April 2011

eywords:hromosome instability

a b s t r a c t

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are muchmore likely to result in large deletions, gross chromosome rearrangements, and chromosome instabil-ity than DSBs at interstitial sites within chromosomes. In the present study, we investigated whetherthis response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing thefrequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at inter-stitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitoredthe frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR

ouble-strand break

onhomologous end joiningelomere

at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBsnear telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurringat interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres aredeficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that causeDSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and

arran

eventual chromosome re

. Introduction

DNA double-strand breaks (DSBs) are a critical DNA lesion,esponsible for both the toxic effects of ionizing radiation andadiation-induced chromosome rearrangements leading to cancer1]. The repair of DSBs occurs through either homologous recombi-ation repair (HRR) or nonhomologous end joining (NHEJ). HRR inammalian cells primarily uses the sister chromatid as a template,

nd is therefore limited to DSBs that occur after DNA replication [2].HEJ involves the joining of broken ends, and therefore can occur atnytime during the cell cycle. There are two forms of NHEJ, classi-al (C-NHEJ) and alternative (A-NHEJ). C-NHEJ has been extensivelytudied and many of the proteins that are involved are known,hereas much less is known about A-NHEJ [3,4]. A-NHEJ has pri-arily been observed in cells deficient in C-NHEJ, and has therefore

een proposed to serve as a backup mechanism for repair of DSBs.oth C-NHEJ and A-NHEJ produce mutations at the site of a DSB

aused by ionizing radiation, however, A-NHEJ is commonly asso-iated with large deletions [5,6] and chromosome rearrangements5,7–10]. Another characteristic of A-NHEJ is that repair commonlyccurs at sites with microhomology [6,8,11–13].

∗ Corresponding author. Tel.: +1 415 476 9083; fax: +1 415 476 9069.E-mail address: jmurnane@radonc.ucsf.edu (J.P. Murnane).

568-7864/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2011.03.001

gements by alternative NHEJ.© 2011 Elsevier B.V. All rights reserved.

Not all DSBs are repaired equally well. Most DSBs generatedby ionizing radiation are repaired within a few hours; however,some DSBs require many hours to be repaired [14,15]. One factorthat can influence the efficiency of repair of DSBs is their locationin the genome. Goodarzi et al. [16] found that DSBs within hete-rochromatin are repaired much more slowly than DSBs occurring ineuchromatin. Moreover, they also found that unlike DSBs occurringin euchromatin, the repair of DSBs in heterochromatin is dependenton ATM.

The ends of chromosomes, called telomeres, are another struc-tural feature that can influence DSB repair. Telomeres are composedof the TTAGGG repeat sequence and associated proteins thattogether form the T-loop structure that keep the ends of chromo-somes from appearing as DSBs and prevent chromosome fusion[17,18]. Telomeres are maintained in human germ line cells bytelomerase, but shorten during cell division in somatic cells dueto insufficient telomerase expression [19]. The gradual telomereshortening that occurs during growth of somatic cells that donot express sufficient telomerase normally results in telomere-associated DSB repair foci and cell senescence [20,21]. However,

in cells that are unable to senesce, telomeres continue to shorten,eventually resulting in extensive chromosome fusion [22].

The role of the telomere in protecting chromosome ends andpreventing chromosome fusion can influence the response to DNAdamage within subtelomeric regioins. Ricchetti et al. [23] found

Repair 10 (2011) 536–544 537

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Fig. 1. The structure of the plasmids used in this study. The pNCT-tel plasmid islocated adjacent to the telomere on chromosome 16p in clone B3 of the EJ-30 tumorcell line, and was previously used to demonstrate the sensitivity of subtelomericregions to DSBs. The pNCT-tel plasmid contains a �-lactamase gene for resistanceto ampicillin and bacterial origin of replication (Amp/ori), a Neo gene for resistanceto G418, and a gene for Herpes simplex virus thymidine kinase (HSV-tk). The pEJ5-GFP-tel and pDR-GFP-tel plasmids were generated from the pEJ5-GFP and pDR-GFPplasmids by insertion of telomeric repeat sequences. The telomeric repeat sequenceswere added to seed the formation of a new telomere following targeted integrationthrough the shared homology in the Amp/ori sequences. The pDR-GFP-tel plasmidwas used to monitor the frequency of HRR and contains a GFP gene that is defec-tive due to an I-SceI site in the coding sequence, a puro gene for selection withpuromycin, and a complimentary fragment of the GFP gene for repair of the I-SceI-induced DSB. The pEJ5-GFP-tel plasmid was used to monitor the frequency of NHEJand contains a puro gene flanked by I-SceI sites, which is located between the GFPcoding sequence and promoter. NHEJ between the two I-SceI-induced DSBs resultsin activation of the GFP gene. The pGFP-ISceI-tel plasmid was used to determine

D. Miller et al. / DNA

hat I-SceI-induced DSBs near telomeres in yeast are much moreikely to result in gross chromosome rearrangements (GCRs) thanSBs occurring at interstitial locations, and concluded that thisifference in response to DSBs near telomeres was due to differ-nces in DSB repair. We have also reported that I-SceI-induced DSBsccurring near telomeres in a human tumor cell line are much moreikely to result in large deletions, GCRs, and chromosome instabilityhan DSBs occurring at interstitial locations within a chromosome24]. This increased likelihood of telomere loss and GCRs as a resultf DSBs within subtelomeric regions is not limited to human cancerells, because I-SceI-induced DSBs near telomeres in mouse ES cellsesults in similar types of rearrangements and chromosome insta-ility [25,26]. The likelihood of telomere loss and GCRs as a resultf DSBs within subtelomeric regions has important implicationsor the chromosome instability associated with cancer. We havereviously proposed that the sensitivity of subtelomeric regionso DSBs plays an important role in the high rate of spontaneouselomere loss commonly observed human cancer cells [24,27,28].he chromosome fusions resulting from telomere loss initiate chro-osome instability through breakage/fusion/bridge (B/F/B) cycles

29], which can generate many of the chromosome rearrangementseading to cancer [30]. The importance of B/F/B cycles resulting fromelomere loss in cancer was demonstrated by the high rate of car-inomas in mice that are deficient in both telomerase and p53 [31].he sensitivity of subtelomeric regions to DSBs could also be impor-ant in ionizing radiation-induced carcinogenesis, because we haveemonstrated that the sensitive region extends at least 100 kb fromtelomere and therefore poses a large target for DSB-induced chro-osome instability [32].In the current study, we have investigated whether the

ncreased frequency of large deletions and GCRs that result fromSBs within subtelomeric regions in mammalian cells is due todeficiency in DSB repair. These studies involve the analysis of

he frequency of DSB repair by HRR and NHEJ through the acti-ation of the gene for green fluorescent protein (GFP) followinghe introduction of DSBs with the I-SceI endonuclease. The repairf I-SceI-induced-DSBs through activation of GFP has been usedxtensively in mammalian cells to study the mechanisms of DSBepair and DSB-induced chromosome rearrangements [12,33–37].

e compared the frequency of repair of I-SceI-induced DSBs in aFP gene integrated at telomeric sites, interstitial sites, and inter-titial sites containing telomeric repeat sequences, to determinehether telomeres or telomeric repeat sequences influence the

fficiency DSB repair.

. Materials and methods

.1. Plasmids

The pNCT-tel plasmid has been previously described [38]. pNCT-el contains a neomycin-resistance (Neo) gene for positive selectionith G418, a herpes simplex virus thymidine kinase (HSV-tk) gene

or negative selection with ganciclovir, an I-SceI recognition siteor introducing DSBs with the I-SceI endonuclease, and 0.8 kb ofelomeric repeat sequences for seeding the formation of new telom-res (Fig. 1). The pQCXIH-ISceI retroviral vector that was usedor expression of the I-SceI gene was constructed as previouslyescribed [24].

The pDR-GFP-tel plasmid was generated from the pDR-GFP plas-id [12,39] by the insertion of telomeric repeat sequences (Fig. 1).

he first step in construction of pDR-GFP-ISceI was to remove thenique NotI restriction site in pDR-GFP. This was accomplishedy first digesting the plasmid with NotI, and using the Klenowragment of E. coli DNA polymerase (Invitrogen) to fill in the single-tranded NotI overhang prior to ligation. The next step was to

the frequency of large deletions (>50 bp) and GCRs. The cell clones containing thepGFP-ISceI-tel plasmid were generated from the clones containing the pEJ5-GFP-telplasmid by I-SceI-mediated deletion of the puro gene, and contain an active GFPgene with an I-SceI site located between the coding sequence and promoter.

insert a linker containing the NotI and XhoI restriction sites into theunique SspI restriction site located between the �-lactamase geneand chicken �-actin promoter for the GFP gene. A NotI/XhoI restric-tion fragment containing 0.8 kb of telomeric repeat sequences fromthe pNTP-tel plasmid [40] was then inserted into the NotI/XhoI sitesin the pDR-GFP plasmid.

The pEJ5-GFP-tel plasmid was generated from the pEJ5-GFPplasmid [12] by the insertion of telomeric repeat sequences (Fig. 1).The first step was to eliminate two NotI restriction sites flankinga 300 bp fragment containing the polyA addition sequences on theGFP gene. This was accomplished by first digesting the plasmid withNotI, then using the Klenow fragment of E. coli DNA polymerase(Invitrogen) to fill in the single-stranded NotI overhang prior to lig-ation. We next eliminated a unique SacI restriction site, by digestingthe plasmid with SacI, then using the T4 DNA polymerase (Invitro-gen) to remove the single-stranded SacI overhang prior to ligation.We then inserted a linker containing the NotI and SacI restrictionsites between two PvuII restriction sites located between the betalactamase gene and the GFP gene. The 0.8 kb NotI/SacI fragmentcontaining telomeric repeat sequences in the pNTP-tel plasmid[40] was then inserted into the NotI/SacI sites in the pEJ5-GFPplasmid. We then inserted a SmaI/XbaI restriction fragment con-taining the PolyA addition sequences from the pPGKpuro plasmidinto complementary FseI/NheI restriction sites at the end of the GFPgene.

2.2. Cell lines

All of the cell lines used in this study were derived from cloneB3 of the EJ-30 human bladder cell carcinoma cell line. EJ-30 is a

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ubclone of the EJ-30 cell line, which is also called MGH-U1 [41].he cells were grown in �MEM media (UCSF Cell Culture Facility)upplemented with 5% fetal calf serum (Invitrogen-Gibco), 5% new-orn calf serum with iron (Invitrogen-Gibco), 1 mM l-glutamineInvitrogen-Gibco), and were propagated at 37 ◦C in humidifiedncubators.

Cell clone B3 was derived from EJ-30 following transfectionith the linearized pNCT-tel plasmid [38], which seeded the for-ation of a new telomere 4.3 Mb from the original telomere on

he short arm of chromosome 16 (Fig. 1). Rescue of the integratedNCT-tel plasmid and sequencing of adjacent cellular DNA demon-trated that the plasmid integrated in the first intron at the 5′ endf the sarcalumenin gene (Genbank Accession number GQ475285)26].

The targeting of the pDR-GFP and pEJ5-GFP plasmids to a telom-re was accomplished through the addition of telomeric repeatequences to generate the plasmids, pDR-GFP-tel and pEJ5-GFP-el, respectively (Fig. 1). Targeting by homologous recombinationnto the pNCT-tel plasmid at the telomere on chromosome 16 inlone B3 [30,38,42] was facilitated by the presence of an identical-lactamase and replication origin (Amp/ori) in the pNCT-tel plas-id and transfected plasmids. Following integration, the telomeric

epeat sequences in the transfected plasmids are in the proper ori-ntation to seed the formation of a new telomere. Screening foruccessful targeting was performed using positive selection forhe puro gene in the pDR-GFP-tel or pEJ5-GFP-tel plasmids, andelection with ganciclovir for loss of the HSV-tk gene in the pNCT-el plasmid. The puror/Ganr clones were then selected and theirenomic DNA analyzed by Southern blot analysis using a varietyf restriction enzymes to identify clones in which the transfectedlasmid had replaced the pNCT-tel plasmid (data not shown).uccessful targeting of the plasmids was then confirmed by theescue and sequencing of the integrated plasmid sequences anddjacent cellular DNA (data not shown), as was used previously32].

A similar protocol was also used to identify cell clones thatontain the pEJ5-GFP-tel plasmid integrated at interstitial sites.outhern blot analysis using a variety of restriction enzymes washen used to identify clones that contained a single copy of thelasmid (data not shown). PCR was used to determine whetherhe integrated pEJ5-GFP plasmid had retained telomeric repeatequences, using the TRP primer (5′-AACCCTAACCCTAACCCT-3′)ithin the telomeric repeat sequences and the PIM-2R primer

5′-TCCTCCAGAGTGGATTCG-3′) within the adjacent plasmid DNAdata not shown). PCR involved 94 ◦C for 2 min, then 40 cycles of4 ◦C for 30 s, 60 ◦C for 30 s, and 72 ◦C for 30 s.

Cell clones containing the plasmids integrated at interstitialites without telomeric repeat sequences were obtained by trans-ection of clone B3 with the pDR-GFP-tel and pEJ5-GFP-tel plasmidequences after removal of the telomeric repeat sequences withither NotI and XhoI, or NotI and SacI, respectively. The linearizedlasmids were trasfected into clone B3 using Lipofectamine 2000,s recommended by the manufacturer (Invitrogen). The transfectedells were grown in the presence of 2 �g/ml puromycin (Sigma) toelect for cells containing the integrated plasmids. Southern blotnalysis was then used to identify clones that contained a singleopy of the plasmid (data not shown).

Cell clones containing active GFP with an I-SceI site betweenhe GFP coding sequence and promoter (Fig. 1) were gener-ted from clones containing the EJ5-GFP-tel plasmid integratedt a telomeric site (EJ5-6D), interstitial site without telomeric

epeat sequences (EJ5-7F), or interstitial site with telomeric repeatequences (EJ5-7C). Following transient transfection with theCMV-ISceI expression vector, the cells were plated at low density.fter two weeks, the plates were observed using a GFP fluorescentashlight with GFP filter eyeglasses (NightSea) to detect GFP-

10 (2011) 536–544

positive colonies. The GFP-positive colonies were then selected,expanded in culture, and genomic DNA was isolated. As describedbelow for the analysis of small deletions, the genomic DNA wasthen amplified by PCR using the GFP-1 and GFP-3 primers, and thePCR product analyzed by digestion with I-SceI to identify cloneswith an intact I-SceI site.

2.3. Virus preparation and selection for virally infected cells

Packaging of the pQCXIH-I-SceI retroviral vectors and infec-tion of cell cultures was performed as previously described [24].The selection for cells infected with pQCXIH-ISceI was achieved bygrowth in medium containing 50 �g/ml hygromycin (Sigma) for10 days with medium changes every 2 days, to allow for expressionof I-SceI endonuclease and the generation of DSBs. After 10 days,the cells were trypsinized, pooled together, and either analyzed forthe frequency of GFP-positive cells, or replated for preparation ofgenomic DNA. For analysis of the frequency of GFP-positive cells,the cells were aliquoted into a counting chamber slide, and totalcells and GFP-positive cells counted using a Cellometer Vision (Nex-elcom). Two counting chambers were used for each sample, witheach chamber being counted two times. All experiments were per-formed in triplicate, with a minimum of 1000 cells counted for eachsample. The results with the Cellometer Vison were confirmed forsome of the samples using a fluorescent microscope and manualcounting.

2.4. PCR and analysis of small deletions

The presence of small deletions, either at a single I-SceI-induced DSB, or when joining two I-SceI-induced DSBs, wasanalyzed by first generating PCR products spanning an I-SceIsite in the integrated pEJ5-GFP plasmid, and then digesting thePCR products with I-SceI endonuclease. PCR was performed ongenomic DNA isolated from the pooled hygromycin-resistantcell cultures following infection with the pQCXIH-ISceI retrovi-ral vector. PCR was performed using Taq 2X Master Mix (NewEngland Biolabs) and primers GFP-1 (5′-GCGGGGTTCGGCTTCTGG-3′) and GFP-3 (5′-CGCTTCCATTGCTCAGCGG-3′) for small deletionsresulting from a single I-SceI-induced DSBs, and GFP-1 andGFP-2 (5′-TCTTTCATGGCGGACTTG-3′) for small deletions result-ing from joining two I-SceI-induced DSBs. PCR involved 94 ◦Cfor 2 min, then 40 or 41 cycles (single or two I-SceI-inducedDSBs, respectively) of 94 ◦C for 30 s, 62 ◦C for 30 s, and 72 ◦Cfor 30 s. 25 �l of the PCR product was then digested with 20units of I-SceI endonuclease at 37 ◦C overnight, and the productswere run on 4% agarose gels. After staining with ethidium bro-mide, digital images were analyzed using free ImageJ software(http://www.versiontracker.com/dyn/moreinfo/macosx/37303) tocalculate the intensity of the bands. The percentage of cells contain-ing small deletions at the I-SceI site was determined by dividingthe intensity of the uncut band by the combined intensity of thecut and uncut bands. The values for small deletions at a singleI-SceI site were corrected for the fraction of cells that had expe-rienced inactivation of the GFP gene (see Fig. 2), because these cellswould not produce a PCR product due to large deletions, GCRs orfailure to repair the DSB, and would therefore cause an overes-timation of the fraction of cells containing small deletions. Thiscorrection involved multiplying by 0.6 (1–0.4) for clones containing

subtelomeric integration sites, and 0.95 (1–0.05) for clones con-taining interstitial integration sites. The validity of this correctionwas previously demonstrated by the analysis of the frequency ofsmall deletions in 100 individual subclones selected at random[24].

D. Miller et al. / DNA Repair

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Fig. 2. Increased frequency of loss of the GP gene as a result of DSBs near telomeresand interstitial sites containing telomeric repeat sequences. The percentage of GFP-positive cells with (+) or without (−) constitutive expression of I-SceI endonucleasewI7E

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as used to monitor large deletions and GCRs in cell clones containing the pGFP-SceI plasmid adjacent to a telomere (6D1 and 6D6), at an interstitial site (7F1 andF3), and an interstitial site containing telomeric repeat sequences (7C1 and 7C2).xperiments were performed in triplicate and standard deviations are shown.

. Results

.1. Generation of cell lines used for monitoring DSB repair

DSB repair at interstitial and telomeric sites was compared usinghe pDR-GFP and pEJ5-GFP plasmids (Fig. 1), which were previouslysed to monitor the frequency of repair of DSBs generated with the

-SceI endonuclease [12,39]. The frequency of HRR was determinedsing the pDR-GFP plasmid, which contains a GFP gene that is inac-ive due to the presence of an I-SceI recognition site inserted in theoding sequence. The activation of GFP in pDR-GFP results fromhe repair of the I-SceI-induced DSB by HRR using a complimen-ary fragment of the GFP gene that is also present in the plasmid.he frequency of NHEJ was determined using the pEJ5-GFP plas-id, in which the GFP gene is inactive due to the presence of a puro

ene located between the GFP coding sequence and promoter. Theuro gene is flanked at either end by I-SceI recognition sites in theame orientation, so that NHEJ between the two I-SceI sites resultsn deletion of the puro gene and activation of GFP.

Clone B3 of the EJ-30 human tumor cell line was used to isolateell clones that contain a single copy of the pDR-GFP and pEJ5-FP plasmids stably integrated at interstitial sites or at a telomeren the end of the short arm of chromosome 16 (see Section 2).he telomere on the short arm of chromosome 16 in clone B3emonstrated sensitivity to DSBs, similar to other telomeres thatere analyzed in this cell line [24,32]. Following transfection of

he plasmids, we identified three clones containing the interstitialDR-GFP plasmid, DR-3, DR-4, and DR-8, and six clones that containhe interstitial pEJ5-GFP plasmid, EJ5-7F, EJ5-AB, EJ5-AD, EJ5-AE,J5-AI, and EJ5-AL. The targeting of the pDR-GFP and pEJ5-GFPlasmids to a telomeric site was achieved by inserting telomericepeat sequences into the plasmids in the proper orientation toeed the formation of a new telomere (see Section 2). The plasmidsontaining the telomeric repeat sequences, pDR-GFP-tel and pEJ5-FP-tel, were then transfected into clone B3 to target and replace

he pNCT-tel plasmid located at a telomere (Fig. 1). We identifiedhree clones containing the telomeric pDR-GFP-tel plasmid, DR-1,R-2, and DR-7, and three clones that contain the telomeric pEJ5-FP-tel plasmid, EJ5-6D, Ej5-6F, and EJ5-6J. Using a similar protocol,e also identified six cell clones that contain a single copy of the

EJ5-GFP-tel plasmid containing telomeric repeat sequences inte-rated at interstitial sites, EJ5-6B, EJ5-7C, EJ5-7G, EJ5-AC, EJ5-AF,nd EJ5-AJ.

We also isolated cell clones containing the plasmid, pGFP-ISceI,n which the GFP gene is active, with the I-SceI site located between

10 (2011) 536–544 539

the coding sequence and the promoter (Fig. 1). The sequences forthe translational start site and start codon for the GFP gene arelocated 20 bps from the I-SceI site, and therefore the inactivation ofGFP requires deletions of at least 20 bps. This construct thereforeallowed us to determine the frequency of deletions greater than20 bps as a result of DSBs at telomeric and interstitial sites. Thesecell clones were generated from cell clones containing the pEJ5-GFP plasmid following transient expression of I-SceI and selectionfor GFP-positive colonies. Using this protocol, we identified twoclones containing the active GFP gene integrated at a telomeric site,GFP-6D1 and GFP-6D6, at an interstitial site, GFP-7F1 and GFP-7F3,and at an interstitial site containing telomeric repeat sequences,GFP-7C1 and GFP-7C2.

3.2. Comparison of large deletions at telomeric and interstitialsites

Our previous studies demonstrated that DSBs near telomereswere much more likely to result in large deletions and GCRs(Del/GCR) than DSBs at interstitial sites [24,32]. Specifically, theseDel/GCR events involve the complete loss of the integrated plasmidsequences that constitute the subtelomeric region immediatelyadjacent to the telomere. These previous studies were performedusing multiple clones and two different assay systems, both withand without selection. To confirm whether the telomeres contain-ing the GFP gene also showed this increase in large deletions inresponse to DSBs, we compared the frequency of inactivation ofthe GFP gene at telomeric and interstitial integration sites usingcell clones containing the pGFP-ISceI plasmid. DSBs were generatedby infecting the cells with the pQCXIH-ISceI retrovirus, followedby growth in medium containing hygromycin for 10 days to allowtime to generate DSBs and allow for turnover of the GFP protein. Theresults demonstrated a 40% decrease in GFP-positive cells as a resultof DSBs near telomeres, while DSBs at interstitial sites were foundto result in only a 5% decrease in GFP-positive cells (Fig. 2). Theseresults are consistent with earlier studies showing that Del/GCRevents are rare at I-SceI-induced DSBs at interstitial sites [33–35],and that DSBs near telomeres are much more likely to result inDel/GCR events than DSBs at interstitial sites [24,32]. Interestingly,the clones that contain the pGFP-ISceI plasmid located at interstitialsites with telomeric repeat sequences showed a greater decrease inGFP-positive cells than the clones containing the pGFP-ISceI plas-mid located at telomeric sites. However, without further analysis,whether the frequency of Del/GCRs as a result of DSBs near intersti-tial telomeric repeat sequences is consistently greater than at DSBsnear telomeres is not known. Regardless, these results demonstratethat the increase in large deletions resulting from DSBs within sub-telomeric regions is due to the presence of the telomeric repeatsequences.

3.3. The frequency of small deletions at subtelomeric andinterstitial DSBs

We previously found that unlike the frequency of Del/GCRevents, the frequency of small deletions was similar at subtelom-eric and interstitial DSBs [24,32]. To determine whether this isalso true for clones containing the pEJ5-GFP plasmid, we com-pared the frequency of small deletions in clones with interstitialand subtelomeric DSBs. Following infection with the pQCXIH-ISceIretrovirus and selection with hygromycin for 10 days, genomic DNAwas isolated and PCR was performed to amplify a DNA fragment

spanning one of the two I-SceI sites (Fig. 3A). The percentage ofcells in the population that contain small deletions at the I-SceI sitewas then determined by digesting the PCR fragment with I-SceI.The percentage of the PCR fragment that is not cut by I-SceI is thenused to determine the percentage of cells in the population that

540 D. Miller et al. / DNA Repair 10 (2011) 536–544

Fig. 3. No difference in small deletions due to DSBs at interstitial and subtelomeric sites. (A) Cell clones with the pEJ5-GFP plasmid integrated at telomeric (EJ5-6D, EJ5-6F,EJ5-6J) or interstitial (EJ5-7F, EJ5-AB, EJ5-AE) sites were used to determine the percentage of cells in the population that contain small deletions resulting from I-SceI-induced DSBs. Genomic DNA from pooled populations of cells infected with the pQCXIH-ISceI retrovirus and selected with hygromycin for 10 days was amplified by PCRusing oligonucleotide primers that span one of the I-SceI sites in pEJ5-GFP (small black arrows). (B) The PCR products were then digested with I-SceI endonuclease, and thep sing Imt the pt rcenti

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We next compared the frequency of HRR at I-SceI-inducedDSBs using the clones containing the DR-GFP plasmid (Fig. 5). Thefrequency of GFP-positive cells was similar in all of the clones,demonstrating that similar to small deletions, but unlike NHEJ

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Fig. 4. Decreased NHEJ in repair of DSBs near telomeres and interstitial sites con-taining telomeric repeat sequences. The appearance of GFP-positive cells following

ercentage of cut and uncut DNA was determined from the intensity of the bands uhen determined by correcting the fraction of uncut DNA for the number of cells inhese cells would not amplify and would therefore cause an overestimation of the pen triplicate and standard deviations are shown.

ave small deletions (Fig. 3B). This number must first be correctedor the fraction of cells in the population that have undergone inac-ivation of the GFP gene (see Fig. 2), because the DNA from theseells would not generate a PCR product, and therefore would causen overestimation of the fraction of cells containing small deletions.s in our earlier studies, we again saw little if any difference in the

requency of small deletions at the interstitial and subtelomericSBs (Fig. 3C). Therefore, the analysis of small deletions at single

-SceI-induced DSBs, which are preferentially generated by A-NHEJ6,8,12], does not indicate a defect in A-NHEJ near telomeres.

.4. Comparison of DSB repair at telomeric and interstitial sites

The mechanism responsible for the high frequency of large dele-ions and GCRs in response to DSBs in subtelomeric regions wasnvestigated by comparing the frequency of repair of I-SceI-inducedSBs at interstitial and telomeric sites. DSBs were generated by

nfection with the pQCXIH-ISceI retrovirus vector and selectionith hygromycin for 10 days. The frequency of DSB repair by NHEJ

t interstitial and telomeric sites was analyzed using the clones con-aining the pEJ5-GFP plasmid (Fig. 4). Only 0.8–1.2% (average 1.0%)f the cells expressing I-SceI were GFP-positive in the clones withubtelomeric DSBs. In contrast, 4.2–9.9% (average 6.8%) of the cellsxpressing I-SceI were GFP-positive in clones with interstitial DSBs.herefore, despite the large increase in inactivation of the GFP geneesulting from I-SceI-induced DSBs near telomeres (Fig. 2), the fre-uency of NHEJ-mediated rejoining of two different I-SceI-induced

SBs is decreased near telomeres. Importantly, the cell clones with

nterstitial plasmids containing telomeric repeat sequences alsohowed a much lower frequency of rejoining the two I-SceI-inducedSBs than clones with interstitial DSBs without telomeric repeat

equences, consistent with our observation that DSBs occurring at

age J. (C) The percentage of cells in the population containing small deletions wasopulation that contained large deletions or GCRs (see Fig. 1), since the DNA from

age of cells containing small deletions (see Section 2). Experiments were performed

interstitial sites containing telomeric repeat sequences also have ahigh frequency of inactivation of the GFP gene compared to inter-stitial sites without telomeric repeat sequences (see Fig. 2). Thedecrease in NHEJ in the proximity of telomeric repeat sequencestherefore correlates with an increase in large deletions and GCRs.

infection with the pQCXIH-ISceI retrovirus and selection with hygromycin for10 days was used to monitor the frequency of NHEJ in cell clones containing thepEJ5-GFP plasmid at telomeric sites (EJ5-6D, EJ5-6F, EJ5-6J), interstitial sites (EJ5-7F, EJ5-AB, EJ5-AD, EJ5-AE, EJ5-AI, EJ5-AL) or interstitial sites containing telomericrepeat sequences (EJ5-6B, EJ5-7C, EJ5-7G, EJ5-AC, EJ5-AF, EJ5-AJ). Experiments wereperformed in triplicate and standard deviations are shown.

D. Miller et al. / DNA Repair

% g

fp p

osi

tive

cell

s

8

7

6

5

4

3

2

1

0 1 2 7 3 4 8

Telomeric Interstitial

Fig. 5. No difference in HRR in repair of DSBs at interstitial and subtelomeric sites.The appearance of GFP-positive cells following infection with the pQCXIH-ISceIrqDt

bqs

3t

tdwscItiasobgttSsuoliuA

4

dcTotqD

etrovirus and selection with hygromycin for 10 days was used to monitor the fre-uency of HRR in cell clones containing the pDR-GFP plasmid at telomeric (DR-1,R-2, DR-7) or interstitial (DR-3, DR-4, DR-8) sites. Experiments were performed in

riplicate and standard deviations are shown.

etween two different I-SceI sites, there is no difference in the fre-uency of HRR detected at I-SceI-induced DSBs at interstitial andubtelomeric sites.

.5. The frequency of loss of the I-SceI site during DSB repair nearelomeres

Mammalian cells that are deficient in C-NHEJ have been showno repair DSBs by A-NHEJ, which is typically associated with largeeletions and GCRs at I-SceI-induced DSBs [6,12,43,44], similar tohat we have observed as a result of DSBs near telomeres. Several

tudies have reported that rodent and human cells that are defi-ient in C-NHEJ are much more likely to experience the loss of the-SceI site during DSB repair [6,8,12,13,45]. We therefore comparedhe relative frequency of the loss of the I-SceI site during NHEJ atnterstitial and subtelomeric DSBs to determine whether there wasdifference in the likelihood of deletion of the I-SceI site. This analy-is was performed using primers distal to the two I-SceI sites, so thatnly repair events involving the joining of the two I-SceI sites woulde amplified (Fig. 6A). Although most cells would not have under-one NHEJ between the two I-SceI sites, the genomic DNA fromhese cells will not generate a PCR product. Approximately 40% ofhe PCR product spanning the interstitial DSBs was not cut with I-ceI, i.e., had small deletions when joining the two different I-SceIites (Fig. 6B and C). In contrast, approximately 60% of the PCR prod-ct spanning the subtelomeric DSB was not cut with I-SceI. In viewf the earlier studies demonstrating an increase in the frequency ofoss of the I-SceI site in cells deficient in C-NHEJ [6,8,12,13,45], thisncreased frequency of loss of the I-SceI site suggests that the resid-al NHEJ occurring in subtelomeric regions preferentially involves-NHEJ.

. Discussion

The results presented here demonstrate that the joining of twoifferent I-SceI-induced DSBs by NHEJ is decreased near telomeresompared to interstitial sites in the EJ-30 human tumor cell line.

his decrease in NHEJ near telomeres does not appear to be a resultf a decrease in the frequency of formation of DSBs by I-SceI atelomeric sites. Although we were not able to determine the fre-uency of DSBs directly due to the fact that there is only a singleSB that is not present in all of the cells at the same time, the com-

10 (2011) 536–544 541

parison of other types of events served as important controls. Wedid not observe a difference in the frequency of small deletions(Fig. 3) or HRR (Fig. 5) at subtelomeric and interstitial sites. Theseobservations alone are not conclusive, because we cannot rule outthat an increase in small deletions and HRR might compensate fora decrease in the frequency of DSBs near telomeres. However, adecreased frequency of I-SceI-induced DSBs near telomeres wouldbe entirely inconsistent with the increased inactivation of the GFPgene (Fig. 2) and increased frequency of Del/GCR events [24,32] thatwe have observed near telomeres. This increased frequency of largedeletions and GCRs in response to DSBs in subtelomeric regions isnot limited to the EJ-30 human cancer cell line, because the sametypes of rearrangements i.e., large deletions, GCRs, and chromo-some healing, were also observed in the SCC61 human squamouscell carcinoma cell line (unpublished results) and mouse ES cells asa result of DSBs near telomeres [25,26,40]. Moreover, the increasein large deletions, GCRs, and chromosome instability in response toDSBs in subtelomeric regions is typical of most telomeres, since ithas been observed at multiple telomeres in the EJ-30 human tumorcell line [24,38] and mouse ES cells [25,26,40].

In view of earlier studies demonstrating that a deficiency in C-NHEJ results in an increased frequency of loss of the I-SceI siteduring DSB repair [6,8,12,13,45], the increased frequency of lossof the I-SceI site near telomeres compared to interstitial sitessuggests that A-NHEJ is the preferred mechanism of NHEJ neartelomeres. A-NHEJ has previously been proposed as the mecha-nism of chromosome fusion following telomere loss in mammaliancells [24,26,50,51]. The predominance of A-NHEJ near telomereswould be consistent with the increase in large deletions, GCRs,and use of microhomology that we have observed as a result ofDSBs within subtelomeric regions [24,26,32], since these eventsare all typically associated with A-NHEJ [5–13,43,44]. However,it is important to point out that other studies have found thatA-NHEJ, not C-NHEJ, is more likely to preserve overhangs gener-ated by restriction enzymes, both in mycobacterium [46] and in anextra-chromosomal system in mammalian cells [47]. Thus, whilethe restoration of I-SceI sites has been shown to be promoted byKu and XRCC4, precise rejoining of overhangs can be independentof these factors under some circumstances. In addition, the role ofATM and BRCA1 in the regulation of processing of DSBs by MRE11is also important in restoration of I-SceI sites during DSB repair[48,49]. Proof that subtelomeric regions rely predominantly on A-NHEJ will therefore require a more detailed genetic approach. Thisis an important goal, because the predominance of A-NHEJ for DSBrepair near telomeres would have dramatic consequences for chro-mosome stability due to the increase in Del/GCR events that areassociated with A-NHEJ [6,9,10,12,43,44].

The deficiency in NHEJ within subtelomeric regions may be aresult of the role of telomeric proteins in preventing chromosomefusion. Our results show that the presence of telomeric repeatsequences near interstitial DSBs is sufficient to promote large dele-tions (Fig. 2) and inhibit NHEJ (Fig. 4). An important function oftelomeric proteins is to prevent the telomere from appearing as aDSB and thereby prevent chromosome fusion. TRF2 is a key pro-tein that is involved in telomere maintenance and cap formation[18,52–54]. TRF2 binds to and inhibits ATM, a key protein in thecellular response to DSBs [55,56]. Consistent with this observa-tion, a study in yeast demonstrated that the presence of telomericrepeat sequences near DSBs could prevent the initiation of MEC1-mediated cell cycle checkpoints [57]. The inhibition of ATM couldbe directly involved in the deficiency in C-NHEJ near telomeres,

because ATM is required for NHEJ within heterochromatin [14], andsubtelomeric regions are composed of heterochromatin [58,59]. Inaddition, the role of ATM in limiting MRE11-mediated process-ing of DSBs is also important in promoting C-NHEJ [48,49,60].Alternatively, the deficiency in NHEJ near telomeres could be a

542 D. Miller et al. / DNA Repair 10 (2011) 536–544

Fig. 6. Increased loss of the I-SceI site during NHEJ of DSBs near telomeres. The frequency of loss of the I-SceI site during NHEJ between the two I-SceI sites in the pEJ5-GFPplasmid was compared for cell clones containing interstitial and telomeric integration sites. (A) The pEJ5-GFP plasmid integrated at telomeric (EJ5-6D, EJ5-6F, EJ5-6J) ori ntagec 0 dayi SceI ei stand

rDst(si[trf[TfttPatlstiAicN

iTptfe

nterstitial (EJ5-7F, EJ5-AB, EJ5-AE) sites were used to determine the relative perceells infected with the pQCXIH-ISceI retrovirus and selected with hygromycin for 1n pEJ5-GFP (small black arrows). (B) The PCR products were then digested with I-ntensity of the bands using Image J. Experiments were performed in triplicate and

esult of the role of TRF2 in regulating DNA resection. FollowingNA replication, the leading strand on the end of the chromo-

ome is blunt ended, and therefore must be processed to generatehe 3′ single-stranded overhang required for T-loop formationFig. 7A). TRF2 has been proposed to facilitate the formation of the 3′

ingle-stranded overhang by stimulating the 5′-3′ nuclease activ-ty of MRE11 and inhibiting the 3′-5′ nuclease activity of MRE1161]. The TPP1 and POT1 proteins then bind to the single strandelomeric DNA and facilitate T-loop formation, preventing furtheresection of the 5′ strand. Deficiencies in TPP1 or POT1 there-ore result in large single-stranded tails due to excessive resection62,63]. As a result, chromosome fusions resulting from the loss ofRF2 occur by ATM/53BP1-dependent C-NHEJ, while chromosomeusions resulting from the loss of TPP1, POT1, or naturally shortenedelomeres occur by ATM/53BP1-independent A-NHEJ [51]. Impor-antly, chromosome fusions resulting from loss of TRF2, TPP1, orOT1 are largely eliminated in cells deficient in MRE11 nucleasectivity [51,61]. Based on these results, Rai et al. [51] proposedhat TRF2 prevents chromosome fusion by C-NHEJ, and that theoss of telomere function in the presence of TRF2 results in exten-ive resection/degradation and chromosome fusion by A-NHEJ. Ashey point out, there are several mechanisms by which TRF2 couldnhibit C-NHEJ, one possibility being that TRF2 binds to and inhibitsTM [55], and a second possibility being the role of TRF2 in regulat-

ng MRE11 to promote the formation of 3′ overhangs [61], therebyreating single-stranded substrates that are not amenable to C-HEJ.

Unlike the MRE11-mediated resection of DNA ends contain-ng telomeric repeat sequences, which results in the binding of

PP1/POT1 and T-loop formation that limits resection [62,63], weropose that the resection of DSBs in subtelomeric DNA will con-inue unabated due to the inability of TPP1/POT1 to bind andorm a T-loop (Fig. 7B). Although these long single-stranded 3′

nds would facilitate homologous recombination, this would not

of loss and restoration of the I-SceI site. Genomic DNA from pooled populations ofs was amplified by PCR using oligonucleotide primers distal to the two I-SceI sitesndonuclease. (C) The percentage of cut and uncut DNA was determined from theard deviations are shown.

be possible for cells experiencing DSBs prior to DNA replica-tion when the sister chromatid is not available for HRR. DSBs insubtelomeric regions would therefore behave like telomeres inTPP1/POT1-deficient cells, which undergo extensive resection [62]that promotes homologous recombination [63] and chromosomefusions involving A-NHEJ [51]. Although earlier studies demon-strated that the inhibition of C-NHEJ by TRF2 was specific fortelomeric repeat sequences [64], it is important to point out thatthese studies were conducted in vitro, and did not involve the pro-cessing of DNA ends. As a result, these studies could not rule out thepossibility that TRF2 may influence the processing of DSBs withinsubtelomeric regions as proposed in our model. Similarly, in vitrostudies would not rule out the possibility that the inhibition of ATMby TRF2 would prevent the chromatin modification that might berequired for repair of DSBs within subtelomeric DNA.

The increased likelihood of large deletions and GCRs due toDSBs within subtelomeric regions has important implications withregard to chromosome instability and cancer. As we have previ-ously proposed, the sensitivity of subtelomeric regions to DSBs islikely to be an important factor in the high frequency of sponta-neous telomere loss in cancer cells [65], which leads to many ofthe types of chromosome rearrangements associated with humancancer [66–68]. Cancer cells often experience a high frequency ofDSBs due to oncogene-mediated cell stress, which generates DSBsat regions that are difficult to replicate, called fragile sites [69].Telomeric regions are fragile sites [70] and therefore would be oneof the regions that would experience an increased frequency ofDSBs in cancer cells. However, unlike DSBs at most fragile sites,which are repaired, DSBs near telomeres result in telomere loss and

chromosome instability. The increased frequency of large deletionsand GCRs in response to DSBs in subtelomeric regions may alsobe important in ionizing radiation-induced carcinogenesis. Ioniz-ing radiation is now known to initiate chromosome instability,which has been proposed to play an important role in radiation-

D. Miller et al. / DNA Repair

Fig. 7. A model to explain the deficiency in C-NHEJ near telomeres. (A) The process-ing of the leading strand of the telomere following DNA replication is regulated bythe telomeric protein TRF2, which inhibits the 3′–5′ nuclease activity of MRE11, andstimulates the 5′–3′ nuclease activity of MRE11. Further resection is prevented byTPP1 and POT1, which bind to the single-stranded telomeric repeat sequences andpromote T-loop formation. (B) The processing of DSBs near telomeres is also regu-lated by TRF2 and involves the resection of the 5′ strand by MRE11 to generate the3′ single strand overhang. However, because the DNA is not composed of telomericrepeat sequence, TPP1 and POT1 cannot bind and prevent further resection, result-ing in large single stranded regions, as are found in cells deficient in TPP1 and POT1[etl

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62]. Further resection then occurs through EXO1 and DNA2, similar to HRR. How-ver, cells that have not undergone DNA replication, and therefore do not contain aemplate for HRR, cannot repair the break and experience large deletions, telomereoss, and GCRs.

nduced carcinogenesis [71]. The region that is sensitive to DSBsxtends at least 100 kb from the telomere [24]. Therefore, in viewf the fact that there are 96 telomeres in a diploid human cell, theombined size of the sensitive regions (9.6 Mbps), such that onen every 625 DSBs would cause telomere loss and chromosomenstability.

onflict of interest statement

None declared.

cknowledgement

This work was supported by National Institutes of Health grantsA120205 for J.P.M. and CA120954 for J.M.S.

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