article: Telomerase-independent ALT (alternative lengthening of telomeres) cells are characterized by high frequency of telomeric homologous recombination (HR), C-rich extrachromosomal circles (C-circles) and C-rich terminal 5' overhangs (C-overhangs). However, underlying mechanism is poorly understood. Here, we show that both C-circle and C-overhang form when replication fork collapse is induced by strand break at telomeres. We find that endogenous DNA break predominantly occur on C-rich strand of telomeres in ALT cells, resulting in high frequency of replication fork collapse. While collapsed forks could be rescued by replication fork regression leading to telomeric homologous recombination, those unresolved are converted to C-circles and C-overhang at lagging and leading synthesized strand, respectively. Meanwhile, multiple hallmarks of ALT are provoked, suggesting that strand break-induced replication stress underlies ALT. These findings provide a molecular basis underlying telomeric HR and biogenesis of C-circle and C-overhang, thus implicating the specific mechanism to resolve strand break-induced replication defect at telomeres in ALT cells.
  Linear chromosome ends are capped by telomeres, which are composed of TTAGGG/CCCTAA tandem DNA repeats and a protein complex called shelterin [1–3]. Because of end replication problem [4] and possible DNA resection by Exo I (Exonuclease I) and Apollo to form single-stranded overhang [5,6], telomeres shorten with every cell division until the critically short telomere length is reached that induces cell senescence or apoptosis [7–9]. To counteract telomere shortening, approximately 85% of human cancer cells express telomerase, while those that don’t express telomerase induce alternative lengthening of telomeres (ALT) pathway [10–12]. As a typical fragile site, telomere of ALT cells experiences a high frequency of homologous recombination (HR), which may contribute to lengthening of telomeres [13]. ALT cells are characterized by high heterogeneity of telomere length [10], an elevated frequency of telomere-sister chromatid exchanges (T-SCEs) [13,14], the presence of APBs (ALT-associated promyelocytic leukemia nuclear bodies) [15] and abundant extrachromosomal circular telomeric DNA (C-circles) and C-rich terminal 5' overhangs (C-overhangs) [10,16–18]. The biogenesis of C-circles and C-overhangs is not clear and their functions in cells are largely unknown. It has been proposed that telomeric DNA damage, particularly double-stranded breaks (DSBs), promotes C-circles generation in ALT cells [19,20], and that defects in telomere replication related proteins, such as SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent, regulator of chromatin subfamily A-like 1) or the CST (CTC1/STN1/TEN1), changes the level of C-circles in ALT cells [21–23]. These results imply a potential connection between C-circles formation and DNA damage repair and/or replication defect at telomeres. Regarding to C-overhang, it appears that telomeric DNA damage is not sufficient to induce 5' C-overhangs, rather, the production of C-overhangs is associated with rapid cleavage of telomeres [24]. The question regarding whether and how the formation of C-circle and C-overhang is coordinated and their relationship with high frequency of telomeric HR and ALT remains to be elucidated. The tandemly repeated G-rich DNA in human telomeres has a relatively high tendency to form highly compacted G-quadruplex [25]. In addition, telomeric DNA is susceptible to ultraviolet light-induced [23] and oxidative DNA damage, leading to a relatively high frequency of single- and double-stranded DNA breaks (SSBs and DSBs) and other DNA lesions in telomeric DNA [26–28]. G-quadruplex and DNA lesions frequently block replication fork progression [29–32]. In ALT cells, telomeres may experience particularly high frequency of telomeric DNA damage [17,33,34], leading to replication fork stalling and/or collapse. In addition, it has also been reported that ALT is linked to mutations in the ATRX/DAXX chromatin remodeling complex and histone variant H3. 3, which interfere with nucleosome assembly at telomeres and likely increase replication stress [33,35,36]. With such replication stress, it has been interpreted that ALT cells are competent to replication defect at telomeres. Alternatively, ALT cells may develop a mechanism to cope with unsuccessfully replicated telomeres and to maintain the integrity of chromosome ends. This study provides evidences that C-circles and C-overhangs are produced during replication of lagging and leading strand of telomeres, respectively, and that their production is associated with DNA break-induced replication fork collapse in ALT cells. Replication fork regression, which facilitates HR-dependent replication fork restart, is utilized to rescue collapsed replication fork. However, unsuccessful rescue results in the formation of C-circles and C-overhangs. Meanwhile, multiple hallmarks of ALT were raised by DNA break-induced replication fork collapse, including increased frequency of telomeric HR, formation of ALT associated PML body as well as high abundance of C-circles and C-overhangs.
  C-circle is an extrachromosomal circular telomeric DNA composed of full C-rich strand and notched G-rich complementary strand that is a quantitative biomarker of the ALT mechanism [10,37]. Here, the cell cycle dependence of the appearance of C-circles was explored in ALT-positive U2OS cells. Specifically, U2OS cells were synchronized at G1/S by double-thymidine block, released for 0,3, 6,9 or 12h, corresponding to G1/S, early S, middle S, late S/G2 and G1, respectively (Fig 1A), and then assayed for the presence of C-circles. Φ29 DNA polymerase-based C-circle assay was used to determine the abundance of C-circles in cells. Reliability of method was validated by experiments in which lack of Φ29 leads to no amplified product and C-circle signal is well proportional to the amount of input DNA (R2 = 0. 96 in linear regression of standard curve) (S1A and S1B Fig) [37]. The results showed that the abundance of C-circles increased gradually during S phase, peaked (doubled) at late S/G2 (9h after release) and decreased when cells re-entered G1 (Fig 1B). Since telomeric DNA replicates throughout S phase [38,39], this result suggests that C-circles may be produced during telomere replication and subsequently degraded during or after G2 [40]. It has been reported that RPA2 (replication protein A2) colocalizes with telomeric DNA in human ALT cells [40,41]. We observed that the abundance of telomeric RPA2 foci also gradually increased during S phase and decreased during or after G2 (Fig 1C). Given that RPA2 is a sensor of single-stranded DNA that might be produced during DNA damage repair and/or replication process, the specific correlation between appearance of C-circles and telomeric RPA2 foci implies that C-circle formation may associated with DNA damage response (DDR) and/or DNA replication at telomeres. The mechanism by which C-circles form was further explored by BrdU pulse-labeling synchronized U2OS cells for 12 h after release from G1/S [42], isolating nascent C-circle and analizing its composition by CsCl density gradient ultracentrifugation (Fig 1D). The results showed that while the leading and lagging telomeric DNA was synthesized with similar efficiency (0. 96 vs 1. 11 in amount) (Fig 1E), BrdU-labeled C-circles were dominently enriched in lagging strand telomeric DNA (0. 88 vs 1. 64 for leading vs lagging synthesized C-circles after normalizing with total amount of leading or lagging telomeres) (Fig 1F), in which C-rich strand is newly synthesized and therefore BrdU-labeled. This result is consistent with the previous observation [43], the mechanism underlying the production of C-cirlce from lagging strand would be exlored below. A similar procedure was used to determine whether 5' C-overhangs arise preferentially during leading or lagging strand DNA replication. 12h or 6h BrdU-labeled DNA was fractionated by CsCl gradient ultracentrifugation, fractions corresponding to leading, lagging and unreplicated telomeric DNA were collected, and divided into two parts, one of which was incubated with RecJf, a 5' →3' exonuclease for ssDNA, to specifically degrade 5' overhang DNA to validate telomeric C-rich ssDNA polarity [18]. The resulting samples were analyzed by neutral-neutral 2D agarose gel electrophoresis in which DNA fragments are resolved first by size in one dimension, and then by conformation in second dimension. In combination with in-gel hybridization under native or denatured conditions, 2D agarose gel electrophoresis is able to separate and distinguish linear ssDNA (G-rich or C-rich), linear dsDNA (with or without single-stranded G/C-rich overhangs) and open circular DNA (Fig 1G). C-overhangs were sensitive to RecJf digestion, but resistant to Exo I (a 3' →5' exonuclease for ssDNA), demonstrating that C-overhang is in the 5' to 3' direction (Fig 1H, S1C Fig) [18]. Both 12h and 6h BrdU labeling experiments showed that RecJf sensitive 5' C-overhangs are preferentially generated on telomeres replicated by leading strand (leading: lagging = 1. 00: 0. 36 for 12h labeling sample and 1. 00: 0. 29 for 6h labeling sample) (Fig 1H, S1D Fig). To examine a potential relationship between replication-blocking and formation of C-circle and C-overhang, U2OS cells were treated with agents that result in replication fork stalling. To this end, exponentially growing U2OS cells were treated with HU (hydroxyurea) that blocks replication fork by inducing dNTP pool deficiency or aphidicolin that inhibits B-family DNA polymerase leading to replication fork stalling [44,45]. Interestingly, both treatments resulted in no increase of RPA2 foci or DNA damage foci (p53-binding protein 1,53BP1 foci) on telomeres (termed as TIFs: telomere dysfunction induced foci) (S2A and S2B Fig). In addition, the number of C-circles slightly decreased (Fig 2A) and the abundance of C-overhangs and G-overhangs was not significantly changed when U2OS cells were treated with HU or aphidicolin (Fig 2B, S3A Fig). These results suggested that replication fork stalling per se is not sufficient to stimulate the formation of C-circles and C-overhangs. When U2OS cells were exposed to zeocin, a radio-mimetic chemical that induces oxidative DNA damage including ssDNA and dsDNA breaks [46], increased level of DDR at telomeres was detected, as expected (S2B Fig). Meanwhile, we observed increased abundance of C-circles and C-overhangs (Fig 2C and 2D). In contrast, zeocin treatment led to slight decrease of G-overhangs (S3B Fig). Importantly, we also found that the increase of C-circle and C-overhang upon zeocin treatment was restricted to S-phase (when cells were treated during S-phase), and was abrogated when cells were synchronized at G1 and exposured to zeocin (S3C–S3F Fig). Altogether, these results suggested that C-circles and C-overhangs are produced in ALT cells during telomere replication encountering DNA damages. To imitate the situation in which replication fork progress is blocked by DNA damage, U2OS cells were treated with CPT (camptothecin), a specific inhibitor of Topo I (topoisomerase I) that induces protein-linked ssDNA break at the front of replication fork, leading to fork collapse [47,48]. Strikingly, CPT strongly stimulated the formation of C-circles and C-overhangs in U2OS cells (Fig 2E and 2F) and the abundance of G-overhangs decreased accordingly (S3B Fig). Increased C-circle and C-overhang by zeocin or CPT treatment was also observed in other ALT VA13 cells (S3G and S3H Fig). In a similar experiment, U2OS cells were treated with inhibitors of Topo II (topoisomerase II), VP-16 (etoposide) or ICRF-187 (dexrazoxane) [49–51]. VP-16 induces protein-linked dsDNA breaks in replicating DNA leading to replication fork collapse, while ICRF-187 inhibits the cleavage activity of Topo II leading to replication fork stalling [52]. Interestingly, VP-16 stimulated formation of C-circles and C-overhangs, whereas the abundance of G-overhangs decreased. However, ICRF-187 treatment showed a limited effect on C-circle, C-overhang and G-overhangs (S4A–S4C Fig). This result further demonstrated that DNA damage-induced replication fork collapse rather than replication fork stalling promotes formation of C-circles and C-overhangs. This conclusion was further confirmed by duplicating the experiments in other ALT positive VA13 cells (S4D–S4F Fig). Given a high abundance of C-circles and C-overhangs in ALT cells, we then asked whether intrinsic DNA strand breaks exist in telomeres that induce replication fork collapse. For this purpose, genomic DNA was digested with HinfI and RsaI, followed by digestion with Exo III (Exonuclease III), a 3' to 5' exonuclease that remove nucleotides from blunt end or break/gap in double stranded DNA to generate single stranded DNA (Fig 3A). The digested DNA were hybridized with C-rich or G-rich probe under native or denatured condition [53] (Fig 3A). The rationale was to determine whether endogenous ssDNA breaks and gaps occur in the G-rich or C-rich strand of telomeric DNA in ALT cells. If such lesions were enriched in the C-rich strand, it would be preferentially degraded by Exo III, leaving primarily G-rich ssDNA to hybridize with a C-rich probe, while the reverse specificity, or lack of specificity would be observed in the absence of preferential endogenous ssDNA breaks in the C-rich strand of the telomere (Fig 3A). We detected much more G-rich ssDNA than C-rich ssDNA (Fig 3B). This suggests that endogenous ssDNA breaks and gaps are present predominantly on the C-rich strand of telomeric DNA in U2OS cells. MMS (Methyl-methanesulfonate) is a DNA damaging agent that preferentially creates mutagenic lesions in cytosine of ssDNA [54,55]. Here, we showed that exposure to MMS significantly stimulates formation of C-circles and C-overhangs in ALT cells (Fig 3C and 3D). To further confirm that ssDNA breaks in the C-rich strand of telomeric DNA induce formation of C-circle and C-overhang, we expressed CRISPR-Cas9 with a D10A mutation in the RuvC nuclease domain of Cas9 (Cas9-D10A), which specifically generates ssDNA breaks in the strand complementary to sgRNA (Fig 3E) [56,57]. Indeed, when sgRNA with telomeric G-rich sequence (sgTel) was co-expressed with Cas9-D10A in U2OS cells, we observed significant number of C-rich, but not G-rich DNA fragments (smear on gel) that are released from telomeres and detected by alkaline constant-field gel electrophoresis (alkaline plug assay, see Method for detail) (S5A and S5B Fig), indicating specific induction of DNA breaks by Cas9-D10A at C-rich strands. As expected, expression of wild-type Cas9 (wtCas9, WT) induced double-stranded breaks at telomeres [58], leading to increase of both G- and C-rich fragments (S5A and S5B Fig). However, expression of nuclease-deficient mutant Cas9 (dCas9) led to no increase of G- or C-rich fragments (S5A and S5B Fig). We observed significant increase of C-circles and C-overhangs in cells expressing wtCas9 or Cas9-D10A but not in cells expressing dCas9 (S5C and S5D Fig). Similar experiments were also performed in non-ALT human HEK 293T cells. Western blot analysis demonstrated that wtCas9, dCas9 and Cas9-D10A protein were expressed at a similar level (Fig 3F). The results showed that expression of both wtCas9 and Cas9-D10A stimulated formation of C-circles (Fig 3G). The formation of C-overhangs was strongly stimulated by expression of Cas9-D10A (Fig 3H). And the expression of wtCas9 also slightly increased the abundance of C-overhangs, consistent with our previous finding that telomeric DSB initiates homologous recombination mediated repair that produces 3′ C-rich overhang [58]. Collectively, these results suggest that endogenous breaks/gaps or extraneously induced ssDNA breaks in C-rich strand of telomeric DNA stimulates formation of C-circles and C-overhangs. To explore how ALT cells respond to replication fork collapse, CPT-treated U2OS cells were analyzed using IF-FISH (immunofluorescence and fluorescence in situ hybridization) to determine the proteins enriched at telomeres in response to replication fork collapse induced by CPT treatment. Strikingly, the abundance of PML foci and ALT associated PML bodies (APBs) increased in cells exposed to CPT (Fig 4A and 4B). In addition, we observed a significant increase of 53BP1 foci genome-wide and at telomeres when cells were treated with CPT (Fig 4C and 4D). Moreover, RPA2 foci at telomeres increased in CPT treated cells (Fig 4E and 4F). We also found that SMARCAL1, Rad51 and SLX4 were recruited to telomeres in U2OS cells and their foci at telomeres were significantly increased when cells were challenged with CPT (Fig 4G–4L). Importantly, RPA2/Rad51/SMARCAL1/SLX4 compose a machinery termed replication fork regression [21,31,59–61], a mechanism that rescues collapsed replication fork. Evidences presented above suggest that replication fork collapse in telomeric DNA is tightly linked to formation of C-circle and C-overhang structures. Therefore, it was predicted that replication fork regression, mediated by RPA2-Rad51-SMARCAL1-SLX4 axis, might rescue collapsed replication fork and thus suppress formation of C-circles and C-overhangs. To test this, U2OS cells were treated with RPA2- or SMARCAL1-targeted siRNA and the abundance of C-circles and C-overhangs was examined (Fig 5A and 5D). Indeed, C-circles and C-overhangs were more abundant in RPA2 or SMARCAL1-deficient U2OS cells (Fig 5B, 5C, 5E and 5F). Accordingly, G-overhangs were slightly decreased (S6A Fig). Moreover, when Rad51 was inhibited by B02, a specific inhibitor of Rad51 [62], the abundance of C-circles and C-overhangs also increased, while G-overhangs decreased (Fig 5G and 5H, S6B Fig). These results suggested that replication fork regression prevents the formation of C-circles and C-overhangs in U2OS cells. The same experiments were also repeated in VA13 cells. Consistently, we observed that both depletion of RPA2 (or SMARCAL1) and inhibition of Rad51 by B02 stimulated formation of C-circles and C-overhangs, but reduced the abundance of G-overhangs (S6C–S6I Fig). Meanwhile, we observed that pDNA-PKcs (DNA-dependent protein kinase, catalytic subunit), a key sensor in the NHEJ (non-homologous end-joining) pathway, localizes to telomeres in U2OS cells, and that telomeric pDNA-PKcs (S2056) foci are more abundant in CPT-treated cells (S7A and S7B Fig). To explore whether NHEJ plays a role in production of C-circles or C-overhangs, U2OS cells were treated with DNA-PKcs inhibitor NU7441. Previous report demonstrated that inhibition of ATR by VE-821 leads to decrease of C-circles [23]. Our results showed that NU7441 treatment decreased the level of C-circles (S7C Fig), whereas the abundance of C-overhangs and G-overhangs remained largely unchanged (S7D and S7E Fig). These findings implied that NHEJ machinery promotes circularization of lagging strand DNA at collapsed telomeric replication fork, thus enabling formation of C-circles. Previous studies suggest that replication fork regression is coupled with HR to reinitiate replication [19,31,61]. Consistent with this, we observed that telomere sister chromatid exchange (T-SCE), which indicates HR occurring at telomeres, was inhibited (i. e. , reduced frequency) in SMARCAL1-knockdown U2OS cells (Fig 6B and 6C). In addition, when replication fork collapse was induced by CPT treatment in U2OS cells, we observed increased frequency of T-SCE (Fig 6D and 6E). These results supported the hypothesis that replication fork collapse at telomeres, which is rescued by replication fork regression-mediated process, leads to telomeric recombination. Rad51 plays a key role in both replication fork regression and telomeric recombination [19,31]. When U2OS cells were treated with B02, a specific inhibitor of Rad51 [62], telomeric PCNA and RPA2 foci increased (S8A–S8D Fig), likely due to the accumulation of collapsed replication fork [50]. Consistently, less fully synthesized telomeric DNA were detected (S8E Fig). After treatment with B02 for 4 days, short telomeres were accumulated in U2OS cells (S8F Fig).
  The results show that replication fork collapse leading to C-circles and C-overhangs can be induced by exogenous agents that generate ssDNA or dsDNA breaks in telomeric DNA (zeocin, CPT, VP16, MMS and CRISPR-Cas9 system). In addition, we found that endogenous lesions occur preferentially in the C-rich strand of telomeres in ALT cells (Fig 3B). During replication progression, the C-rich strand templates leading strand DNA synthesis, while the G-rich strand templates lagging strand DNA synthesis [63]. Replication fork collapse, induced by a break or gap on the C-rich strand, has different consequences for leading and lagging replication (Fig 7). For leading synthesis, long single-stranded C-rich DNA remains unreplicated, forming C-overhangs at the end of the chromosome; lagging strand synthesis still proceeds, but would likely lead to “looping-out” during which the stalled replication fork is cut out and cyclized to form C-circles [50]. The evidence supporting this model include: 1) C-circles are primarily derived from the lagging strand, whereas C-overhangs are primarily derived from the leading strand (Fig 1); 2) replication fork collapse, but not conventional replication fork stalling, stimulates production of C-circles and C-overhangs (Fig 2); 3) endogenous breaks/gaps are present in the C-rich telomeric strand and agents that introduce ssDNA breaks in the C-rich telomeric strand (i. e. , MMS treatment and CRISPR-Cas9 (D10A) ) stimulate formation of C-circles and C-overhangs (Fig 3); 4) the formation of C-circles is NHEJ-dependent (S7 Fig). Spontaneous telomeric DNA damage response and telomeric RPA2 foci are often observed in ALT cells [40,64]. We found that RPA2 accumulate in telomeric DNA of CPT-treated cells (Fig 4E and 4F) but not in HU- or aphidicolin-treated cells (S2A Fig), suggesting that replication fork collapse, but not replication fork stalling, is the signal that recruits RPA2. Binding of RPA2 to ssDNA activates the ATR pathway, leading to replication fork protection and restoration [23,65]. DNA damage-induced replication fork collapse can be rescued by replication fork regression [48]. And, SMARCAL1, which is activated by ATR-dependent phosphorylation, plays an essential role in replication fork regression [66]. Indeed, it has been previously reported that the deficiency of SMARCAL1 leads to fragile telomeres [60]. Our results also demonstrated that components of fork regression machinery including Rad51, SMARCAL1 and SLX4 are recruited to telomeres in CPT-treated cells (Fig 4E–4L). This, however, does not exclude the possibility that the mechinary other than fork regression might also be adopted to rescue collapsed replication fork. In fact, it has been reported that translesion synthesis, which is composed of FANCJ, RAD18, ubPCNA and Polη, is engaged in bypassing DNA lesions on replication fork [67] [68]. Interestingly, depletion or inhibition of each component of fork regression machinery stimulated formation of C-circles and C-overhangs (Fig 5). We thus proposed that endogenous breaks/gaps in C-rich strand of telomeric DNA in ALT cells either induce replication fork collapse, leading to C-circle and C-overhang structures, or, fork regression or other fork rescue machinary restores the collapsed replication fork, and C-circle and C-overhang formation is suppressed (Fig 7). In supporting this model, it has been recently discovered that SMARCAL1 loss-of-function mutations in cancers link to the ALT mechanism of telomere maintenance, resulting in ultrabright telomeric foci and the generation of C-circles [69]. In additon, deficiency of Polη, which is essential for translesion synthesis, increases replication stress at telomeres and stimulates the formation of C-circles [68]. ALT cells are characterized by high frequency telomeric recombination [13]. It has been found that a DNA damage at ALT telomeres triggers long-range movement and clustering, resulting in homology-directed telomere synthesis between sister and non-sister chromatids [19,70]. Here, we demonstrated that DNA strand break in the C-rich strand of the telomere leads to replication fork collapse, followed by replication fork regression and telomeric HR (Fig 6). Whether or not this recombination contributes to telomere elongation remains to be elucidated. However, homologous searching and recombination could occur anywhere along the telomere due to its repetitive nature, creating the possibility for telomere extension (Fig 7). In the presence of Rad51 inhibitor B02, short telomeres accumulate (S8F Fig), supporting the idea that Rad51-dependent HR promotes telomere extension. Furthermore, it is worth noting that APBs accumulate in CPT-treated ALT cells (Fig 4). Therefore, replication fork collapse provokes multiple hallmarks of ALT, including telomeric HR, APBs, C-circles and C-overhangs. While C-circles and C-overhangs are associated with telomere trimming, telomeric HR might contribute to telomere elongation. Additional studies are needed to understand how these events are coordinated in order to maintain chromosome end integrity and telomere length homeostasis in ALT cells.
  U2OS, HEK 293T and VA13 cells were grown in Dulbecco’s modified Eagles’ medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin at 37°C in 5% CO2. Unless otherwise indicated, cell lines were treated with HU (2mM, Sigma) or aphidicolin (1μg/mL, Sigma) or zeocin (100μg/mL, Thermo Fisher) or CPT (0. 25μM, MCE) or MMS (0. 25mM, Sigma) or VP-16 (10μM, Sigma) or ICRF-187 (50μg/mL, Selleck) or B02 (27. 4μM, EMD Millipore) or VE821 (10μM, MCE) or NU7441 (250nM, Selleck). Knockdown experiments were performed with the Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) using following siRNA targets: siNC/UUCUCCGAACGUGUCACGUdTdT/ACGUGACACGUUCGGAGAAdTdT; siRPA2-1/GGCUCCAACCAACAUUGUUdTdT/AACAAUGUUGGUUGGAGCCdTdT; siRPA2-2/GCCUGGUAGCCUUUAAGAUdTdT/AUCUUAAAGGCUACCAGGCdTdT; siSMARCAL1-1/CCAAGAGACACCAGCUCAUdTdT/ AUGAGCUGGUGUCUCUUGG dTdT; siSMARCAL1-2/UUGCUAAGAAGGUCAAAGCdTdT/ GCUUUGACCUUCUUAGCAAdTdT. Cells were collected 60h after transfection for experiments. Lenti-CRISPRv2 (Addgene plasmid # 52961) was used in this study [71]. Scaffold sequence of sgRNA was modified to 5' -NNNNNNNNNNNNNNNNNNGUUUAAGAGCUAUGCUGGAAACAGCAUA GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3′, as previously described [58,72]. sgSCR (5′-TGCTCCGTGCATCTGGCATC-3' ), sgTel (5' -GTTAGGGTTAGGG TTAGGGTTA-3' ) [58] were cloned as described previously [73]. Cas9 mutations, including D10A and dead-nuclease (D10A, H840A), were constructed by mutagenesis kit (Fast Mutagenesis System, Transgen Biotech). The transfection was carried out with Lipofectamine 2000 Reagent (Thermo Fisher Scientific). U2OS cells were synchronized at G1/S by" double thymidine block" method as described previously [42]. Briefly, exponentially growing U2OS cells were blocked with 2mM thymidine for 19h, washed three times with prewarmed PBS and released into fresh medium for 10h, and then blocked again with 2mM thymidine for another 14h. For 5-bromo-2-deoxyuridine (BrdU, Sigma) labeling, cells were incubated with fresh medium containing 100μM BrdU for 12 h after release from G1/S. FACS analysis was carried out as described previously [42]. All genomic DNA was extracted and purified using AxyPrep Blood Genomic DNA Miniprep Kit (Axygen) according to manufacturer’s instructions. DNA concentration was measured by Nanodrop-2000. For 2D agarose gel analysis, 10μg DNA was digested overnight at 37°C with 10U HinfI (Thermo Fisher), 10U RsaI (Thermo Fisher) and 2μg/mL RNase A (Takara). The reaction was terminated with EDTA and analyzed by 2D agarose gel electrophoresis. 30U RecJf (New England Biolabs) was added for removing 5' single-stranded DNA. For internal gaps/nicks analysis, 5μg genomic DNA was digested overnight at 37°C with 5U HinfI (Thermo Fisher), 5U RsaI (Thermo Fisher) and 1μg/mL Ribonuclease A (RNase A, Takara) and purified with QIAquick PCR Purification kit (Qiagen). Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0. 7% agarose gel electrophoresis and in gel hybridization. CsCl gradient ultracentrifugation and DNA purification were performed as described previously [5,42]. DNA was purified and dissolved in 60μL ddH2O. One half of each sample was incubated with RecJf prior to analysis by 2D agarose gel electrophoresis. C-circle assay was performed as described previously [37]. The concentration of genomic DNA was determined by fluoremetry based method (Qubit 3. 0 Fluorometer, Thermo Fisher Scientific). Exactly the same amount of genomic DNA was input for C-circle assay (30ng for U2OS, 100ng for HEK 293T and VA13 cells). Each assay was repeated three times to obtain the quantitative result. To determine C-circles in CsCl fractions, 1μL of each fraction was incubated in 40μL reaction containing 19μL ddH2O and 20μL C-circle amplification master buffer (0. 2mg/mL BSA, 0. 1% Tween 20,1mM dATP, dGTP and dTTP each, 1× Φ29 Buffer and 7. 5U Φ29 DNA polymerase (Thermo Fisher) ) for 8h at 30°C, and then subjected to slot-blot and hybridization with C-probe. Neutral-Neutral 2D agarose gel electrophoresis was performed as described previously [74,75]. Briefly, enzyme digested DNA samples were loaded into a 0. 4% agarose gel for first dimension and electrophoresis was performed at 1V/cm for 12 h at room temperature in TBE buffer. Lanes were excised, soaked in TBE containing 0. 3μg/mL ethidium bromide (EB) (Sigma) for 30min, embedded in 1% agarose gel containing EB. Second dimension electrophoresis was performed at 4°Cfor 6 h at 3V/cm. The gel was then dried at room temperature by vacuum drier, and hybridized with G-/C-probe under native or denatured condition. The telomere length assay was performed as previously described [42]. 5μg genomic DNA was digested overnight at 37°C with 5U HinfI (Thermo Fisher), 5U RsaI (Thermo Fisher) and 1μg/mL RNase A (Takara). Digested DNA samples were subjected to conventional 0. 7% agarose gel in TAE buffer at 2V/cm for 16h at room temperature. The gel was dried at 42°C with vacuum drier, and hybridized with C-probe. IF-FISH was performed as previously described [50,58]. Fluorescent probe is Cy3- (TTAGGG) 3 (Panagene). Primary antibodies include anti-53BP1 (Novus Biologicals), anti-RPA2 (EMD Millipore), anti-PCNA (Genetex), anti-PML (Santa Cruz), anti-SMARCAL1 (Santa Cruz), anti-SLX4 (Novus Biologicals), anti-pDNA-PKcs (S2056) (Abcam) and anti-Rad51 (Santa Cruz). Secondary antibodies include DyLight488 conjugated anti-rabbit (Multisciences), DyLight488 conjugated anti-mouse (Multisciences). The telomeric sister chromatid exchange (T-SCE) was determined by CO-FISH, which is performed as described previously [58,76]. Fluorescent probes are Cy3- (TTAGGG) 3 (Panagene) and FITC- (CCTAAA) 3 (Panagene). The alkaline constant-field gel electrophoresis was performed as described previously described [77]. Briefly, 1×106 cells were rinsed with 1×PBS, resuspended in 50μl 0. 7% 45°C pre-warmed agarose (made with 1×TE, pH 8. 0), and solidified in 1ml decapitated injector. Agarose plugs were incubated in fresh-made lysis-buffer (30mM Tris-HCl pH8. 0,300mM NaCl, 25mM EDTA, 0. 5% SDS, 0. 1mg/ml Protease K, 0. 1mg/ml RNase A) overnight. The plugs were then denatured in 100mM NaOH with 1mM EDTA, placed into the wells of 0. 7% alkaline agarose gel (50mM NaOH with 1mM EDTA) and sealed with 0. 7% alkaline agarose gel. Electrophoresis was carried out at 1V/cm for 12h at 4°C. The gel was subjected to in gel hybridization with telomeric probe. In-gel hybridization was performed as described previously [50,78]. For native in gel hybridization, gels were hybridized in Denhart’s hybridization buffer with 32P-labeled C-/G- telomeric probe. The telomeric probes were prepared as described previously [79]. Gels were washed 3 times with 2×SSC and 0. 5% SDS, exposed to PhosphorImager screen (GE Healthcare) and scanned on Typhoon imager (GE Healthcare). Image Quant software was used for data analysis. For denatured in-gel hybridization, gels were denatured with 0. 5 M NaOH, neutralized with 1 M Tris–HCl (pH 8. 0) and then followed the procedure for native hybridization. Western blots were performed with antibodies against Flag (Monoclonal ANTI-FLAG M2 antibody, F1804, Sigma), SMARCAL1 (Santa Cruz), RPA2 (EMD Millipore), or β-actin (Proteintech). Two-tailed unpaired student’s t-test was used for statistical analysis (Graphpad Prism). Error bars represent the mean± SEM of three biological repeats/independent experiments. * P<0. 05, ** P<0. 005, *** P<0. 001.
summary: 10 to 15% human cancers utilize telomerase-independent alternative lengthening of telomeres (ALT) to maintain their telomere length. Unexpectedly, we find that endogenous C-strand breaks predominantly exist in telomeres of ALT cells, which induce high frequency of replication fork collapse. While collapsed fork triggers fork regression machinery to restart the replication, leading to telomeric homologous recombination; those unresolved are converted to C-circle and C-overhang. These findings suggest that the formation of C-circle and C-overhang represents a unique manner for ALT cells to prevent chromosome instability induced by replication defect at telomeres. Moreover, multiple hallmarks of ALT are provoked during this process, demonstrating that DNA strand break at telomeres underlies ALT mechanism.
keywords: chromosome structure and function
  molecular probe techniques
  purification techniques
  dna electrophoresis
  dna damage
  telomeres
  dna replication
  agarose gel electrophoresis
  dna
  molecular biology techniques
  gel electrophoresis
  research and analysis methods
  electrophoretic techniques
  probe hybridization
  chromosome biology
  molecular biology
  biochemistry
  cell biology
  nucleic acids
  genetics
  biology and life sciences
  dna purification
  chromosomes
title: Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination