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Open Full Page Published OnlineFirst December 17, 2014; DOI: 10.1158/1541-7786.MCR-14-0381 Cell Cycle and Senescence Molecular Cancer Research DNA-Directed Polymerase Subunits Play a Vital Role in Human Telomeric Overhang Processing Raffaella Diotti, Sampada Kalan, Anastasiya Matveyenko, and Diego Loayza Abstract Telomeres consist of TTAGGG repeats bound by the shelterin could also show that the CST, shelterin, and polymerase com- complex and end with a 30 overhang. In humans, telomeres plexes interact, revealing contacts occurring at telomeres. We shorten at each cell division, unless telomerase (TERT) is found that the polymerase complex could associate with telome- expressed and able to add telomeric repeats. For effective telomere rase activity. Finally, depletion of p180 by siRNA led to increased maintenance, the DNA strand complementary to that made by overhang amounts at telomeres. These data support a model in telomerase must be synthesized. Recent studies have discovered a which the polymerase complex is important for proper telomeric link between different activities necessary to process telomeres overhang processing through fill-in synthesis, during S phase. in the S phase of the cell cycle to reform a proper overhang. These results shed light on important events necessary for efficient Notably, the human CST complex (CTC1/STN1/TEN1), known to telomere maintenance and protection. interact functionally with the polymerase complex (POLA/pri- mase), was shown to be important for telomere processing. Here, Implications: This study describes the interplay between DNA focus was paid to the catalytic (POLA1/p180) and accessory replication components with proteins that associate with chro- (POLA2/p68) subunits of the polymerase, and their mechanistic mosome ends, and telomerase. These interactions are proposed to roles at telomeres. We were able to detect p68 and p180 at be important for the processing and protection of chromosome telomeres in S-phase using chromatin immunoprecipitation. We ends. Mol Cancer Res; 13(3); 402–10. Ó2014 AACR. Introduction suggesting coordination between telomerase and the C-strand synthesis machinery. In humans, the mechanism of C-strand Telomeres are structures located at the end of linear chromo- processing is tightly regulated, as demonstrated by the observa- somes, essential for their stability and integrity. They consist of 0 0 0 tion that the 5 ends in AATC-5 80% of the times and in AATCC-5 stretches of repetitive DNA sequences and the protein complex 0 15% of the times (6). The terminal 5 nucleotide, however, bound to them, known in mammals as shelterin. Their role is to becomes randomized upon the depletion of POT1 (7), the protect the chromosome ends from being inappropriately overhang binding protein in the shelterin complex. Different recognized by the DNA damage machinery (1). The ends of 0 models for the creation of the 3 G-strand overhang have been mammalian telomeres exhibit a 30 overhang of 50 to 300 proposed and, in general, they invoke an interplay of leading and nucleotides, produced by competing actions of telomerase, the lagging strand processing due to the intrinsic properties of extending it by repeat additions, by ExoI and Apollo, which DNA replication and telomerase activity (see for example ref. 8). create the 50 resected end (2), and by C-strand fill-in processing. Recently, it has been elegantly shown that the two strands are in Another complex, called the CST complex, is known to limit fact differentially processed during replication (9). The leading telomerase activity in S phase (3), and to associate with the overhang is initially a blunt end that is processed later in S phase RNA primer synthesizing complex Pola–primase (4), thereby for the creation of the overhang. The lagging strand overhang contributing an additional activity likely to participate in over- length is first determined by the placement of the last RNA primer, hang processing. which is then removed before further processing (9). Final over- In human cells, telomeres shorten at each cell division, unless hang processing occurs in late S–G . This study establishes the telomerase is present and active in adding TTAGGG repeats. 2 basis for a differential processing of the two strands without During active telomere elongation in telomerase-positive cells, excluding other additional processing events for the C-strand. no significant change is observed in mean overhang length (5), Additional indication of differential metabolism for the two terminal strands was described in the mouse system, in which the exonucleases Apollo and ExoI, were found to be important for Department of Biological Sciences, Hunter College and CUNY Grad- overhang production through 50 end resection at the leading and uate Center, New York, New York. lagging strands, respectively (2). This work also elucidated an Note: Supplementary data for this article are available at Molecular Cancer important role for POT1b in both leading and lagging strand Research Online (http://mcr.aacrjournals.org/). overhang formation through the coordination of the nucleases Corresponding Author: Diego Loayza, Hunter College, City University of New action and the C-strand fill-in through association with the CST York, 695 Park Avenue, New York, NY 10065. Phone: 212-772-5312; Fax: 212-772- complex. 5227; E-mail: [email protected] In Saccharomyces cerevisiae, as well as in most other eukar- doi: 10.1158/1541-7786.MCR-14-0381 yotes, the CST complex, composed by three RPA-like proteins, Ó2014 American Association for Cancer Research. Cdc13-Stn1-Ten1 (CTC1-OBFC1-Ten1 in humans), was found 402 Mol Cancer Res; 13(3) March 2015 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 17, 2014; DOI: 10.1158/1541-7786.MCR-14-0381 Pola and Human Telomeres to be important for telomere protection and replication into rabbits, as per the protocol set by the manufacturer (BioSyn- (10, 11). The role of the CST complex in the coordination of thesis). The peptides were: NH2-GCKGRQEALERLKKAKAGEK- telomere elongation in humans was corroborated by the find- OH for p180, and NH2-GCRLYLRRPAADGAERQSP-OH for p68. ings that it contributes to limiting telomerase activity at extend- The peptide for FEN1 NH2-GCSTKKKAKTGAAGKFKRGK–OH, ing telomeres through complex interactions with telomerase for TRF1 NH2-GCGSIEKEHDKLHEEIQNLI-OH (as described itself and TPP1/POT1 (3). In addition, it was recently shown in ref. 24), for POT1 NH2-CYGRGIRVLPESNSDVDQLKKDLES that CST plays a role in both general telomere replication and (as described in ref. 26), for TPP1 NH2-GCTGPRAGRPRA- overhang processing, particularly for longer telomeres that QARGVRGR-OH, and for OBFC1 NH2-GCKTKIEIGDTIRVRG- represent a challenge for the replication machinery. Specifically, SIRT-OH. The p53BP1 antibody was purchased from Novus C-strand fill in was affected in OBFC1-depleted cells, delaying (NB100-304). The TRF2 antibody used for immunofluorescence the processing that leads to the final, mature G-overhang was purchased from Millipore, clone 4A794 (05-521). The Chk1- (12, 13). The human CST complex, in addition, was isolated p-Ser345 antibody was purchased from Cell Signaling Technol- as a set of accessory factors for the POLa–primase complex (4). ogy (#2348). The p68 and p180 antibodies used for Western blot In S. cerevisiae, Cdc13 is able to interact with POL1, the POLa analyses and TRAP assays were purchased from Abcam (Ab57002 D homolog, and STN1 interacts with POL12, the regulatory and Ab65009). The POT1 OB and FLAG-TRF1 constructs and cell subunit of Pola–primase, which is named POLA2 in humans lines are described in refs. 24 and 26. (14). The POLa–primase complex is believed to be important for replication of the lagging strand and the C-strand fill in for Plasmids both strands after resection. The complex is composed by the The cDNA for p68 (gene name POLA2) was purchased as a full- catalytic subunit, Pola, also termed p180, two primase sub- length clone from the EST collection maintained by Invitrogen. units, and the regulatory subunit POLA2, or p68 (see ref. 15). The full-length cDNA was amplified by PCR using primers with Although it has recently been established that telomeres, not- appropriate cloning sites (50 BamHI and 30 EcoRI) and cloned into withstanding their reported nature as fragile sites of DNA pLPC-MYC (25) to generate a MYC-tagged version driven by the replication on chromosomes (16), do not undergo a telo- CMV promoter. The PCR oligonucleotides were: 50-TGCTTAG- mere-specific replication program, but mainly a chromo- GATCCGCATCCGCCCAGCAGCTG-30 and 50-TGGAGAGAATT- some-specific one (17), it has been found that partial altering CTCAGATCCTGACGACCTGCACAG-30 corresponding to target of the replication machinery can have telomere-specific effects. sites for codons 2–7 at the 50 end and the last 7 codons of the Mouse cells with a temperature-sensitive p180 exhibit overhang cDNA, including the stop codon. The OBFC1 cDNA was PCR extension and concomitant overall telomere elongation (18). cloned from a complete EST purchased from ATCC as a template, Therefore, although defects in DNA replication and in partic- and with the following two oligonucleotides: 50-ATAACACA- ular in Pola–primase activity are predicted to affect general GATCTCAGCCTGGATCCAGCCGGTGTG-30 and 50-TTCACCTC- chromosomal DNA replication, it is possible that this activity TCGAGTCAGAACGCTGTGTAGTAGTGC-30, yielding a BglII-XhoI displays noncanonical, telomere-specificroles,inparticularin
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