DNA-Directed Polymerase Subunits Play a Vital Role …mcr.aacrjournals.org/content/molcanres/13/3/402.full.pdfCell Cycle and Senescence DNA-Directed Polymerase Subunits Play a Vital

Cell Cycle and Senescence

DNA-Directed Polymerase Subunits Play a VitalRole in Human Telomeric Overhang ProcessingRaffaella Diotti, Sampada Kalan, Anastasiya Matveyenko, and Diego Loayza

Abstract

Telomeres consist of TTAGGG repeats bound by the shelterincomplex and end with a 30 overhang. In humans, telomeresshorten at each cell division, unless telomerase (TERT) isexpressed and able to add telomeric repeats. For effective telomeremaintenance, the DNA strand complementary to that made bytelomerase must be synthesized. Recent studies have discovered alink between different activities necessary to process telomeresin the S phase of the cell cycle to reform a proper overhang.Notably, the humanCST complex (CTC1/STN1/TEN1), known tointeract functionally with the polymerase complex (POLA/pri-mase), was shown to be important for telomere processing. Here,focus was paid to the catalytic (POLA1/p180) and accessory(POLA2/p68) subunits of the polymerase, and their mechanisticroles at telomeres. We were able to detect p68 and p180 attelomeres in S-phase using chromatin immunoprecipitation. We

could also show that the CST, shelterin, and polymerase com-plexes interact, revealing contacts occurring at telomeres. Wefound that the polymerase complex could associate with telome-rase activity. Finally, depletion of p180 by siRNA led to increasedoverhang amounts at telomeres. These data support a model inwhich the polymerase complex is important for proper telomericoverhang processing through fill-in synthesis, during S phase.These results shed light on important events necessary for efficienttelomere maintenance and protection.

Implications: This study describes the interplay between DNAreplication components with proteins that associate with chro-mosome ends, and telomerase. These interactions are proposed tobe important for the processing and protection of chromosomeends. Mol Cancer Res; 13(3); 402–10. �2014 AACR.

IntroductionTelomeres are structures located at the end of linear chromo-

somes, essential for their stability and integrity. They consist ofstretches of repetitive DNA sequences and the protein complexbound to them, known in mammals as shelterin. Their role is toprotect the chromosome ends from being inappropriatelyrecognized by the DNA damage machinery (1). The ends ofmammalian telomeres exhibit a 30 overhang of 50 to 300nucleotides, produced by competing actions of telomerase,extending it by repeat additions, by ExoI and Apollo, whichcreate the 50 resected end (2), and by C-strand fill-in processing.Another complex, called the CST complex, is known to limittelomerase activity in S phase (3), and to associate with theRNA primer synthesizing complex Pola–primase (4), therebycontributing an additional activity likely to participate in over-hang processing.

In human cells, telomeres shorten at each cell division, unlesstelomerase is present and active in adding TTAGGG repeats.During active telomere elongation in telomerase-positive cells,no significant change is observed in mean overhang length (5),

suggesting coordination between telomerase and the C-strandsynthesis machinery. In humans, the mechanism of C-strandprocessing is tightly regulated, as demonstrated by the observa-tion that the 50 ends in AATC-50 80%of the times and inAATCC-50

15% of the times (6). The terminal 50 nucleotide, however,becomes randomized upon the depletion of POT1 (7), theoverhang binding protein in the shelterin complex. Differentmodels for the creation of the 30 G-strand overhang have beenproposed and, in general, they invoke an interplay of leading andthe lagging strand processing due to the intrinsic properties ofDNA replication and telomerase activity (see for example ref. 8).Recently, it has been elegantly shown that the two strands are infact differentially processed during replication (9). The leadingoverhang is initially a blunt end that is processed later in S phasefor the creation of the overhang. The lagging strand overhanglength isfirst determined by the placement of the last RNAprimer,which is then removed before further processing (9). Final over-hang processing occurs in late S–G2. This study establishes thebasis for a differential processing of the two strands withoutexcluding other additional processing events for the C-strand.Additional indication of differential metabolism for the twoterminal strands was described in the mouse system, in whichthe exonucleases Apollo and ExoI, were found to be important foroverhang production through 50 end resection at the leading andlagging strands, respectively (2). This work also elucidated animportant role for POT1b in both leading and lagging strandoverhang formation through the coordination of the nucleasesaction and the C-strand fill-in through association with the CSTcomplex.

In Saccharomyces cerevisiae, as well as in most other eukar-yotes, the CST complex, composed by three RPA-like proteins,Cdc13-Stn1-Ten1 (CTC1-OBFC1-Ten1 in humans), was found

Department of Biological Sciences, Hunter College and CUNY Grad-uate Center, New York, New York.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Diego Loayza, Hunter College, City University of NewYork, 695 Park Avenue, NewYork, NY 10065. Phone: 212-772-5312; Fax: 212-772-5227; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-14-0381

�2014 American Association for Cancer Research.

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to be important for telomere protection and replication(10, 11). The role of the CST complex in the coordination oftelomere elongation in humans was corroborated by the find-ings that it contributes to limiting telomerase activity at extend-ing telomeres through complex interactions with telomeraseitself and TPP1/POT1 (3). In addition, it was recently shownthat CST plays a role in both general telomere replication andoverhang processing, particularly for longer telomeres thatrepresent a challenge for the replication machinery. Specifically,C-strand fill in was affected in OBFC1-depleted cells, delayingthe processing that leads to the final, mature G-overhang(12, 13). The human CST complex, in addition, was isolatedas a set of accessory factors for the POLa–primase complex (4).In S. cerevisiae, Cdc13 is able to interact with POL1, the POLahomolog, and STN1 interacts with POL12, the regulatorysubunit of Pola–primase, which is named POLA2 in humans(14). The POLa–primase complex is believed to be importantfor replication of the lagging strand and the C-strand fill in forboth strands after resection. The complex is composed by thecatalytic subunit, Pola, also termed p180, two primase sub-units, and the regulatory subunit POLA2, or p68 (see ref. 15).Although it has recently been established that telomeres, not-withstanding their reported nature as fragile sites of DNAreplication on chromosomes (16), do not undergo a telo-mere-specific replication program, but mainly a chromo-some-specific one (17), it has been found that partial alteringof the replication machinery can have telomere-specific effects.Mouse cells with a temperature-sensitive p180 exhibit overhangextension and concomitant overall telomere elongation (18).Therefore, although defects in DNA replication and in partic-ular in Pola–primase activity are predicted to affect generalchromosomal DNA replication, it is possible that this activitydisplays noncanonical, telomere-specific roles, in particular inoverhang processing.

In addition, telomerase was found physically associated withprimase and the lagging strand replicationmachinery in the ciliateEuplotes crassus (19). This association appeared to be develop-mentally regulated as it occurred specifically in mated cells, andnot in vegetatively growing cells. Genetic evidence in fission andbudding yeast also implicates primase subunits in telomerereplication (14, 20, 21), although themechanisms at play remainunclear.

Our study investigates these possible roles at human telo-meres, and focuses on p68 (POLA2) and p180 (POLA1). Weshow that they are present at telomeres in S phase, interactwith the shelterin complex and with the CST subunit OBFC1,as well as with telomerase itself, and that they are importantfor the regulation of telomeric overhang amounts in humancells.

Materials and MethodsCell lines and antibodies

The HeLaII line is a subclone of HeLa S3 (ATCCCCL-2.2), withtelomere length in the 3- to 6.5-kb range (22), and used in ref.(23). The HTC75 cell line is a HT1080 derivative described inref. 24. The cells were grown in DMEM/10% BCS, and theretroviral transduction protocol was identical to that describedin ref. 25. Cells were selected for the plasmid with 2 mg/mgpuromycin, where applicable. All rabbit sera used were generatedagainst a peptide conjugated to KLH and used for immunization

into rabbits, as per the protocol set by the manufacturer (BioSyn-thesis). The peptides were: NH2-GCKGRQEALERLKKAKAGEK-OH for p180, and NH2-GCRLYLRRPAADGAERQSP-OH for p68.The peptide for FEN1 NH2-GCSTKKKAKTGAAGKFKRGK–OH,for TRF1 NH2-GCGSIEKEHDKLHEEIQNLI-OH (as describedin ref. 24), for POT1 NH2-CYGRGIRVLPESNSDVDQLKKDLES(as described in ref. 26), for TPP1 NH2-GCTGPRAGRPRA-QARGVRGR-OH, and for OBFC1 NH2-GCKTKIEIGDTIRVRG-SIRT-OH. The p53BP1 antibody was purchased from Novus(NB100-304). The TRF2 antibody used for immunofluorescencewas purchased fromMillipore, clone 4A794 (05-521). The Chk1-p-Ser345 antibody was purchased from Cell Signaling Technol-ogy (#2348). The p68 and p180 antibodies used for Western blotanalyses and TRAP assays were purchased from Abcam (Ab57002and Ab65009). The POT1DOB and FLAG-TRF1 constructs and celllines are described in refs. 24 and 26.

PlasmidsThe cDNA for p68 (gene name POLA2) was purchased as a full-

length clone from the EST collection maintained by Invitrogen.The full-length cDNA was amplified by PCR using primers withappropriate cloning sites (50 BamHI and30 EcoRI) and cloned intopLPC-MYC (25) to generate a MYC-tagged version driven by theCMV promoter. The PCR oligonucleotides were: 50-TGCTTAG-GATCCGCATCCGCCCAGCAGCTG-30 and 50-TGGAGAGAATT-CTCAGATCCTGACGACCTGCACAG-30 corresponding to targetsites for codons 2–7 at the 50 end and the last 7 codons of thecDNA, including the stop codon. The OBFC1 cDNA was PCRcloned from a complete EST purchased from ATCC as a template,and with the following two oligonucleotides: 50-ATAACACA-GATCTCAGCCTGGATCCAGCCGGTGTG-30 and 50-TTCACCTC-TCGAGTCAGAACGCTGTGTAGTAGTGC-30, yielding a BglII-XhoIfragment as a PCR product. The TPP1 EST was purchased fromInvitrogen and PCR cloned with the following two oligonucleo-tides 50-AGGAGGATCCCCTGGCCGCTGTCAGAGTGACG-30 and50-GAGGACTCGAGTCACATCGGAGTTGGCTCAGAC-30, yieldinga BamHI-XhoI fragment as a PCR product.

Depletions by siRNA or shRNAHeLaII cells were maintained in DMEM (Invitrogen) supple-

mented with 1% penicillin and streptomycin and 10% FBS. ThesiRNAs used were synthesized by Dharmacon RNA Technologies.For p68 RNAi, double-stranded siRNAwere designed to target thefollowing sequences: p68-1 siRNA 50-UGGAAGAAGAAGAG-GAAAUUU-30 (target in the 50 UTR, exon 3) and p68-2 siRNA50-UAUCUGAGCUUAAGGAAUAUU-30 (coding sequence, exon7). For p180: p180-1 siRNA 50-CUGAGUACUUGGAAGUUAA-30

(coding sequence, exon 13); p180-2 siRNA 50-CAGAUCAUGU-GUGAGCUAA-30 (coding sequence, exon 21); p180-3 siRNA 50-GAGAGUAGCUGGAAUGUAA-30 (30UTR, exon 37). HeLaII cellswere transfected using Lipofectamine (Invitrogen) according tothe manufacturer's instructions. Briefly, cells at a confluency ofapproximately 50% to 60%were plated in a 6-well plate 18 to 24hours before transfection. Transfections were done once and cellswere processed 48 hours after transfection for protein extractionor immunofluorescence. As a control, siGFP (Dharmacon) wasused. For shRNA, the LMPvector fromOpenBiosystemswas used,which is based on the miR30 miRNA. The target sequences werePCR cloned according to themanufacturer's protocol basedon theNM_002689.2 sequence for the p68 cDNA. The target sequences

Pola and Human Telomeres

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were: sh50UTR, 50-CTCTGCCACCGTCACTGAGAAGTAGTGAA-GCCACAGATGTACTTCTCAGTGACGGTGGCAGAA-30; sh664,50-CCCTCTTGAACTCTTACACCACTAGTGAAGCCACAGATGTA-GTGGTGTAAGAGTTCAAGAGGA-30; sh1088, 50-ACCTGTCAC-TCTGCTGGGCCAGTAGTGAAGCCACAGATGTACTGGCCCAG-CAGAGTGACAGGC-30; sh30UTR, 50-AATGCTCCGTGTCCAGAA-GTAATAGTGAAGCCACAGATGTATTACTTCTGGACACGGAGC-ATG-30.

ImmunofluorescenceImmunostaining for p53BP1 and TRF2 was performed for

HeLaII cells plated onto glass coverslips and processed for RNAi.After the 48-hour transfection period, cells were extracted with Txbuffer (0.5% Triton X-100, 20 mmol/L Hepes-KOH, pH 7.9, 50mmol/L NaCl, 3 mmol/L MgCl2, 300 mmol/L sucrose) for 10minutes at room temperature (RT). After twoPBSwashes, the cellswere fixed with PBS/3% paraformaldehyde, 2% sucrose for 10minutes at RT. After two PBS washes, cells were permeabilizedwith Tx buffer for 10 minutes at RT, washed twice with PBS, andblocked with PBG (PBS/0.2% fish gelatin. 0.5% BSA) for 30minutes. Coverslips were then incubated with the rabbit anti-p53BP1 antibody (Novus NB100-304A-1), at a concentration of1:500 in PBG overnight. Cover slips were then rinsed three timeswith PBG solution and incubated with secondary TRITC-conju-gated goat anti-rabbit antibody (Jackson Immunoresearch) inPBG at a concentration of 1:500 for 45 minutes at RT. Coverslips were rinsed two times with PBG. Coverslips were thenincubated with PBG and DAPI at 100 ng/mL to visualize thenuclei. Coverslips were mounted onto slides with embeddingmedia. Imageswere collectedwith anOlympusBX61fluorescencemicroscope using a 60� objective connected to a HamamatsuORCA-ER CCD camera, controlled by the SlideBook 5.1 imagecapture software. The telomere FISH shown in Supplementary Fig.S1C was performed as described in ref. 2.

Cell synchronization and FACSHeLaII were plated 106 cells in 10-cm plates. After 24 hours,

thymidine to a final concentration of 2 mmol/L was added to themedia. The cells were treated with thymidine for 14 hours, andthen they were rinsed three times with warm PBS and freshmedium without thymidine. The cells were released for 11 hoursand then thymidine, 2 mmol/L final concentration, was added tothe medium again for the second block. After 14 hours the cells,were released as above and collected at the appropriate timepoints for ChIP and FACS. For FACS, cells were collected andrinsed twice in cold PBS, resuspended in 0.2mL of PBS/2mmol/LEDTA, 2mLof cold70%ethanolwas addeddropwise and the cellswere kept at 4�C 24 hours for fixation. The cells were then spundown and resuspended in 0.5 mL of PBS/2 mmol/L EDTA. Ofnote, 10 mL of heat-inactivated RNase A (10mg/mL) and 25 mL ofpropidium iodide (1 mg/mL) were added and the cells wereincubated at 37�C for 30 minutes. The samples were then ana-lyzed using a FACSCalibur Flow Cytometer.

Cell extracts and immunoprecipitationsCell extracts and immunoprecipitations were performed as

described in ref. (26). The quantitations shown in Fig. 4 weredone using the ImageJ analysis software. For siRNA and shRNAWestern blot analyses, the relative ratios were obtained by nor-malizing the p180 and p68 signal to GAPDH and by dividing by

the untreated sample signal. For the coimmunoprecipitationexperiments, the enrichment values were obtained by subtractingthe signal from the preimmune from the serum signal for eachantibody.

TRAP assaysThe TRAP assays were performed as per the manufacturer's

protocol (Millipore, S7710). For the IP-TRAP assays, the immu-noprecipitations were performed as described above, and thebeads were washed six times in cold lysis buffer. The beads werethen resuspended in 40 mL of lysis buffer, and 2 mL of theresuspended beads were used as input for the TRAP assay. ForRNAse-treated controls, 20 mL of resuspended beads wereremoved and treated with 10 mg of RNAse A at 37�C for 30minutes and washed twice with cold lysis buffer.

Chromatin immunoprecipitationsThe chromatin immunoprecipitations (ChIP) were performed,

as described in ref. 26, on HeLaII cells synchronized by doublethymidine block as described.

In-gel hybridizationGenomic DNA was isolated from cells as described in ref. 25

and processed for in-gel hybridization. The DNA was digestedwith AluI and MboI, and control samples were further digestedwith ExoI to digest the signal derived from the 30 overhang. Ofnote, 4 mg of DNA for each sample was loaded on a 0.7% agarosegel in 0.5X TBE. Following electrophoresis, the gels were dried atRT for at least 3 hours, and then prehybridizedwith inChurchmix(0.5mol/LNa2PO4, pH7.2, 1mmol/L EDTA, pH8.0, 7& SDS, 1%BSA) for 30 minutes at 50�C and hybridized overnight with endlabeled (CCCTAA)4 or (TTAGGG)4 oligonucleotides at 50�C.After hybridization, the gels were washed three times with 4�SCC and one time with 4� SCC, 0.1% SDS at 55�C, and exposedovernight to a PhosphorImager screen. To detect the total telo-mere signal, the gels were then denatured in 0.5mol/L NaOH and1.5 mol/L NaCl for 30 minutes and neutralized (3 mol/L NaCl,0.5mol/L Tris-HCl, pH7.0) twice for 15minutes, rinsedwith dH2

O and hybridized with the end-labeled oligonucleotides. The gelswere then exposed overnight to a PhosphorImager screen, and theG-overhang signal was calculated by dividing the native(CCCTAA)4 signal by that obtained with the denatured gel.

Telomeric 30overhang analysis by double-strand specificnuclease (DSN)

The DSN reaction was performed as previously described (27).Fourmicrograms of genomicDNA in 1�DSNbuffer was digestedwith 0.2 U/mg of DNA of DSN (Axxora cat. EVN-EA001-KI01) at37�C for 2 hours. As a control, 10 U of ExoI were added to thegenomic DNA before DSN and incubated at 37�C for 1 hour todigest terminal single-strandedDNA. The digestionswere stoppedwith 0.5 mL of 0.5 mol/L EDTA and an equal volume of form-amide before heating the samples at 65�C for 5 minutes. Thesampleswere runon a6%denaturing polyacrylamide gel contain-ing 8 mol/L urea. Electrophoresis was performed at 15V/cm in0.5� TBE. The gel was then electroblotted onto a Hybond Nmembrane (Amersham-GE) in 0.5� TBE buffer at 4�C and 30Vfor 90 minutes. The membrane was then air dried, UV cross-linked, and hybridized to a C-rich telomeric probe at 42�C. Themembrane was washed with 0.2 mol/L wash buffer (0.2 mol/L

Diotti et al.

Mol Cancer Res; 13(3) March 2015 Molecular Cancer Research404




Na2HPO4 pH 7.2, 1 mmol/L EDTA, and 2% SDS). The overhangsignal was calculated by subtracting the ExoI signal from the DSNsignal, and by calculating the ratio of each sample over theuntreated condition.

ResultsDepletion of p180 by siRNA, but not of p68, leads to a DNAdamage response

To study the roles of p180 and p68,we performed depletions ofboth proteins by siRNA and p68 by shRNA in HeLaII cells (Fig. 1and Supplementary Fig. S4). Among the three siRNA targets usedfor p180, the depletion was consistent down to about 30% ofcontrol endogenous levels (Fig. 1A and C). For p68, the first targetsite resulted in slightly over 50% depletion, and the second oneyielded a higher level of depletion, of 65% (Fig. 1B and D). Theselevels of depletion correlated with mild effects on cell-cycleprogression, in the case of p180, with a slight delay in S/G2–M(Supplementary Fig. S1A and S1B), and no obvious effect wasseen in the case of p68. The depletions we report in this study aremilder than those previously reported by others (15), and mayexplain our capacity to detect the telomeric effects reported herewithout major inhibition of cell-cycle progression elicited with

more significant reduction of Pola activity. Knockdown of p180generated a broad DNA damage response in the cell, as judged bythe induction of p53BP1 foci (Fig. 2A). This effect was notobserved upon depletion of p68. The average number of nucleiwith three ormore p53BP1 foci went frombelow 8% in control orp68 siRNA, to 35% with our best p180 siRNA targets (Fig. 2C).This observation likely corresponds to a broad induction of DNAdamage, and not telomere deprotection, because no colocaliza-tionwith telomeres was observed in this case (Supplementary Fig.S1C). The DNA damage response seen with p180 depletions iscompatible with replication stress, as a significant activation ofChk1 was observed (Fig. 2B), known to result from induction ofATR. Upon quantitation of the signal detected for Chk1-Ser-345phosphorylation, the levels of activated Chk1 increased by at least5-fold upon p180 depletion (Fig. 2D). We did not detect asignificant effect with p68 depletion, as the signal obtained wasnot significantly different from the siGFP-negative control (Fig. 2Band D).

Thus, disruption of Pola activity led to an apparent ATR-dependent DNA damage response as well as a mild S phase delayin the case of p180 depletion, while no overt effects on cell-cycleprogression or induction of DNA damage in the case of our p68knockdown experiments were observed.

POLa–primase components can be detected at telomeres bychromatin immunoprecipitation

To determine whether p180 and p68 could be detected attelomeres, we performed ChIP, a technique widely used to studyproteins localization at telomeres. The assay was carried out onasynchronous aswell as synchronized cell lines using anti-peptiderabbit sera against p180, p68, and OBFC1, the latter being asubunit of the CST complex. ChIP done on asynchronous HTC75control cell lines yielded a low but reproducible signal for bothp68 and p180 (Fig. 3A and B). We were able to visualize shelterincomponents at telomeres in these cells, such as TRF1 andPOT1, aspreviously reported, as well as FEN1, with a yield of 4% of thetotal telomeric signal, likely representing an S phase population(28, 29). In these cells, we were able to detect a low but repro-ducible signal for p68, with a yield of 3%. The p180 subunit wasnot detected at telomeres in this setting. We also performed theChIP on HTC75 cells with normal or elongated telomeres(through POT1DOB expression) and found that OBFC1 and p68were also present at elongated telomeres (not shown). As the rolesof Pola–primase are primarily related to DNA replication in Sphase, we sought to determine whether a telomeric associationwould be occurring for p68, and perhaps also p180, during thisphase, as has been reported for FEN1 (28). HeLaII cells weresynchronized through a double thymidine block, and the cellswere collected at 0, 2, 4, 6, and 8 hours after release (Fig. 3C andDand Supplementary Fig. S2). We found that, in staged S phasecells, both p68 and p180 could be reliably detected at telomeres,albeit at low levels. Althoughp180peaked in the early S phase andin late S–G2, p68 associated with telomeres more stably through-out S phase. These results could indicate a possible role for Pola attelomeres specifically during S phase.

The POLa–primase complex interacts with shelterinWe then sought to determine whether an association between

the Pola complex and the telomeric shelterin complex could bedetected. We also examined possible interactions with OBFC1,

Figure 1.Depletion of p68 (POLA2) or p180 (POLA) by siRNA inHeLaII cells. A,Westernblot analysis showing p180 depletion with three different target sites. B,depletion of p68, with two different target sites. C, quantitation of thedepletion of p180. D, quantitation of the depletion of p68. The signal obtainedfor p180 or p68was normalized toGAPDH, and divided by the signal detectedfor the untreated control. The values shown are averages and SE on threeindependent experiments.






given the known relationship with the Pola–primase complex(4). To that end, we used HTC75 cells expressing tagged versionsof OBFC1, POT1, TRF1, or TPP1 and asked whether each com-ponent could individually be immunoprecipitated with anti-p68or anti-p180 antibodies.

We were able to detect an association between shelterin sub-units POT1 and TPP1, and OBFC1 (Fig. 4A). In addition, weconfirmed the association between OBFC1 and p180 reportedpreviously (ref. 4; Fig. 4A). We then extended our studies topossible interactions between p180 and p68 with shelterin com-ponents. HTC75 cells overexpressing MYC-TTP1 (Fig. 4B) andboth HTC75 and HeLa 1.2.1.1 overexpressing MYC-p68 (Sup-plementary Fig. S3A and S3B) were used to test the potential

interaction. MYC-TPP1 was found to coimmunoprecipitate withp180 (Fig. 4B), suggesting an interaction between the Pola andshelterin complexes. Similarly, MYC-p68 could be immunopre-cipitated in HTC75 and HeLa 1.2.1.1 with POT1 or TRF2 anti-bodies (Supplementary Fig. S3A and S3B). We confirmed thesefinding by immunoprecipitating endogenous p68 in HeLaII cellswith antibodies against POT1, TRF1, TRF2, and TPP1 (Fig. 4C). Inaddition, we found that FLAG-TRF1 could be pulled down withp68 or p180 antibodies, confirming the interactions betweenshelterin and Pola in a different cell line. Therefore, it is possiblethat the amounts of p180 and p68 observed at telomeres by ChIP(Fig. 3) result from interactions at the protein level with the CSTand shelterin complexes. The protein interactions found did not

Figure 2.Depletion of p180, but not p68, leadsto formation of p53BP1 foci and Chk1activation. A, detection of p53BP1 inHeLaII cells 48 hours after siRNAtreatment for p68 or p180, with theindicated target sites. Blue, DAPI; Red,p53BP1; Green, TRF2. B, Western blotanalysis showingpChk1-Ser345 for thecontrol and siRNAagainst p68 or p180.The top two panels represent differentexposures of the same blot. TheGAPDH loading control is shownbelow. C, quantitation of p53BP1 foci inHeLaII cells treated with p68 and p180siRNAs as indicated. A hundred nucleiwere counted for each sample, and theaverages and SE were calculated fromthree independent experiments. D,quantitation of Chk1 phosphorylation,as shown in B. The relative ratio wasobtained by normalizing the pChk1-Ser345 signal to GAPDH and dividingby the siGFP control (mean � SEM forthree experiments).

Diotti et al.





require cell synchronization as the ChIPs did. Our results there-fore demonstrate interactions between three known complexes,the shelterin, CST, and Pola–primase complexes, in three differ-ent human cell lines, which are likely to reflect associationsoccurring at telomeres.

The POLa–primase complex interacts with telomerase.Our observation that Pola–primase is associated with telo-

meres and shelterin places p180 and p68 in prime position tointeract with the telomerase complex. We thus asked whether anassociation could be detected. To this end, a HeLaII extract wasprepared for immunoprecipitations with p180 antibodies, andthe resulting pulled down material was used as input for TRAPactivity assay to assess a possible association with telomerase.In Fig. 5A, we show that p180 antibodies can specifically pull

down telomerase activity. As controls, no TRAP activity was eitherdetected with preimmune serum, or with RNase-treated material(Fig. 5A and Supplementary Fig. S4B).We found similar yields forFEN1, which was previously shown to associate with telomeraseactivity (30), and could precipitate TRAP activity with p68 anti-bodies as well (Supplementary Fig. S4A). Such an interaction wasalso described by others for TPP1 (31). OBFC1, in our hands, didnot pull down TRAP activity (Supplementary Fig. S4A). We couldconfirm the associationbetweenPola–primase and telomerase bycoimmunoprecipitation between p180 and hTERT, the catalyticsubunit of telomerase (Fig. 5B): p180 antibodies could precipitateMYC-hTERT from a HTC75 extract, whereas OBFC1 antibodiesdid not. Thus, we are able to show the telomerase–Pola–primaseinteraction in two ways, by the IP-TRAP assay and bycoimmunoprecipitation.

Figure 3.Association of p68 and p180 withtelomeres by ChIPs. A, ChIP inunsynchronized HTC75 cells. The Aluprobe was used as a non-telomerecontrol. The antibodies used areindicated on top; PI, preimmuneserum. B, quantitation of the DNAyields for the HTC75 ChIP. The yield foreach sample was divided by the totalDNA value after subtraction of thepreimmune background value. C, ChIPin synchronized HeLaII cells. Cells weresubjected to a double thymidine block,and processed for ChIP at 2-hourintervals after release. Probes areindicated at the bottom of the blots. D,quantitationof the yields for p180, p68,and OBFC1 at indicated time pointsafter release. STY, values obtainedwith the TTAGGG (telomeric) probe.An Alu probe was used as a control.






Depletion of p180 leads to increased telomeric overhangamounts

As both shelterin and CST play important roles in telomereprotection through overhang processing, we looked at the telo-meric overhang amounts in conditions where p180 or p68 weredepleted. We examined both short-term depletion (48 hours) viasiRNA for both p68 and p180 (Fig. 6) and long-term depletionusing shRNAs for p68 (Supplementary Fig. S4). This assayinvolves separating restricted genomic DNA by size on an agarosegel and probing the gel in native conditions with a labeledTTAGGGoligonucleotide (Fig. 6A, left). The amounts of overhangdetected for each cell line are normalized to the total telomericDNA detected in denatured conditions (Fig. 6A, right).

A role for p180 in limiting overhang lengthwas observed in oursiRNA experiments (Fig. 6A and B). Of the three targets sites usedagainst p180, two showed anoverhang increase of 35%(target site2) and 24% (target site 3) representing values of significancebelow P¼ 0.05 on three independent experiments. No significanteffect was observed with the siRNAs against p68.

We also performed this assay on long-term depletions for p68.The p68 shRNAs were introduced in HeLaII cells through retro-viral transduction; p68 was knocked down using four differentshRNAs targeting different sites on the predicted mRNA (Supple-mentary Fig. S4). In this case, we detected a mild effect for one ofthe target site (sh644) with a statistically significant P value(Supplementary Fig. S4C). The increase in overhang amountsaveraged 15% in this case. No obvious effect on telomere lengthwas observed. To confirm these results with a different technique,we applied the DSN assay (27) to p180-depleted HelaII cells(Supplementary Fig. S6). The DSN assay allows the detection ofundigested single-stranded telomeric overhangs by hybridizationto a labeled C-rich oligonucleotide. We found that the telomericoverhangs increased in amounts in p180-depleted cells (Supple-mentary Fig. S6A), to an extent similar to that seen with the nativehybridization assay (Supplementary Fig. S6B).

We conclude that a decrease in p180 leads to increased over-hang amounts, suggesting a role in the regulation of the length ofthe telomeric overhang without affecting the resection processcreating the telomeric 50 end. It is possible that p68 is alsoinvolved in this process, although the effect appears weaker.

DiscussionThe results presented here provide a link between the Pola–

primase complex, and two other telomeric complexes, shelterinand the CST complex. Although shelterin and CST have well-documented roles in telomere function, pertaining to the protec-tion and maintenance of telomeres, the implication of Pola–primase in these processes poses a question: are its roles attelomeres related to conventional chromosome DNA replication,or are there telomere-specific, noncanonical roles for Pola–pri-mase at telomeres? Although the final answer requires additionalwork, we would argue based on our findings that the observedeffect on overhang processing reflects a telomere-specific role forPola not related to origin-initiated DNA replication. First, wereport here for the first time an interaction between p180, andp68with shelterin. These types of associations were described inbudding and fission yeast (20, 21), but never, to our knowledge,in human cells. Second, we did not observe major defects in thecell-cycle profiles of p180- or p68-depleted cells in our study,arguing for mild effects, if any, in chromosomal DNA replication(Fig. 2 and Supplementary Fig. S2). These observations could besimply explained by arguing that the remaining amounts of p180or p68 in our depletion experiments are sufficient to support DNAreplication, but limiting for effective overhang processing. And,

Figure 4.Interactions between Pola and shelterin components detected by IP-Western. A–C, extracts prepared from HTC75 cells expressing taggedconstructs, indicated on the right, were used for immunoprecipitations withthe antibodies indicated at the top of the lanes. The input fractions andamounts precipitated with beads only (no antibody) are shown on theleftmost lanes of each blot. The numbers below the blots represent the foldenrichment for a specific antibody as compared with its preimmune serum.The antibodies used to probe the Western blot analyses were anti-MYC 9E10antibody (A and B) and an anti-POLA2 serum (C). The antibodies used forimmunoprecipitations are indicated at the top of the lanes. The blot wasprobed with a commercial mouse anti-p68 antibody.

Figure 5.Interaction between p180 and telomerase. A, TRAP assay performed onHelaIIextract (left lane), and on immunoprecipitated material with p180 serum (S)or a p180monoclonal purchased fromAbcam. Controls includeRNase treatedlysates, as well as beads only, as indicated. B, IP-Western showingimmunoprecipitation of hTERT with p180 serum. The lysate was preparedfrom HTC75 cells expressing MYC-tagged hTERT. The blot was probed withthe 9E10 anti-MYC antibody.

Diotti et al.





finally, we documented an association between telomerase andthe p180 and p68 subunits of Pola–primase. This association canbedetected at theprotein levelwithhTERT, the catalytic subunit oftelomerase, and likely reflects a functional role because we canprecipitate telomerase activity with primase subunits p68 andp180. This idea is in our view interesting in that it could uncover atelomere-specific set of events implicating Pola–primase, whichcould be targeted in tumor cells, for instance, to limit theirproliferation. Such a role could be highly conserved in evolution,and be related to the finding that a special mutant allele in thebudding yeast POL12 gene, which represents the p68 ortholog,displays dysregulated telomere function, including longer telo-mere length, and telomere deprotection (14). In addition,the yeast POL12 gene interacts genetically with STN1, the OBFC1ortholog in this organism (14). In human cells, dominant-

negative approaches could allow for a finer analysis of telo-mere-specific functions for Pola–primase.

An important future direction will be to understand the mech-anism of action of Pola–primase at telomere, if different from ormore complex than mere association with the replication forkprogressing through the telomere. At present, we think it valuableto interpret these results in light of recent work showing that theCST complex, in particular OBFC1, limits telomerase activity,thereby participating in telomere length homeostasis (3) and hasa specific role in C-strand fill-in (12). Knowing that OBFC1 is partof an "alpha activating factor" complex (4), but has no obviousrole in the recruitment of the complex to telomeres, leads us tosuggest that Pola–primase is recruited through telomere-specificinteractions, perhaps involving shelterin, to lay down the terminalRNA primer and restrict the length of the overhang. Depletingamounts of Pola–primase would render this step limiting andresult in increasing overhang length overall. It will be interestingto analyze the nature of the potential direct contacts betweenshelterin and Pola–primase, as well as between Pola–primaseand the telomerase complex.

In addition, althoughwedid not observe any significant changein the terminal 50 nucleotide in our conditions using the STELAassay (not shown), 50 end resection may be altered upon strongerinactivation of the Pola complex. This would present an inter-esting synergy with POT1, which does influence the terminal 50

nucleotide in human cells (7). As a result of the interactionsreported here, the overhang phenotype we describe is predicted tobe dependent on telomerase activity, and it would thus beinteresting and important to perform the depletion experimentsfor p68 and p180 in telomerase-negative primary cells. In thiscontext, targeting Pola–primase, or the specific interactions withshelterin, would possibly result in immediate or premature senes-cence in primary cells, thereby activating an important tumorsuppressor system. It is relevant tonote that inmouse cells, POT1bis the recruiting activity for the CST complex, itself mediating fill-in synthesis, likely through Pola–primase recruitment (2). Ourdata are compatible with these findings, and it would be valuableto test the possibility that POT1 is the recruiting factor for CST andPola in human cells. Two issues remain of note in our opinion.First, as we did not detect obvious telomere deprotection (bylooking at telomeric p53BP1 foci), this pathway for overhangprocessing is not expected to lead to immediate effects such asapoptosis or premature senescence. However, one could hypoth-esize that lack of effective fill-in synthesis would exacerbate the so-called "end-replication problem" and limit the proliferativepotential of cells, and perhaps even of telomerase-positive tumorcells. Longer term experiments than those reported here in pri-mary human cells would be required to examine this possibility.The second issue iswhether the overhang phenotype requires cellsto be passing through S phase, or whether the specific function ofPola–primase suggested here is independent of actual DNAreplication. Even though we did detect an increase in p68 andp180 at telomeres in S phase, it remains possible that some ofthese interactions occur outside of the context of chromosomalDNA replication and be important for telomere protection.Therefore, it would be valuable to assess the possible effects ofPola–primase in cells that are in G0, which do not experienceprogression through the cell cycle, S phase, or proliferation.

Our view, based on the results presented here, is that telo-mere function relies on multiple interactions among threeimportant complexes, shelterin, CST, and Pola–primase. In

Figure 6.Increased overhang amounts upon siRNA depletion of p180. A, in-gelhybridization for genomic DNA from HeLaII cells treated with siRNA for p68andp180. Left, native gel probedwith the labeled oligonucleotide (CCCTAA)4hybridizing to the telomeric overhang. Right, same gel reprobed afterdenaturation to detect all telomeric sequences. Samples treated with ExoIwere run alongside to control for detection of single-strand DNA in the nativegel. B, overhang intensity is indicated as the ratio of the native signal over thedenatured, normalized to the siGFP control. Error bars represent the SE forthree independent experiments. The two-tailed Student t test for the twosignificant values (<0.05) is shown.






addition, Pola–primase appears to associate with the telomerasecomplex, presumably at telomeres. Shelterin is known to bequantitatively associated with telomeres, whereas the CST andPola–primase complexes could be viewed as telomere-associatedfactors, acting only transiently at telomeres in S phase.We provideevidence here for Pola–primase as an important complex toconsider in the context of telomere protection and maintenance,which potentially offers additional targets to counteract celltransformation and proliferation during tumorigenesis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Diotti, D. LoayzaDevelopment of methodology: R. Diotti, D. LoayzaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Diotti, S. Kalan, A. Matveyenko

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Diotti, S. Kalan, A. Matveyenko, D. LoayzaWriting, review, and/or revision of the manuscript: R. Diotti, S. Kalan,A. Matveyenko, D. LoayzaStudy supervision: D. Loayza

AcknowledgmentsThe authors thank theD.L. laboratory for help and advice, especially S.Uddin

and C. Vuong for excellent technical support, and for comments on the article.

Grant SupportThis work was funded by a SC3 score award # 1SC3GM094071-01A1 from

the NIGMS.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 8, 2014; revised November 10, 2014; accepted December 5,2014; published OnlineFirst December 17, 2014.

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Diotti et al.




2015;13:402-410. Published OnlineFirst December 17, 2014.Mol Cancer Res Raffaella Diotti, Sampada Kalan, Anastasiya Matveyenko, et al. Telomeric Overhang ProcessingDNA-Directed Polymerase Subunits Play a Vital Role in Human

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