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, 539 (7627), 54-58

Break-induced Telomere Synthesis Underlies Alternative Telomere Maintenance

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Break-induced Telomere Synthesis Underlies Alternative Telomere Maintenance

Robert L Dilley et al. Nature.

Abstract

Homology-directed DNA repair is essential for genome maintenance through templated DNA synthesis. Alternative lengthening of telomeres (ALT) necessitates homology-directed DNA repair to maintain telomeres in about 10-15% of human cancers. How DNA damage induces assembly and execution of a DNA replication complex (break-induced replisome) at telomeres or elsewhere in the mammalian genome is poorly understood. Here we define break-induced telomere synthesis and demonstrate that it utilizes a specialized replisome, which underlies ALT telomere maintenance. DNA double-strand breaks enact nascent telomere synthesis by long-tract unidirectional replication. Proliferating cell nuclear antigen (PCNA) loading by replication factor C (RFC) acts as the initial sensor of telomere damage to establish predominance of DNA polymerase δ (Pol δ) through its POLD3 subunit. Break-induced telomere synthesis requires the RFC-PCNA-Pol δ axis, but is independent of other canonical replisome components, ATM and ATR, or the homologous recombination protein Rad51. Thus, the inception of telomere damage recognition by the break-induced replisome orchestrates homology-directed telomere maintenance.

Conflict of interest statement

The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper.

Figures

Extended Data Figure 1
Extended Data Figure 1. An inducible system for studying break-induced telomere synthesis
a, Schematic of inducible TRF1–FokI system. bd, Characterization of U2OS inducible TRF1–FokI system by western blot (b), immunofluorescence (c), and telomere ChIP (d). e, Agarose gel of sonicated DNA prepared for BrdU pulldown. f, g, BrdU pulldown dot blot for telomere content (f) from asynchronous or G2-enriched U2OS cells induced (Ind) with TRF1–FokI for 2 h, with cell-cycle profiles by propidium iodide staining (g). Images were captured at 60× magnification. Dox, doxycycline; S, Shield-1; T, 4-hydroxytamoxifen; DD, destabilization domain; ER, oestrogen receptor; rtTA, reverse tetracycline transactivator; TRE3G, tetracycline response element; WT, wild-type; D450A, nuclease-null mutant.
Extended Data Figure 2
Extended Data Figure 2. Visualization of spontaneous ALT telomere synthesis
ac, BrdU immunofluorescence assay to visualize spontaneous ALT telomere synthesis, with representative images of VA13 cells (a) and quantification of a panel of ALT and ALT+ cell lines (b) and U2OS cells induced with TRF1–FokI for 2 h (c). d, e, Representative images of BrdU immunofluorescence of metaphases from spontaneous GM847 cells (d) and U2OS induced (+Ind) with TRF1–FokI for 2 h upon release from RO-3306 (e). Images were captured at 60× magnification. Data represent mean ± s.e.m. of three independent experiments. ***P ≤0.001.
Extended Data Figure 3
Extended Data Figure 3. Break-induced telomere synthesis occurs independently of telomere maintenance mechanism
a, A panel of ALT and ALT+ inducible TRF1–FokI cell lines tested for TRF1–FokI and ATRX expression by western blot and nascent telomere synthesis by BrdU pulldown dot blot for telomere content after induction (Ind) with TRF1–FokI for 2 h. b, c, BrdU pulldown dot blot for telomere content (b) from HeLa 1.3 cells induced (Ind) with TRF1–FokI for 2 h, with quantification (c). Data represent mean ± s.e.m. of two independent experiments. *P ≤0.05.
Extended Data Figure 4
Extended Data Figure 4. Hop2 contributes to telomere clustering but is dispensable for telomere length maintenance
a–h, CRISPR/Cas9-mediated excision of HOP2 (sgHOP2) in VA13 cells, with western blot of populations (a). Analysis of Hop2 co-localization with telomere foci by IF-FISH (b), telomere focus size by FISH (c), APBs by PML co-localization with telomere foci (d, e), and telomere exchanges by CO-FISH (f) from sgHOP2 #2 population. Analysis of clones (c1–c6) by western blot (g) and TRF pulsed-field gel at ~PD 25 (h). Peak intensity of telomere length is indicated by red dot. Images were captured at 60× magnification. Data represent mean ± s.e.m. of at least two independent experiments. ***P <0.0005, **P <0.005, *P <0.05.
Extended Data Figure 5
Extended Data Figure 5. Requirements for break-induced and spontaneous ALT telomere synthesis
a, BrdU pulldown dot blot timecourse for telomere content from U2OS induced (Ind) with TRF1-FokI for indicated times and treated with indicated siRNAs. b, c, BrdU pulldown dot blots for telomere content from U2OS induced (Ind) with TRF1–FokI for 2 h and treated with indicated siRNAs. d, BrdU pulldown dot blot for telomere content from HeLa 1.3 induced (Ind) with TRF1–FokI for 2 h and treated with indicated siRNAs, with quantification. ei, Analysis of spontaneous ALT telomere synthesis using BrdU immunofluorescence from VA13 (eh) and GM847 and LM216J (i) treated with indicated siRNAs. Images were captured at 60× magnification. Data represent mean ± s.e.m. of two (d) or three (ei) independent experiments. ****P ≤0.0001, **P ≤0.01, *P ≤0.05.
Extended Data Figure 6
Extended Data Figure 6. Pol δ predominates at ALT telomeres
a, Representative images of replisome components (green) and telomere foci (red) from U2OS induced with TRF1–FokI for 2 h. b, western blot of Pol δ complex from cell lines treated with TRF1–FokI. Asterisk denotes non-specific band. c, d, Quantification of co-localized POLD3 (c) or POLD1 (d) with telomere foci from U2OS induced with TRF1–FokI for 2 h. e, f, Representative images (e) of co-localized RFC1-PCNA-POLD3 (green) and telomere foci (red) from U2OS induced with TRF1–FokI for 2 h, with quantification (f). WT =wild-type, D450A =nuclease-null mutant. Images were captured at 60× magnification. Data represent mean ± s.e.m. of three independent experiments. ****P ≤0.0001, ***P ≤0.001.
Extended Data Figure 7
Extended Data Figure 7. POLD3 is critical for telomere maintenance in ALT-dependent cells
a, b, analysis of transient POLD3 depletion by C-circle dot blot (a) from U2OS and CO-FISH (b) from VA13 (n =1780 ends for siCtrl, n = 1637 ends for siPOLD3). c, TRF analysis from U2OS populations at ~PD 25. Peak intensity of telomere length is indicated by red dot. d, schematic of U2OS POLD3 CRISPR (sgPOLD3) cloning strategy with western blot. eg, analysis of POLD3 expression from U2OS clones c1–c4 by qPCR (e), POLD1 Co–IP (f), and darker exposure of western blot from Fig. 4a (g). Asterisk denotes non-specific band. h, Quantification of relative telomere content by dot blot from U2OS clones c1–c4. i, Heat map summarizing decreases (blue), increases (red), or no change (white) in telomere maintenance from U2OS clones c1–c4 as compared to U2OS control. j, k, POLD3-reconstituted CRISPR clones analysed for C-circles by dot blot (j) and Pol δ expression by western blot (k). EV, empty vector; WT, reconstituted POLD3. Data represent mean ± s.e.m. of two independent experiments. **P ≤0.01, *P ≤0.05.
Extended Data Figure 8
Extended Data Figure 8. Extended analysis of POLD3 CRISPR clones
a, b, TRF analysis by pulsed-field gel (a) and C-circle dot blot (b) from U2OS POLD3 CRISPR (sgPOLD3) clones with normal POLD3 expression (c5–c9) at ~PD 25. c, U2OS POLD3 CRISPR clones from an independent guide RNA (sgPOLD3 #2) analysed by TRF and western blot at ~PD 25. d, TRF analysis by pulsed-field gel from HeLa 1.3 populations at ~PD 25. e, Schematic of HeLa 1.3 POLD3 CRISPR cloning strategy with western blot. f, TRF analysis by pulsed-field gel from HeLa 1.3 clones c1–c11 at ~PD 25. Peak intensity of telomere length is indicated by red dot.
Extended Data Figure 9
Extended Data Figure 9. Knockdown efficiencies
a–n, Western blots of U2OS or VA13 cells treated with indicated siRNAs. Asterisk denotes non-specific band.
Figure 1
Figure 1. Break-induced telomere synthesis occurs by long-tract unidirectional telomeric recombination
a, Schematic of BrdU pulldown. IP, immunoprecipitation. b–d, BrdU pulldown dot blot for telomere content using a 32P-labelled telomere oligonucleotide (b) from U2OS cells induced (Ind) with TRF1–FokI for 2 h, with quantification (c) and time course of C- and G-rich telomere strands compared to Alu repeats (d). e, Schematic of telomere SMARD. f–h, Representative images (f) of telomere (blue) labelled with IdU (red) and CldU (green) from U2OS cells induced (Ind) with TRF1–FokI, with quantification (g) and length of telomere fibres (h). Median length quantified ± 95% C.I. WT, wild-type; D450A, nuclease-null mutant. Data represent mean ± s.e.m. of three (c) or two (g) independent experiments. **P ≤0.01, *P ≤0.05.
Figure 2
Figure 2. Break-induced telomere synthesis occurs by alternative HDR and utilizes a non-canonical replisome defined by Pol δ
a, Quantification of co-localized Hop2 and telomere foci in TRF1–FokI expressing VA13 cells treated with the indicated small interfering RNAs (siRNAs) or inhibitors. b, Mean squared displacement (MSD) analysis of live-cell telomere movement from U2OS cells following TRF1–FokI induction. c–f, BrdU pulldown dot blots for telomere content (c, d, e, f) from U2OS cells induced (Ind) with TRF1–FokI for 2 h, with rescue experiment (e), and quantifications (c, d, f). g, Quantification of telomere SMARD from U2OS cells induced with TRF1–FokI and treated with the indicated siRNAs. EV, empty vector; WT, reconstituted POLD3. Data represent mean ± s.e.m. of at least two independent experiments. ***P ≤0.001, **P ≤0.01, *P ≤0.05.
Figure 3
Figure 3. Rapid loading of PCNA acts as the initial sensor of telomere damage
a, Quantification of co-localized GFP-tagged replisome subunits and telomere foci from U2OS cells induced with TRF1–FokI for 2 h. b, Schematic of POLD3 and representative images of POLD3 deletion mutants. c, Quantification of co-localized endogenous PCNA and POLD3 and telomere foci from U2OS cells induced with TRF1–FokI for 2 h. d, BrdU pulldown dot blot for telomere content from U2OS cells induced (Ind) with TRF1–FokI for 2 h. e, Kinetics of PCNA and POLD3 loading at damaged telomeres in relation to Rad51, γH2AX, and DNA synthesis in U2OS cells. IF, immunofluorescence. f, Representative live-cell imaging of PCNA recruitment to damaged telomeres before telomere clustering in U2OS cells. Time in minutes shown in upper left corners. g, Quantification of the requirements for PCNA loading at damaged telomeres in U2OS cells. h, Quantification of spontaneous co-localized RFC1, PCNA, and POLD3 and telomere foci from a panel of ALT and ALT+ cell lines. Fixed cell and live cell images were captured at 60× and 100× magnification, respectively. Data represent mean ± s.e.m. of two (a) or three (c, e, g, h) independent experiments. ****P ≤0.0001.
Figure 4
Figure 4. POLD3 is critical for telomere maintenance in ALT-dependent cells
a, Analysis of Pol δ protein expression by western blot from U2OS CRISPR clones (sgPOLD3) with decreased POLD3 expression (c1–c4) compared to empty guide control (sgEmpty). GAPDH is used as a loading control. Darker exposure shown in Extended Data Fig. 7g. b, TRF analysis using pulsed-field gel electrophoresis from U2OS clones c1–c4 at ~PD 25. Peak intensity of telomere length is indicated by red dot. c, Quantification of relative mean telomere length in 31 pooled sg POLD3 clones from two independent guide RNAs with normal and decreased POLD3 protein expression. d, e, Analysis of telomere maintenance by TIF quantification (co-localized 53BP1 and telomere foci) (d) and C-circle assay (e) from U2OS clones c1–c4. f, Model. Data represent mean ±s.e.m. of three independent experiments. ****P ≤0.0001, ***P ≤0.001, *P ≤0.05.

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