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• W2913360660 abstract "•An assay to visualize ALT telomere DNA synthesis in APBs•APBs are functionally important for ALT DNA synthesis•RAD52 promotes telomere D-loops in vitro and ALT DNA synthesis in APBs•A RAD52-independent BIR pathway is responsible for C-circle formation Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe an assay to visualize ALT-mediated telomeric DNA synthesis in ALT-associated PML bodies (APBs) without DNA-damaging agents or replication inhibitors. Using this assay, we find that ALT occurs through two distinct mechanisms. One of the ALT mechanisms requires RAD52, a protein implicated in break-induced DNA replication (BIR). We demonstrate that RAD52 directly promotes telomeric D-loop formation in vitro and is required for maintaining telomeres in ALT-positive cells. Unexpectedly, however, RAD52 is dispensable for C-circle formation, a hallmark of ALT. In RAD52-knockout ALT cells, C-circle formation and RAD52-independent ALT DNA synthesis gradually increase as telomeres are shortened, and these activities are dependent on BLM and BIR proteins POLD3 and POLD4. These results suggest that ALT occurs through a RAD52-dependent and a RAD52-independent BIR pathway, revealing the bifurcated framework and dynamic nature of this process. Alternative lengthening of telomeres (ALT) is a telomerase-independent but recombination-dependent pathway that maintains telomeres. Here, we describe an assay to visualize ALT-mediated telomeric DNA synthesis in ALT-associated PML bodies (APBs) without DNA-damaging agents or replication inhibitors. Using this assay, we find that ALT occurs through two distinct mechanisms. One of the ALT mechanisms requires RAD52, a protein implicated in break-induced DNA replication (BIR). We demonstrate that RAD52 directly promotes telomeric D-loop formation in vitro and is required for maintaining telomeres in ALT-positive cells. Unexpectedly, however, RAD52 is dispensable for C-circle formation, a hallmark of ALT. In RAD52-knockout ALT cells, C-circle formation and RAD52-independent ALT DNA synthesis gradually increase as telomeres are shortened, and these activities are dependent on BLM and BIR proteins POLD3 and POLD4. These results suggest that ALT occurs through a RAD52-dependent and a RAD52-independent BIR pathway, revealing the bifurcated framework and dynamic nature of this process. The maintenance of telomeres is critical for the genomic stability and sustained survival of proliferating cells (Artandi and DePinho, 2010Artandi S.E. DePinho R.A. Telomeres and telomerase in cancer.Carcinogenesis. 2010; 31: 9-18Crossref PubMed Scopus (593) Google Scholar, Hanahan and Weinberg, 2011Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (42734) Google Scholar, Palm and de Lange, 2008Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1384) Google Scholar, Verdun and Karlseder, 2007Verdun R.E. Karlseder J. Replication and protection of telomeres.Nature. 2007; 447: 924-931Crossref PubMed Scopus (379) Google Scholar). Telomerase, an RNA-templated enzyme that extends telomeres, plays a crucial role in telomere maintenance. To bypass replicative senescence during tumorigenesis, telomerase is activated in the majority of human cancers (Shay, 2016Shay J.W. Role of telomeres and telomerase in aging and cancer.Cancer Discov. 2016; 6: 584-593Crossref PubMed Scopus (365) Google Scholar). However, about 10%–15% of human cancers use a telomerase-independent but recombination-dependent pathway to maintain telomeres (Dilley and Greenberg, 2015Dilley R.L. Greenberg R.A. ALTernative telomere maintenance and cancer.Trends Cancer. 2015; 1: 145-156Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Heaphy et al., 2011Heaphy C.M. Subhawong A.P. Hong S.M. Goggins M.G. Montgomery E.A. Gabrielson E. Netto G.J. Epstein J.I. Lotan T.L. Westra W.H. et al.Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes.Am. J. Pathol. 2011; 179: 1608-1615Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, Reddel, 2014Reddel R.R. Telomere maintenance mechanisms in cancer: clinical implications.Curr. Pharm. Des. 2014; 20: 6361-6374Crossref PubMed Scopus (57) Google Scholar). This pathway, which is referred to as alternative lengthening of telomeres (ALT), is a potential therapeutic target in cancers lacking telomerase activity. Although a number of DNA repair and recombination proteins have been implicated in ALT, the molecular process through which ALT occurs is still poorly understood (Cesare and Reddel, 2010Cesare A.J. Reddel R.R. Alternative lengthening of telomeres: models, mechanisms and implications.Nat. Rev. Genet. 2010; 11: 319-330Crossref PubMed Scopus (673) Google Scholar, Sobinoff and Pickett, 2017Sobinoff A.P. Pickett H.A. Alternative lengthening of telomeres: DNA repair pathways converge.Trends Genet. 2017; 33: 921-932Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, although several common features of ALT-positive (ALT+) cells are widely used to assess the ALT status, whether and how these ALT features are mechanistically linked to the process of ALT remains largely unclear. A better understanding of the framework of the ALT pathway and the molecular mechanisms underlying the hallmarks of ALT will greatly facilitate the characterizations and targeting of ALT+ cancers. One of the hallmarks of ALT is ALT-associated PML bodies (APBs) (Yeager et al., 1999Yeager T.R. Neumann A.A. Englezou A. Huschtscha L.I. Noble J.R. Reddel R.R. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res. 1999; 59: 4175-4179PubMed Google Scholar). In ALT+ cells, APBs containing both telomeres and PML are enriched in the G2 phase of the cell cycle (Grobelny et al., 2000Grobelny J.V. Godwin A.K. Broccoli D. ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle.J. Cell Sci. 2000; 113: 4577-4585Crossref PubMed Google Scholar). High-resolution imaging studies revealed telomere clusters around PML bodies (Draskovic et al., 2009Draskovic I. Arnoult N. Steiner V. Bacchetti S. Lomonte P. Londoño-Vallejo A. Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination.Proc. Natl. Acad. Sci. USA. 2009; 106: 15726-15731Crossref PubMed Scopus (110) Google Scholar). Furthermore, a number of DNA repair and recombination proteins, including RPA, RAD51, RAD52, BLM, and others, were detected in APBs, raising the possibility that APBs provide a “recombinogenic microenvironment” to promote ALT (Acharya et al., 2014Acharya S. Kaul Z. Gocha A.S. Martinez A.R. Harris J. Parvin J.D. Groden J. Association of BLM and BRCA1 during telomere maintenance in ALT cells.PLoS ONE. 2014; 9: e103819Crossref PubMed Scopus (23) Google Scholar, Lillard-Wetherell et al., 2004Lillard-Wetherell K. Machwe A. Langland G.T. Combs K.A. Behbehani G.K. Schonberg S.A. German J. Turchi J.J. Orren D.K. Groden J. Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2.Hum. Mol. Genet. 2004; 13: 1919-1932Crossref PubMed Scopus (124) Google Scholar, Nabetani et al., 2004Nabetani A. Yokoyama O. Ishikawa F. Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body.J. Biol. Chem. 2004; 279: 25849-25857Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, O’Sullivan et al., 2014O’Sullivan R.J. Arnoult N. Lackner D.H. Oganesian L. Haggblom C. Corpet A. Almouzni G. Karlseder J. Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.Nat. Struct. Mol. Biol. 2014; 21: 167-174Crossref PubMed Scopus (167) Google Scholar, Potts and Yu, 2007Potts P.R. Yu H. The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins.Nat. Struct. Mol. Biol. 2007; 14: 581-590Crossref PubMed Scopus (280) Google Scholar, Stavropoulos et al., 2002Stavropoulos D.J. Bradshaw P.S. Li X. Pasic I. Truong K. Ikura M. Ungrin M. Meyn M.S. The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis.Hum. Mol. Genet. 2002; 11: 3135-3144Crossref PubMed Scopus (160) Google Scholar, Wu et al., 2000Wu G. Lee W.H. Chen P.L. NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres.J. Biol. Chem. 2000; 275: 30618-30622Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, Yeager et al., 1999Yeager T.R. Neumann A.A. Englezou A. Huschtscha L.I. Noble J.R. Reddel R.R. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res. 1999; 59: 4175-4179PubMed Google Scholar). Despite these tantalizing observations, it still remains unclear whether ALT DNA synthesis occurs specifically in APBs and whether APBs are essential for ALT DNA synthesis. In addition to APBs, ALT+ cells are also characteristic for harboring higher levels of extrachromosomal telomeric DNA circles, especially single-stranded C-rich circles (C-circles) (Cesare and Griffith, 2004Cesare A.J. Griffith J.D. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops.Mol. Cell. Biol. 2004; 24: 9948-9957Crossref PubMed Scopus (247) Google Scholar, Henson et al., 2009Henson J.D. Cao Y. Huschtscha L.I. Chang A.C. Au A.Y. Pickett H.A. Reddel R.R. DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity.Nat. Biotechnol. 2009; 27: 1181-1185Crossref PubMed Scopus (315) Google Scholar, Nabetani and Ishikawa, 2009Nabetani A. Ishikawa F. Unusual telomeric DNAs in human telomerase-negative immortalized cells.Mol. Cell. Biol. 2009; 29: 703-713Crossref PubMed Scopus (83) Google Scholar, Ogino et al., 1998Ogino H. Nakabayashi K. Suzuki M. Takahashi E. Fujii M. Suzuki T. Ayusawa D. Release of telomeric DNA from chromosomes in immortal human cells lacking telomerase activity.Biochem. Biophys. Res. Commun. 1998; 248: 223-227Crossref PubMed Scopus (73) Google Scholar, Tokutake et al., 1998Tokutake Y. Matsumoto T. Watanabe T. Maeda S. Tahara H. Sakamoto S. Niida H. Sugimoto M. Ide T. Furuichi Y. Extra-chromosomal telomere repeat DNA in telomerase-negative immortalized cell lines.Biochem. Biophys. Res. Commun. 1998; 247: 765-772Crossref PubMed Scopus (92) Google Scholar, Wang et al., 2004Wang R.C. Smogorzewska A. de Lange T. Homologous recombination generates T-loop-sized deletions at human telomeres.Cell. 2004; 119: 355-368Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). C-circle levels correlate with the levels of telomere DNA synthesis in ALT+ cells, and high C-circle abundance is widely used as a marker for ALT activation (O’Sullivan et al., 2014O’Sullivan R.J. Arnoult N. Lackner D.H. Oganesian L. Haggblom C. Corpet A. Almouzni G. Karlseder J. Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.Nat. Struct. Mol. Biol. 2014; 21: 167-174Crossref PubMed Scopus (167) Google Scholar, Sobinoff et al., 2017Sobinoff A.P. Allen J.A. Neumann A.A. Yang S.F. Walsh M.E. Henson J.D. Reddel R.R. Pickett H.A. BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.EMBO J. 2017; 36: 2907-2919Crossref PubMed Scopus (84) Google Scholar, Yu et al., 2015Yu E.Y. Pérez-Martín J. Holloman W.K. Lue N.F. Mre11 and Blm-dependent formation of ALT-like telomeres in Ku-deficient Ustilago maydis.PLoS Genet. 2015; 11: e1005570Crossref PubMed Scopus (16) Google Scholar). Nonetheless, how C-circles are generated during ALT remains elusive. ALT has been long speculated to be a recombination-based process (Dunham et al., 2000Dunham M.A. Neumann A.A. Fasching C.L. Reddel R.R. Telomere maintenance by recombination in human cells.Nat. Genet. 2000; 26: 447-450Crossref PubMed Scopus (707) Google Scholar). In the budding yeast, the survival of telomerase null cells relies on two distinct recombination pathways (types I and II survivors) (Le et al., 1999Le S. Moore J.K. Haber J.E. Greider C.W. RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase.Genetics. 1999; 152: 143-152Crossref PubMed Google Scholar). Although both pathways require Rad52, only one (type I survivors) depends on Rad51 (Chen et al., 2001Chen Q. Ijpma A. Greider C.W. Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events.Mol. Cell. Biol. 2001; 21: 1819-1827Crossref PubMed Scopus (219) Google Scholar). Both of the yeast pathways also require Pol32, a subunit of DNA polymerase δ critical for break-induced DNA replication (BIR) (Lydeard et al., 2007Lydeard J.R. Jain S. Yamaguchi M. Haber J.E. Break-induced replication and telomerase-independent telomere maintenance require Pol32.Nature. 2007; 448: 820-823Crossref PubMed Scopus (367) Google Scholar). Recent studies in human cells further revealed that ALT is a replication stress-associated and BIR-related process. Depletion of ASF1 induces replication stress at telomeres and a spectrum of ALT-associated phenotypes (O’Sullivan et al., 2014O’Sullivan R.J. Arnoult N. Lackner D.H. Oganesian L. Haggblom C. Corpet A. Almouzni G. Karlseder J. Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.Nat. Struct. Mol. Biol. 2014; 21: 167-174Crossref PubMed Scopus (167) Google Scholar). Induction of DNA double-strand breaks (DSBs) at telomeres elicits robust DNA synthesis through a process requiring POLD3, the counterpart of the yeast Pol32 (Dilley et al., 2016Dilley R.L. Verma P. Cho N.W. Winters H.D. Wondisford A.R. Greenberg R.A. Break-induced telomere synthesis underlies alternative telomere maintenance.Nature. 2016; 539: 54-58Crossref PubMed Scopus (236) Google Scholar). In ALT+ cells, overexpression of BLM promotes extension of telomeres in a POLD3-dependent manner (Sobinoff et al., 2017Sobinoff A.P. Allen J.A. Neumann A.A. Yang S.F. Walsh M.E. Henson J.D. Reddel R.R. Pickett H.A. BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.EMBO J. 2017; 36: 2907-2919Crossref PubMed Scopus (84) Google Scholar). Depletion of POLD3 or it associated partner POLD4 in ALT+ cells led to a reduction in conservatively replicated telomeres, indicating their involvement in ALT (Roumelioti et al., 2016Roumelioti F.M. Sotiriou S.K. Katsini V. Chiourea M. Halazonetis T.D. Gagos S. Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication.EMBO Rep. 2016; 17: 1731-1737Crossref PubMed Scopus (96) Google Scholar). Furthermore, the levels of mitotic DNA synthesis (MiDAS) at telomeres are elevated in ALT+ cells (Min et al., 2017Min J. Wright W.E. Shay J.W. Alternative lengthening of telomeres mediated by mitotic DNA synthesis engages break-induced replication processes.Mol. Cell. Biol. 2017; 37 (e00226-17)Crossref PubMed Scopus (115) Google Scholar). Telomeric MiDAS is dependent on RAD52, which is also implicated in BIR (Bhowmick et al., 2016Bhowmick R. Minocherhomji S. Hickson I.D. RAD52 facilitates mitotic DNA synthesis following replication stress.Mol. Cell. 2016; 64: 1117-1126Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, Min et al., 2017Min J. Wright W.E. Shay J.W. Alternative lengthening of telomeres mediated by mitotic DNA synthesis engages break-induced replication processes.Mol. Cell. Biol. 2017; 37 (e00226-17)Crossref PubMed Scopus (115) Google Scholar, Özer et al., 2018Özer Ö. Bhowmick R. Liu Y. Hickson I.D. Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism.Oncotarget. 2018; 9: 15836-15846Crossref PubMed Scopus (48) Google Scholar, Sotiriou et al., 2016Sotiriou S.K. Kamileri I. Lugli N. Evangelou K. Da-Ré C. Huber F. Padayachy L. Tardy S. Nicati N.L. Barriot S. et al.Mammalian RAD52 functions in break-induced replication repair of collapsed DNA replication forks.Mol. Cell. 2016; 64: 1127-1134Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Despite the similarities between ALT and BIR, some BIR factors are not required for telomere DNA synthesis in ALT+ cells when analyzed with different assays (Özer et al., 2018Özer Ö. Bhowmick R. Liu Y. Hickson I.D. Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism.Oncotarget. 2018; 9: 15836-15846Crossref PubMed Scopus (48) Google Scholar, Sobinoff et al., 2017Sobinoff A.P. Allen J.A. Neumann A.A. Yang S.F. Walsh M.E. Henson J.D. Reddel R.R. Pickett H.A. BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.EMBO J. 2017; 36: 2907-2919Crossref PubMed Scopus (84) Google Scholar). Together, these findings suggest that ALT is likely triggered by replication-associated telomere breaks and occur through a BIR-like process. Although ALT is linked to BIR, it is still poorly understood as a pathway. Importantly, naturally occurring DNA synthesis at ALT telomeres is temporally and spatially regulated during the cell cycle. Telomeric DNA synthesis was observed in G2 and mitotic ALT+ cells, presenting opportunities to detect ALT activity (Dilley et al., 2016Dilley R.L. Verma P. Cho N.W. Winters H.D. Wondisford A.R. Greenberg R.A. Break-induced telomere synthesis underlies alternative telomere maintenance.Nature. 2016; 539: 54-58Crossref PubMed Scopus (236) Google Scholar, Min et al., 2017Min J. Wright W.E. Shay J.W. Alternative lengthening of telomeres mediated by mitotic DNA synthesis engages break-induced replication processes.Mol. Cell. Biol. 2017; 37 (e00226-17)Crossref PubMed Scopus (115) Google Scholar, Nabetani et al., 2004Nabetani A. Yokoyama O. Ishikawa F. Localization of hRad9, hHus1, hRad1, and hRad17 and caffeine-sensitive DNA replication at the alternative lengthening of telomeres-associated promyelocytic leukemia body.J. Biol. Chem. 2004; 279: 25849-25857Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Özer et al., 2018Özer Ö. Bhowmick R. Liu Y. Hickson I.D. Human cancer cells utilize mitotic DNA synthesis to resist replication stress at telomeres regardless of their telomere maintenance mechanism.Oncotarget. 2018; 9: 15836-15846Crossref PubMed Scopus (48) Google Scholar, Wu et al., 2000Wu G. Lee W.H. Chen P.L. NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres.J. Biol. Chem. 2000; 275: 30618-30622Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Assays that directly visualize the ALT DNA synthesis at unperturbed telomeres are critical for delineating the mechanisms of ALT and the cellular circuitries regulating this process. In this study, we developed an assay to visualize telomere DNA synthesis in APBs formed in G2 human cells. The DNA synthesis detected by this assay is specific to ALT+ cells and regulated by known ALT factors. Using this assay, we found that ALT DNA synthesis occurs exclusively in APBs and is dependent on PML. Furthermore, RAD52 but not RAD51 is required for efficient ALT DNA synthesis. RAD52 is not only required for maintaining telomeres in ALT+ cells but also sufficient to promote telomeric D-loop formation in vitro, explaining its role in BIR at ALT telomeres. Surprisingly, however, RAD52 is not required for C-circle formation, suggesting the presence of a RAD52-independent ALT pathway. In RAD52-knockout cells, telomeres were progressively shortened, but C-circle formation and residual ALT DNA synthesis were gradually increased. These RAD52-independent ALT activities are dependent on BLM and POLD3/4, suggesting that the second ALT pathway is also mediated by BIR. Together, these results suggest that ALT is mediated by two BIR-related pathways, one RAD52 dependent and the other RAD52 independent. These findings establish a bifurcated framework of the ALT pathway, setting the stage for future investigations to dissect the molecular events in this process. To understand the process of ALT telomere DNA synthesis, we sought to visualize this process at its natural cellular location and in a cell-cycle window when it normally occurs. In particular, we sought to develop an assay to specifically monitor telomere DNA synthesis in APBs. Because in G2 cells DNA replication is largely completed throughout the genome, and APBs are readily detectable, we synchronized cells in G2 with thymidine and CDK1 inhibitor or with CDK1 inhibitor alone (Figure 1A). Using synchronized G2 cells, we performed 5-ethynyl-2′-deoxyuridine (EdU) labeling, telomere fluorescence in situ hybridization (FISH), and PML immunostaining. In ALT+ U2OS cells, a fraction of telomeres colocalized with PML, confirming the presence of APBs (Figures 1B and S1A). In addition, a fraction of telomeres colocalized with EdU foci, showing the DNA synthesis at ALT telomeres (Figures 1B and S1A). Notably, virtually all the EdU foci in G2 U2OS cells colocalized with both telomeres and PML, suggesting that the DNA synthesis in these cells occurred primarily in APBs (Figures 1B and S1A). Similar observations were made in two other ALT+ cell lines, SAOS2 and SKLU (Figure S1B). In contrast to that in ALT+ cells, although PML bodies were detected in ALT− HeLa 1.3 cells synchronized in G2, they rarely colocalized with telomeres (Figures 1B and S1A). In G2 HeLa cells, EdU foci were sparse, and they very rarely colocalized with telomeres and PML (Figures 1B and S1A). Thus, by monitoring telomeres, PML and DNA synthesis simultaneously in G2 cells, we have established an assay to visualize ALT telomere DNA synthesis in APBs. We termed this assay ATSA (ALT telomere DNA synthesis in APBs). The ATSA assay allowed us to examine the association of APBs with ALT telomere DNA synthesis. In U2OS cells, although most of the EdU+ telomeres were in APBs, a significant fraction of APBs did not display detectable EdU signals (Figure 1C), suggesting that telomere DNA synthesis is either not initiated or insufficient in some APBs. Interestingly, EdU+ APBs displayed more telomere FISH signals than did EdU− APBs (Figure S1C). Furthermore, in EdU+ APBs, the signals of telomere FISH, PML, and EdU all positively correlated with one another (Figure S1D). These results suggest that the extents of telomere clustering in APBs are likely linked to the levels of ALT telomere DNA synthesis. Because EdU+ telomeres were almost exclusively detected in APBs (Figure 1C), we asked whether the lack of EdU+ telomeres outside of APBs is due to the low abundance of telomeric DNA at non-APB telomere foci. We selected a group of telomere foci of similar sizes in or out of APBs and compared their EdU signals (Figure S1E). Even in this size-normalized population of telomere foci, EdU signals were still exclusively detected in APBs, suggesting that telomeres outside of APBs do not synthesize DNA efficiently. The strong association of EdU+ telomeres with APBs indicates that APBs may be involved in ALT activity. To test this possibility, we used two independent small interfering RNAs (siRNAs) to knock down PML, a key structural component of APBs (Figure S1F). Depletion of PML eliminated APBs and drastically reduced EdU+ telomeres (Figures 1D and 1E), showing that APBs are indeed functionally important for ALT telomere synthesis. To further validate the ATSA assay, we used it to test several known regulators of ALT. The BLM helicase is implicated in APB formation and ALT DNA synthesis (O’Sullivan et al., 2014O’Sullivan R.J. Arnoult N. Lackner D.H. Oganesian L. Haggblom C. Corpet A. Almouzni G. Karlseder J. Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1.Nat. Struct. Mol. Biol. 2014; 21: 167-174Crossref PubMed Scopus (167) Google Scholar, Sobinoff et al., 2017Sobinoff A.P. Allen J.A. Neumann A.A. Yang S.F. Walsh M.E. Henson J.D. Reddel R.R. Pickett H.A. BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.EMBO J. 2017; 36: 2907-2919Crossref PubMed Scopus (84) Google Scholar). In U2OS cells, depletion of BLM with two independent siRNAs drastically reduced APBs and DNA synthesis at telomeres in G2 (Figures 1F, S1G, and S1H). The BIR proteins POLD3 and PODL4 are also involved in ALT (Roumelioti et al., 2016Roumelioti F.M. Sotiriou S.K. Katsini V. Chiourea M. Halazonetis T.D. Gagos S. Alternative lengthening of human telomeres is a conservative DNA replication process with features of break-induced replication.EMBO Rep. 2016; 17: 1731-1737Crossref PubMed Scopus (96) Google Scholar, Sobinoff et al., 2017Sobinoff A.P. Allen J.A. Neumann A.A. Yang S.F. Walsh M.E. Henson J.D. Reddel R.R. Pickett H.A. BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.EMBO J. 2017; 36: 2907-2919Crossref PubMed Scopus (84) Google Scholar). Knockdown of POLD3 and PODL4 in U2OS cells significantly reduced EdU+ APBs but only modestly decreased total APB levels (Figures 1G, S1I, and S1J), supporting the idea that POLD3/4 are important for ALT telomere synthesis in APBs. Thus, the ATSA assay is not only capable of confirming the known genetic requirements of ALT but also revealing previously unknown regulatory mechanisms. Both RAD51 and RAD52 are among the DNA recombination proteins detected in APBs (Yeager et al., 1999Yeager T.R. Neumann A.A. Englezou A. Huschtscha L.I. Noble J.R. Reddel R.R. Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res. 1999; 59: 4175-4179PubMed Google Scholar), but their functions in APBs have not been tested directly. To assess the functions of RAD51 and RAD52 in APBs, we first examined their localization in G2 U2OS cells. RAD52 but not RAD51 was detected in the majority of APBs and EdU+ telomeres (Figures 2A, S2A, and S2B). To test whether RAD51 and RAD52 are functionally required for the DNA synthesis in APBs, we analyzed RAD51 and RAD52 knockdown U2OS cells with the ATSA assay (Figures 2B and S2C). The numbers of EdU+ APBs, but not total APBs, were significantly reduced by knockdown of RAD52 (Figures 2B–2D). In contrast, RAD51 knockdown did not affect EdU+ APBs or total APBs (Figures 2B–2D). To confirm these results, we generated independent RAD52-knockout (KO) cell lines using CRISPR/Cas9 (Figure S2D). These RAD52-KO cell lines did not show significant cell-cycle alterations before extensive passaging (Figure S2E). Similar to that in RAD52 knockdown cells, the numbers of EdU+ APBs, but not total APBs, were significantly reduced in RAD52-KO cells (Figures 2E, 2F, and S2F). Consistently, RAD52 KO reduced the fractions of APBs that were EdU+ (Figure 2G), suggesting that RAD52 contributes to ALT telomere synthesis in APBs. Together, these results suggest that RAD52 but not RAD51 is important for ALT activity in APBs. To further verify the effects of RAD52 KO on ALT telomere synthesis, we generated RAD52-KO cell lines that inducibly express wild-type RAD52 (Figure S2G). Induction of RAD52 in RAD52-KO cells significantly increased EdU+ APBs (Figures 2H and S2H), confirming that RAD52 is critical for ALT telomere synthesis in APBs. The role of RAD52 in the DNA synthesis in APBs prompted us to investigate whether RAD52 is required for telomere maintenance in ALT+ cells. Using a qPCR-based method (Lau et al., 2013Lau L.M. Dagg R.A. Henson J.D. Au A.Y. Royds J.A. Reddel R.R. Detection of alternative lengthening of telomeres by telomere quantitative PCR.Nucleic Acids Res. 2013; 41: e34Crossref PubMed Scopus (56) Google Scholar), we measured the overall levels of telomeric DNA in wild-type (WT) U2OS cells and cells of multiple RAD52-KO clones (Figure 3A). The levels of telomeric DNA were reduced in newly generated RAD52-KO cells compared with WT cells (Figure 3A). After 3 months of passaging, RAD52-KO cells displayed a further reduction in telomeric DNA (Figure 3A), suggesting that telomeres were progressively shortened in the absence of RAD52. The levels of telomeric DNA were even lower in a RAD52-KO clone (KO #1) that had been passaged for >6 months (Figure 3A). Nonetheless, because of the lack of early passages of KO #1, we do not know whether telomeres were shortened at the same rate as in the other clones. In contrast to that in U2OS cells, KO of RAD52 in HeLa 1.3 cells did not result in a reduction in telomeric DNA relative to WT cells even after 3 months of passaging (Figure S3A). To directly test whether telomeres were shortened in ALT+ cells lacking RAD52, we used the telomere restriction fragment (TRF) assay to analyze cells of multiple RAD52-KO clones that had been passaged for different lengths of time (Figure 3B). Compared with WT U2OS cells, RAD52-KO cells displayed clear reductions in both the amount and length of telomeric DNA (Figure 3B). The reduction of telomeric DNA in RAD52-KO cells correlated with the length of passage time, but telomeres were eventually stabilized after extensive passaging (Figure 3B). Consistent with the TRF data, telomere FISH signals were significantly reduced at individual telomeres in RAD52-KO cells compared with WT cells (Figure 3C). The RAD52-KO cell line that has been extensively passaged also showed r" @default.
• W2913360660 created "2019-02-21" @default.
• W2913360660 creator A5012648780 @default.
• W2913360660 creator A5026961383 @default.
• W2913360660 creator A5084386513 @default.
• W2913360660 creator A5087762808 @default.
• W2913360660 creator A5089425646 @default.
• W2913360660 date "2019-01-01" @default.
• W2913360660 modified "2023-10-17" @default.
• W2913360660 title "Alternative Lengthening of Telomeres through Two Distinct Break-Induced Replication Pathways" @default.
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• W2913360660 doi "https://doi.org/10.1016/j.celrep.2018.12.102" @default.
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