Alternative Lengthening of Telomeres through Two Distinct Break-Induced Replication Pathways

Abstract

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.

Keywords: ALT; ALT DNA synthesis in APBs; ALT-associated PML bodies; APB; ATSA; BIR; BLM; C-circle; POLD3; RAD51; RAD52; Telomere; alternative lengthening of telomeres; break-induced replication.

Figure 1.. An Assay to Monitor DNA Synthesis at ALT Telomeres in APBs

(A) Experimental scheme for visualizing EdU incorporation in APBs in cells enriched in G2 with thymidine and CDK1i or with CDK1i. G2 cells were labeled with EdU for 1 or 3 h. EdU and PML were detected by immunofluorescence (IF), and telomeres were detected by FISH using the TelC PNA probe (left). EdU incorporation and cell-cycle profiles of the cell populations tested (right). (B) Representative images showing the localization of EdU, PML, and telomeres at G2 U2OS (ALT⁺) and HeLa1.3 (ALT⁻) cells. The cells were synchronized in G2 using thymidine and CDK1i. (C) Quantification of telomeres for their presence in APBs and EdU incorporation. The numbers of total, APB⁺, and EdU⁺ telomeres were determined in U2OS cells (2,295 telomeres in 42 cells were analyzed). (D) Representative images showing the localization of EdU, PML, and telomeres in PML knockdown and control U2OS cells. (E and F) U2OS cells were treated with control, PML (E), or BLM (F) siRNAs and synchronized in G2 using CDK1i. Approximately 100 cells were divided into five groups (0, 1 or 2, 3 or 4, 5 or 6, and ≥7) on the basis of the numbers of EdU⁺ telomeres. (G) Cells were treated with control, POLD3, or POLD4 siRNA and synchronized using thymidine and CDK1i, and 100 cells were divided into five groups on the basis of the numbers of EdU⁺ APBs.

Figure 2.. RAD52 but Not RAD51 Is Important for ALT Activity in APBs

(A) Representative images showing the localization of RAD52 to APBs in a G2 U2OS cell. Cells were synchronized in G2 using thymidine and CDK1i. (B) Representative images showing incorporation of EdU in APBs in U2OS cells treated with control, RAD51, or RAD52 siRNA. Cells were synchronized in G2 using CDK1i. (C and D) Approximately 100 cells analyzed in (B) were divided into five groups on the basis of the numbers of EdU⁺ APBs (C) or total APBs (D). (E and F) Wild-type (WT) and newly generated RAD52-KO U2OS cells were synchronized in G2 using thymidine and CDK1i, analyzed as in (B). Approximately 100 cells were divided into five groups on the basis of the numbers of EdU⁺ APBs (E) or total APBs (F). RAD52-KO #2 and #3 are two independently generated cell lines. (0 M) indicates that the RAD52-KO cells were newly generated. (G) Quantification of the EdU⁺ APBs among total APBs in cells analyzed in (E) and (F). (H) WT RAD52 was inducibly expressed in RAD52-KO cells with increasing concentrations of doxycycline (Dox). The total EdU signals of EdU⁺ telomeres in >100 cells were quantified. The cells with total EdU signals of 15 a.u. or more were colored in red, and the fractions of these cells in the indicated cell populations were determined.

Figure 3.. RAD52 Is Required for Telomere Maintenance in ALT⁺ Cells

(A) Relative telomeric DNA contents determined by qPCR in WT U2OS cells, newly generated RAD52-KO cells, and RAD52-KO cells passaged for different lengths of time. 0 M, 3 M, > 6 M, and > 9 M indicates that the RAD52-KO cells have been passaged for 0, 3, >6, or >9 months, respectively. Error bars denote SD; n = 3 (experimental triplicates); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired Student’s t test). (B) Telomere length was determined by TRF assay in WT U2OS cells, newly generated RAD52-KO cells, and RAD52-KO cells passaged for different lengths of time. Telomeres were detected by a 5^’ end-labeled (CCCTAA)₃ probe (top). Input genomic DNA was titrated and analyzed in anti-dsDNA dot blots and shown as loading controls (bottom). (C) Telomere FISH of metaphase chromosomes of WT and extensively passaged RAD52-KO U2OS cells. (D) WT U2OS cells and RAD52-KO cells passaged for different lengths of time were analyzed for senescence by β-galactosidase staining. Representative images of β-galactosidase staining (left) and quantifications of β-galactosidase-positive cells (right) are shown. Error bars denote SD; n = 3 (experimental triplicates); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired Student’s t test).

Figure 4.. RAD52 Overcomes the Inhibition of Telomeric ssDNA Annealing by RPA

(A) End-labeled telomeric ssDNA (ssTelo, 20 nM) was incubated with RAD52 protein (80, 160, 320, and 640 nM) and analyzed for complex formation. The formation of DNA-protein complex was quantified according to the reduction in free ssTelo. Error bars denote SD; n = 3 (independent experiments). (B) ssTelo (20 nM) was incubated with the RPA complex (20, 40, 80, and 160 nM) and analyzed as in (A). (C and D) Telomeric ssDNA complementary to ssTelo (CCCTTA)₁₀ (40 nM) was end labeled, incubated without or with RPA (1 μM), and then incubated without or with RAD52 (1.4 μM). Unlabeled ssTelo (TTAGGG)₁₀ (40 nM) was added to the reactions. Reactions were carried out at 20°C in (C) or 27°C in (D), and stopped at 0, 2, 5, and 10 min. Annealing efficiency was determined by quantifying the remaining free probe after reactions.

Figure 5.. RAD52 but Not RAD51 Promotes Telomeric D-Loop Formation in the Presence of RPA

(A) An ssDNA oligomer with homologous sequences in the pBSDK+ plasmid (oligo 1, 30 nM) was end labeled. The probe was first coated with RPA (400 nM) or left uncoated, then sequentially incubated with RAD52 (1 μM) and the pBSK+ plasmid (25 nM). Error bars denote SD; n = 3 (independent experiments). (B) End-labeled ssTelo (20 nM) was coated with RPA (200 nM) or left uncoated and then sequentially incubated with RAD52 (400 and 800 nM) and a plasmid containing 648 bp telomeric sequence (pTelo, 10 nM). The levels of telomeric D-loop formation were determined by quantifying the remaining free probe after reactions in the short exposure of the gel. Error bars denote SD; n = 3 (independent experiments). (C) Telomeric D-loop formation was carried out as in (B) using the indicated proteins. The levels of telomeric D-loop formation were quantified at 0, 2, 5, and 10 min as in (B). Error bars denote SD; n = 3 (independent experiments).

Figure 6.. RAD52 Is Dispensable for C-Circle Formation

(A) Relative C-circle levels in U2OS cells transfected with control or RAD52 siRNA. Telomeric DNA contents were determined by qPCR with or without rolling-circle amplification (RCA) (see Figure S6B). The RCA+/RCA− ratios of samples reflect the relative levels of C-circle amplification. Error bars denote SD; n = 3 (experimental triplicates); ns, not significant (unpaired Student’s t test). (B) Relative C-circle levels in WT U2OS cells, newly generated RAD52-KO (0 M) cells, and passaged RAD52-KO (3 M) cells. Relative C-circle levels were determined as in (A). Error bars denote SD; n = 3 (experimental triplicates); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired Student’s t test). (C) WT U2OS cells, newly generated RAD52-KO (0 M) cells, and passaged RAD52-KO (3 M) cells were synchronized in G2 using thymidine and CDK1i. Approximately 100 cells were divided into five groups on the basis of the numbers of EdU⁺ APBs. (D) Representative images of the RAD52-KO cells analyzed in (C).

Figure 7.. The RAD52-Independent ALT Pathway Is Mediated by BLM and POLD3/4

(A) Representative images showing EdU incorporation in APBs in extensively passaged RAD52-KO U2OS cells. Cells were treated with control or BLM siRNA and synchronized in G2 using CDK1i. (B) Approximately 100 cells analyzed in (A) were divided into five groups on the basis of the numbers of EdU⁺ telomeres. (C) Relative C-circle levels in extensively passaged RAD52-KO cells transfected with control or BLM siRNA. Error bars denote SD; n = 3 (experimental triplicates); *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired Student’s t test). (D) Extensively passaged RAD52-KO cells were transfected with control, POLD3, or POLD4 siRNA and synchronized in G2 by CDK1i. Approximately 100 cells were divided into five groups on the basis of the numbers of EdU⁺ APBs. (E) Relative C-circle levels in extensively passaged RAD52-KO cells transfected with control, POLD3, or POLD4 siRNA. Quantification was done as in (C). (F) Relative C-circle levels in extensively passaged RAD52-KO cells treated with control, RAD51, or MRE11 siRNA were analyzed as in (C). (G) A model of the bifurcated framework of the ALT pathway.