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Break-induced replication orchestrates resection-dependent template switching

Nature volume 619, pages 201–208 (2023)Cite this article

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Abstract

Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres1,2. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear3,4,5. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS1,6. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.

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Fig. 1: Telomere DSBs activate homology-directed repair, ATR signalling and Fanconi anaemia pathways.
Fig. 2: Break-induced telomere synthesis promotes RAD18-dependent interaction of ubiquitinated PCNA and SNM1A at damaged telomeres.
Fig. 3: RAD18 and SNM1A are required for end resection during break-induced telomere synthesis.
Fig. 4: RAD18 and SNM1A are required for the formation of complex recombination intermediates during break-induced telomere synthesis.
Fig. 5: The RAD18–ubiquitinated PCNA–SNM1A axis mediates template switching-dependent lesion bypass to promote break-induced replication.

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Data availability

All data are available in the main paper or its Supplementary Information. All reagents are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Déjardin (CNRS, France) for guidance on performing PICh experiments. We thank members of the Greenberg laboratory for critical discussion. This work was supported by NIH RO1 GM1101149 and RO1 CA174904 and Bloom Syndrome Grant Program BLOOM-22-004-01 to R.A.G., RO1 ES007061 and R35 CA241801 to P.S. and a Gray Foundation Team Science Award to R.A.G. and P.S.; R50CA265315 to Y.K.; and an AFCRI-funded postdoctoral fellowship to T.Z.

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Authors and Affiliations

  1. Department of Cancer Biology, Penn Center for Genome Integrity, Basser Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Tianpeng Zhang, Haoyang Jiang & Roger A. Greenberg

  2. Department of Biochemistry and Structural Biology and Greehey Children’s Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Yashpal Rawal, Youngho Kwon & Patrick Sung

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  1. Tianpeng Zhang
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Contributions

R.A.G. and T.Z. designed the experiments. T.Z. performed all major cellular experiments with some involvement from H.J. Y.R. carried out protein purification and the nuclease assays. Y.K. and P.S. assisted in the design of the nuclease assays. T.Z. and R.A.G. wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to Roger A. Greenberg.

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R.A.G. is a cofounder and scientific advisory board member of RADD Pharmaceuticals and is on the scientific advisory board of Trevarx Biomedical. Neither engagement directly relates to the substance of this study. All other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 related to Fig. 1. Break induced telomere synthesis triggers replication stress.

a. TRF1-FokI (D450A, WT) was induced with doxycycline for 18 hrs, followed by 4-OHT for 2 hrs. Whole cell extracts were separated with SDS-PAGE and blotted with Flag antibody (for Flag tagged TRF1-FokI), and GAPDH (loading control) antibodies. b. Telomere restriction fragment (TRF) analysis of telomere length from HeLa S3 and U2OS cells induced with TRF1-FokI (D450A, WT) as shown in (a), respectively, using 32P-labeled telomeric C-probe under denaturing condition. c. Silver staining of telomere chromatin bound fractions enriched by PICh with telomeric probe (Telo) or scramble probe (SCR) in HeLa S3 (left) or U2OS (right) induced with TRF1-FokI (D450A, WT) for 2 hrs. d. Gene ontology (GO) terms significantly enriched at damaged telomeres in both HeLa S3 and U2OS. Fold change of (WT+1)/(D450A+1) of total peptide number in both HeLa S3 and U2OS cells were calculated. Greater than two-fold increases in peptide number in both cell lines was analyzed. e. Western blot showing PolD3 depletion, with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3), from U2OS cells induced with TRF1-FokI for 2 h. Whole cell extracts were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), PolD3, and GAPDH (loading control) antibodies. f. Experimental procedure of telomere synthesis in G2/M synchronized U2OS cells +/− TRF1-FokI induction. g. Representative images of EdU (Green) and telomeres (Telo, Red) in S phase and non-S phase U2OS cells that were arrested in G2 by RO-3306 and induced with TRF1-FokI for 2.5 h. h. Quantification of (g) for EdU colocalization with telomeres in PolD3 knocked out (sgPolD3) or control (sgCtrl) U2OS cells following TRF1-FokI induction for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 184, 222, 170, 203 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. i. Representative IF-FISH images of RPA2 (Green) colocalization with telomere (Telo, Red) in U2OS cells with/ without TRF1-FokI for 2 hrs. j. Quantification of (i) for number of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 322, 286, 287, 270 (left to right)). Statistical analyses are done with unpaired two-tailed student’s t-test. p values are shown. k. Representative IF-FISH images of pRPA2(S33) (Green) colocalization with telomeres (Telo, Red) in U2OS cells induced with TRF1-FokI for 2 h. l. Quantification of (k) for number of pRPA2(S33) and telomere foci colocalization events in U2OS cells +/− TRF1-FokI induction for 2 h in cells with sgRNA targeting Rosa (sgCtrl) or PolD3 (sgPolD3). Data represent the mean ± SEM of three independent experiments (n = 306, 392, 371, 350 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 2 related to Fig. 2. BITS triggers RAD18 mediated PCNA-Ub and SNM1A recruitment at damaged telomeres.

a. PICh-WB for telomere associated RAD18, SNM1A, PCNA and PCNA-Ub (Ubiquitylated PCNA (Lys164)) in HeLa S3 and U2OS induced with TRF1-FokI (D450A and WT), respectively. GAPDH, TRF2 and mCherry (mCherry tagged TRF1-FokI) were blotted as experimental controls. b,c. Nuclear extracts from U2OS cells or HeLa S3 stably expressing SNM1A (or mutants) following induction with TRF1-FokI for 2 hrs were subjected to immunoprecipitation (IP) with anti-HA affinity beads. Input and IP samples were separated with SDS-PAGE and blotted with mCherry (for mCherry tagged TRF1-FokI), SNM1A, TRF2, ubiquitinated PCNA, PCNA and H2A (loading control) antibodies. d. Nuclear extracts from HeLa S3 stably expressing SNM1A (or mutants) after treatment with replication stress agents were subjected to immunoprecipitation (IP) with anti-HA affinity beads. Input and IP samples were separated with SDS-PAGE and blotted with pATR (T1989), SNM1A, TRF2, ubiquitinated PCNA (Ubiquityl PCNA (Lys164)), PCNA and H2A (loading control) antibodies. “LE”: long exposure; “SE”: short exposure. e. Experimental procedure for BrdU-IP (for (f)) or PICh (Fig. 2h, i) in HeLa S3 cells induced with TRF1-FokI with aphidicolin at the indicated concentrations. f. BrdU pulldown slot blot for telomere (or Alu) content using 32P-labeled telomeric G-probe or Alu probe, from HeLa S3 cells induced with TRF1-FokI for 2 hrs in presence of aphidicolin (Aphi). g. Quantification of (f). Relative abundance of telomere content enriched by BrdU pulldown are normalized to the uninduced sample. Data represent the mean ± SEM of three independent experiments. The uncropped gel and dot blot images are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 3 related to Fig. 3. RAD18 and SNM1A are required for ssDNA generation at damaged telomeres.

a–d. Representative images (a) and (c) and quantification (b) and (d) of native-FISH by telomeric C-probe (TelC-Cy3, Red) and G-probe (TelG-Cy3, Red) in U2OS cells induced with TRF1-FokI for 2 hrs. Data represents the mean ± SEM of three independent experiments (n = 354, 343 in (b) and n = 335, 288 in (d)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. e. Western blot of RAD18 from U2OS cells induced with TRF1-FokI for 2 hrs. RAD18 was knocked out with sgRNAs (#1, #2). f. Quantification of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs as indicated. Data represent the mean ± SEM of three independent experiments (n = 638, 553, 594, 547, 522, 532 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. g. Western blot of SNM1A from U2OS cells following induction with TRF1-FokI for 2 hrs in cells with sgRNA targeting Rosa (sgCtrl) or SNM1A (sgSNM1A #1, #2, #5). h. Quantification of number of RPA2 and telomere foci colocalization events in U2OS cells following induction with TRF1-FokI for 2 hrs as indicated. Data represent the mean ± SEM of three independent experiments (n = 354, 350, 286, 282, 281, 331, 339, 270 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. i. Quantification of number of RPA2 and telomere foci colocalization events in SNM1A knocked out U2OS cells reconstituted with sgRNA resistant SNM1A or mutants following TRF1-FokI induction for 2 hrs. Data represent the mean ± SEM of three independent experiments (n = 495, 536, 442, 390, 416, 368, 483, 396 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 4 related to Fig. 3. Endonuclease and exonuclease activities assay with full length SNM1A.

a. Left, flow chart summarizing the SNM1A purification scheme. Right, purified SNM1A and SNM1ADH/AA, 300 ng each, were analyzed by Coomassie blue staining and Western-blot. b. SNM1A and SNM1ADH/AA were tested for exonuclease activity using 5′ 32P-labeled 80-nt ssDNA as substrate with MgCl2 and/or MnCl2 as indicated. Data (mean ± SEM) from three independent experiments were quantified and shown in the histogram. c,d. SNM1A and SNM1ADH/AA were tested for exonuclease activity using 5′ 32P-labeled 80-bp dsDNA (c) or fork DNA (d) with MgCl2. Data (mean ± SEM) from three independent experiments were quantified and shown in the histogram. e. SNM1A and SNM1ADH/AA were tested with zeocin treated ϕX174 replicative form DNA with MgCl2. Data (mean ± SEM) from four independent experiments were quantified and shown in the histogram. f. SNM1A and SNM1ADH/AA were tested with ϕX174 replicative form I (supercoiled) DNA and MgCl2. Data (mean ± SEM) from three independent experiments were quantified and shown in the histogram. The uncropped gel images are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 5 related to Fig. 3. Canonical double-strand break exonucleases are dispensable for end resection during break induced telomere synthesis.

a. Western blot showing human ExoI depletion from U2OS cells with sgRNA targeting Rosa (sgCtrl) or ExoI (sgExoI #2, #3). b. Quantification of RPA2 and telomere foci colocalization events in U2OS cells with sgRNA targeting Rosa (sgCtrl) or ExoI following TRF1-FokI induction for 2 h. Data represent the mean ± SEM of three independent experiments (n = 587, 586, 546, 553, 523, 478 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. c. Quantification of the relative C-rich (Left) and G-rich (Right) single-stranded telomere intensity in U2OS cells with sgRNA targeting ExoI. Data represent the mean ± SEM of two or three independent experiments (n = 7 (left) and n = 8 (right) biological replicates in total). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. d. Western blot showing DNA2 depletion from U2OS cells induced with TRF1-FokI for 2 h. e. Quantification of RPA2 and telomere foci colocalization events in U2OS cells, with gRNAs targeting Rosa (sgCtrl) or DNA2 induced with TRF1-FokI for 2 h. Data represent the mean ± SEM of three independent experiments (n = 519, 382, 331, 332, 441, 355, 340, 339 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. f. Quantification of the relative C-rich (Left) and G-rich (Right) single-stranded telomere intensity in U2OS cells that express sgRNA targeting Rosa or DNA2, respectively. Data represent the mean ± SEM of five independent experiments. Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. g. Western blot showing MRE11 depletion from U2OS cells using either spCas9 (left panels) or AsCas12a (right panels). TRF1-FokI was induced for 2 h. h. Quantification of RPA2 and telomere foci colocalization events in U2OS cells induced with TRF1-FokI for 2 hrs. Data represent the mean ± SEM of three independent experiments (n = 556, 459, 510, 544 (left) and n = 495, 691, 479, 588 (right) (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. i. Schematic of the dual CRISPR/Cas (spCas9 and AsCas12a) mediated deletions of ExoI, DNA2 and MRE11. spCas9 was used for single knockouts and spCas9 + AsCas12a were used for triple knockouts. j. Western blot showing MRE11, ExoI and DNA2 knock outs individually or in combination with dual CRISPR/Cas system (spCas9 and AsCas12a) from U2OS cells induced with TRF1-FokI for 2 h. The uncropped gel images are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 6 related to Fig. 4. RAD18 and SNM1A are dispensable for canonical DSBR at telomeres.

a. Representative C-circle slot blot and quantification of the relative intensity of C-circles from U2OS cells that express sgRNA targeting Rosa (sgCtrl) or RAD18 (sgRAD18 #1, #2). TRF1-Fok1 was induced for 2 h. Data represent the mean ± SEM of three independent experiments (three biological replicates for each independent experiments, n = 9, 9, 9, 6, 6, 6). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. b. Representative C-circle slot blot and quantification of the relative intensity of C-circles from U2OS cells that express sgRNA targeting Rosa (sgCtrl) or SNM1A (sgSNM1A #1, #2, #5). TRF1-Fok1 was induced for 2 h. Data represent the mean ± SEM of three independent experiments (three biological replicates for each independent experiments, n = 9, 9, 9, 9, 9, 9). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. The uncropped gel images are provided in Supplementary Fig. 1.

Source data

Extended Data Fig. 7 related to Fig. 5. RAD18 and SNM1A are required for BITS.

a. Western blot of PCNA ubiquitination in RAD18 knocked out VA13 cells. VA13 cells were targeted by the indicated sgRNAs and TRF1-FokI was induced with doxycycline + 4-hydroxytamoxifen. b. Quantification of EdU colocalizing with telomeres in RAD18 knocked out (sgRAD18, #1 and #2 guide RNA) or sgCtrl in VA13 cells following TRF1-FokI for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 340, 341, 291, 323, 365, 277 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. c. Western blot of PCNA ubiquitination (Ubiquitylated PCNA (Lys164)) in U2OS cells stably overexpressing RAD18 (or mutants). d. Quantification of EdU colocalizing with telomeres in U2OS cells stably overexpressing RAD18 (or mutants) following induction with TRF1-FokI for 2.5 hrs in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 164, 123, 149, 134, 143, 148, 125, 125, 136, 134 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. e. Quantification of EdU colocalizing with telomeres in SNM1A knocked out U2OS cells reconstituted with sgRNA resistant SNM1A or mutants. TRF1-FokI was induced for 2.5 h in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 202, 237, 222, 167, 233, 207, 166, 239 (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. f. Western blot showing endogenous SNM1A and RAD18 depletion in U2OS cells by the dual CRISPR/Cas9 system. SNM1A is targeted by spCas9 with gRNA #2, and RAD18 is targeted by saCas9 with gRNA #4 and #8, respectively. Whole cell extracts were separated with SDS-PAGE and blotted with SNM1A, RAD18 and GAPDH (loading control) antibodies. g–j. Quantification of EdU colocalizing with telomeres in MRE11, ExoI, and DNA2 that have been knocked out individually or in combination in U2OS cells. TRF1-FokI was induced for 2.5 h in G2 phase. Data represent the mean ± SEM of three independent experiments (n = 302, 318, 260, 336 (left) and n = 315, 348, 328, 398 (right) in (g); n = 248, 252, 260, 263, 281, 203 in (h); n =  206, 201, 181, 185, 170, 253, 223, 219 in (i); n = 388, 390, 333, 368, 381, 351, 348, 336, 423, 390, 383, 347, 357, 412, 349, 285 in (j) (left to right)). Statistical analyses were performed using an unpaired two-tailed student’s t-test. p values are shown. k. Western blot showing wild type RAD18 and mutants overexpression in SNM1A proficient and depleted U2OS cells. l. Western blot showing wild type Rad18 and mutant overexpression in SNM1A proficient and depleted LM216J cells. m. Telomere Restriction Fragments (TRF) analysis of telomere length in LM216J cells that overexpress wild type RAD18 or RAD18 mutants in context of SNM1A proficient (sgRosa) or knocked out (sgSNM1A) conditions. The uncropped gel images are provided in Supplementary Fig. 1.

Source data

Extended Data Table 1 PICh results for DDR pathways in HeLa S3 and U2OS
Full size table

Supplementary information

Supplementary Fig. 1

Uncropped gel images from western blots, Southern blots and agarose gel images in Figs. 2–5 and Extended Data Figs. 1–7.

Reporting Summary

Supplementary Table 1

PICh results for HeLa S3 U2OS TRF1–FOKI D450A versus WT.

Supplementary Table 2

PICh results for U2OS TRF1–FOKI induction sgRosa versus sgRAD18.

Supplementary Table 3

PICh results for HeLa S3 TRF1–FOKI Ctrl versus aphidicolin.

Supplementary Table 4

Information on sgRNAs, cloning primers, antibodies and software.

Source data

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Zhang, T., Rawal, Y., Jiang, H. et al. Break-induced replication orchestrates resection-dependent template switching. Nature 619, 201–208 (2023). https://doi.org/10.1038/s41586-023-06177-3

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