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POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis

A Publisher Correction to this article was published on 27 October 2021

This article has been updated

Abstract

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.

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Fig. 1: Formation and repair of spontaneous DSBs throughout the cell cycle.
Fig. 2: TMEJ in HR-deficient cells is delayed until mitosis.
Fig. 3: RAD52 locates to resected DSBs to prevent their repair in G2-phase BRCA2 mutants.
Fig. 4: RAD52 prevents the premature usage of TMEJ in BRCA2 mutants.
Fig. 5: RAD52 and BRCA2 both suppress TMEJ.
Fig. 6: RAD52 and BRCA2 inhibit the polymerase function of POLθ.
Fig. 7: RAD52 and BRCA2 inhibit POLθ DNA synthesis by distinct mechanisms.

Data availability

Source data are provided with this study. All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Stark for providing the U2OS EJ2 reporter and POLθ/RAD52 KO cell lines, and A. Löwer and P. Jeggo for helpful discussions. We thank R. Weimer, C. Braun and M. P. Lowery for technical assistance, and D. Deckbar and A. Taubmann for experimental and intellectual contributions during the early stages of the project. Work in the M.L. laboratory is supported by the Deutsche Forschungsgemeinschaft (DFG, project ID 393547839—SFB 1361) and the Bundesministerium für Bildung und Forschung (grant nos 02NUK054C, 02NUK050B and 02NUK042D), work in the W.D.H. laboratory is supported by the National Institutes of Health (grant nos GM58015 and GM137751), and work in the R.D.W. laboratory is supported by the National Institutes of Health (grant nos CA247773 and CA193124) and the J. Ralph Meadows Chair in Carcinogenesis Research. This research was also supported by the National Institutes of Health award CA187561 to J.L. and by core services supported by P30 CA93373.

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M.L.A., M.E., H.P.L., A.G., J.L. and A.C.G. performed experiments and interpreted data. S.B. and R.D.W. provided reagents. M.L. and W.D.H. conceived the experiments and wrote the paper aided by M.L.A., M.E., H.P.L., A.G., J.L., A.C.G., S.B. and R.D.W.

Corresponding author

Correspondence to Markus Löbrich.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Repair of spontaneous breaks by TMEJ in HR-deficient cells is delayed until mitosis.

a, Spontaneous γH2AX foci in G1 and G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU for 1 h and foci were immediately analysed after labelling in EdU cells (microscopic dot blot on the left indicates the evaluated cell populations) (n = 4 independent experiments, except siRAD51 + siPOLθ: n = 3 independent experiments). b, Spontaneous γH2AX foci in HeLa cells treated with a second POLθ siRNA (siPOLθ_6). (i) Cells were labelled as in Fig. 1a and EdUBrdU+ cells were analysed (n = 3 independent experiments, except siCTRL/siBRCA2 at 12 h: n = 4 independent experiments). (ii) Cells were labelled and analysed as in (a) (n = 3 independent experiments, except siCTRL/siBRCA2/siRAD51: n = 4 independent experiments). The data without siPOLθ_6 in (i) are also part of Fig. 2a,b whilst the data without siPOLθ_6 in (ii) are also shown in panel (a) of the present figure. c, Spontaneous γH2AX foci in fibroblasts. The same analysis as in (b) was performed with 82-6 hTERT (WT) cells (for (i): n = 4 independent experiments, for (ii): n = 3 independent experiments). d, Spontaneous chromatid breaks in G2 and mitotic HeLa cells. Cells were transfected with siRNAs and analysed by premature chromosome condensation (PCC) for G2 cells or a 2-h colcemid treatment for mitotic cells. Chromatid breaks were quantified per spread and normalized to 70 chromosomes (n = 3 independent experiments). e, Lagging chromosomes in ana/telophase HeLa cells. Cells were transfected with siRNAs and ana/telophases were identified by their morphology seen in the DAPI and phospho-histone H3(S10) (pH3) stainings (n = 4 independent experiments). f, Micronuclei in G1 HeLa cells. Cells were transfected with siRNAs, labelled as in Fig. 1a and EdUBrdU+ cells were analysed in G1 (n = 4 independent experiments). All data show the mean ± s.e.m. Individual experiments, each derived from 40 (S/G2 foci, G2 spreads and lagging chromosomes), 60 (M spreads), 100 cells (G1 foci) or 200 cells (micronuclei), are shown as dots. *: P < 0.05, **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 2 Repair of exogenous breaks by TMEJ in HR-deficient cells is delayed until mitosis.

a, γH2AX foci after CPT treatment in S/G2 and G1 HeLa cells. Cells were transfected with siRNAs and treated with EdU and 20 nM CPT for 1 h. EdU+ cells were in S during the CPT treatment and progressed to G2 and G1 after 12 h. Foci were analysed in these cells at the indicated times (microscopic dot blot on the left indicates the evaluated cell populations) (n = 4 independent experiments). b, γH2AX foci after IR in G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU for 3 h and irradiated with 2 Gy X-rays, after which BrdU was added. EdU+BrdU cells were in G2 at the time of IR and partially progressed to G1 after 12 h, at which time they were analysed (microscopic dot blot on the left indicates the evaluated cell populations) (n = 3 independent experiments). c, γH2AX foci in HeLa cells treated with PARPi for 24 h. Cells were transfected with siRNAs, treated with a PARP inhibitor (Olaparib, 1 µM) for 24 h, pulse-labelled with EdU for the last hour and γH2AX foci were then analysed in EdU G2, mitotic or G1 cells (n = 3 independent experiments). All data show the mean ± s.e.m. Individual experiments, each derived from 40 (S/G2), 80 (Ana/Telo) or 100 cells (G1), are shown as dots. **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 3 RAD52 locates to resected DSBs to prevent their repair in G2-phase BRCA2 mutants.

a, Diagram of the GFP–RAD52 protein: GFP tag (green), RAD52 self-binding domain (orange), RPA-binding domain (purple), RAD51-binding domain (yellow) and nuclear localization signal (NLS, red). Expression levels of endogenous vs. tagged RAD52 were assessed by immunoblotting. GAPDH was used as loading control. The example immunofluorescence (IF) image shows the variability in GFP–RAD52 expression between different cells (scale bar, 10 µm). For foci analysis, cells with very high or very low GFP–RAD52 expression were excluded. b, Kinetics of γH2AX, RAD51 and GFP–RAD52 foci in G2 HeLa cells stably expressing GFP–RAD52. Cells were labelled with EdU, irradiated with 2 Gy X-rays and EdU G2 cells were analysed (n = 5 independent experiments, except 0.25 h and 5 h: n = 3 or n = 2 independent experiments, respectively). c, Co-localization studies of RAD52, RAD51, pRPA and γH2AX in G2 HeLa GFP–RAD52 cells. Cells were labelled with EdU, irradiated with 2 Gy X-rays and EdU G2 cells were analysed (n = 2 independent experiments, except co-localization of RAD52/RAD51: n = 3 independent experiments). Example IF images are shown in the right panel (scale bar, 5 µm). d, Spontaneous γH2AX foci in S/G2 HeLa cells. Cells were transfected with siRNAs, labelled as in Fig. 1a and EdUBrdU+ cells were analysed (n = 4 independent experiments). e, γH2AX foci after IR in G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU G2 cells were analysed (n = 3 independent experiments). f, γH2AX foci after IR in G2 fibroblast cells. Experiment was performed as in (e), but using 82-6 hTERT (WT) cells (n = 3 independent experiments). Data without siBRCA2 are the same as in Fig. 3e. All data show the mean ± s.e.m, except for (b) and the conditions in (c) with n = 2 independent experiments for which only the mean is shown. Individual experiments, each derived from 40 cells (except (c): 20 cells), are shown as dots. *: P < 0.05; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 4 RAD52 limits the premature usage of TMEJ in BRCA2 mutants to prevent the formation of chromosomal fusions.

a, γH2AX foci after IR in G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU G2 cells were analysed (n = 4 independent experiments). b, γH2AX foci after IR in G1 and G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU G1 and G2 cells were analysed (n = 2 independent experiments for G1 and n = 3 independent experiments for G2, except siLIG4/siLIG4 + siRAD52/siBRCA2 + siLIG4: n = 2 independent experiments). The G2 data without siLIG4 are also shown in Extended Data Fig. 3e. c, γH2AX foci after IR in G2 fibroblasts lacking XLF. 2BN hTERT (XLF deficient: XLF*) cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdU G2 cells were analysed (n = 3 independent experiments). XLF mutants have much higher levels of unrepaired breaks compared with WT fibroblasts due to their defect in canonical non-homologous end joining, a major pathway for repairing IR-induced DSBs in G2 (compare to Extended Data Fig. 3f). d, Chromatid breaks and fusions after IR and CPT in G2 HeLa cells. Cells were transfected with siRNAs and treated with IR (2 Gy X-rays) or CPT (20 nM, 1 h). Chromosome spreads were obtained by PCC 8-10 h post IR/CPT treatment. Chromatid breaks and fusions were quantified per spread and normalized to 70 chromosomes (n = 4 independent experiments, except siBRCA2 + siPOLθ/siBRCA2 + siRAD52 + siPOLθ: n = 3 independent experiments). Examples of chromosome spreads with breaks (red arrows) and fusions (blue arrows) in irradiated HeLa cells are shown (scale bar, 5 µm). All data show the mean ± s.e.m., except for conditions in (b) with n = 2 independent experiments for which only the mean is shown. Individual experiments, each derived from 30 (G2 spreads) or 40 cells (G1 and G2), are shown as dots. *: P < 0.05; **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 5 Elevated end joining frequencies arise after co-depletion of BRCA2 and RAD52.

a, Diagram of the reporter assay monitoring end joining (EJ) events utilizing micro-homologies (modified from ref. 24). The U2OS EJ2 reporter cells carry a GFP cassette which is preceded by an I-SceI recognition sequence and stop codons in all reading frames flanked by two 8 bp regions of homology (shown in red). Repair events that result in the removal of the stop codons and maintain the GFP reading frame will result in expression of functional GFP. The majority of such events use the 8 bp homologous regions for annealing, leading to a deletion of 35 bp and the formation of a XcmI restriction site24. b, Frequency of GFP + U2OS EJ2 reporter cells. Cells were transfected with siRNAs and treated with triamcinolone acetonide (TA) (200 nM) to induce I-SceI localization to the nucleus. (i) Upon break induction, the percentage of GFP + cells was quantified using microscopic analysis and normalized to the siCTRL sample (n = 3 independent experiments). In addition, GFP − and GFP + cells were sorted, followed by DNA extraction and PCR amplification across the break site. The final products were then subjected to a restriction test as follows: (ii) Molecular analysis of control GFP − - and GFP + -sorted cells. For GFP − cells, the PCR products across the break site can be efficiently cut by I-SceI but not by XcmI. For GFP + cells, the products can be cut by XcmI but not by I-SceI, indicating that the original recognition sequence is lost and a XcmI restriction site is formed due to micro-homology usage at the 8 bp repeat. (iii) Molecular analysis of GFP + -sorted cells. For all siRNA conditions, the PCR products across the break site can be efficiently cut by XcmI but not by I-SceI, as seen in the GFP + control in (ii). All data show the mean ± s.e.m. Individual experiments, each derived from 10,000 events, are shown as dots. **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 6 Absent or deregulated TMEJ causes distinct mitotic aberrations.

a, Spontaneous γH2AX foci in G1 and G2 U2OS cells. U2OS WT and KO cells were transfected with siRNAs, labelled with EdU for 1 h and foci were immediately analysed after labelling in EdU cells in G1 and G2 (n = 3 independent experiments). b, Alternative representation of the data in Fig. 4e. U2OS WT and KO cells were transfected with siRNAs and ana/telophase cells were analysed for the presence of lagging chromosomes and anaphase bridges. Cells were identified by their morphology seen in the DAPI and phospho-histone H3(S10) (pH3) stainings. The percentage of cells showing 0 to ≥4 spontaneous lagging chromosomes or 0 to ≥3 anaphase bridges is represented. (combined raw data from n = 3 independent experiments). c, Spontaneous anaphase bridges in mitotic HeLa cells. Cells were transfected with siRNAs and anaphase cells were analysed for the presence of anaphase bridges. Cells were identified by their morphology seen in the DAPI and phospho-histone H3(S10) (pH3) stainings (n = 3 independent experiments). All data show the mean ± s.e.m. Individual experiments, each derived from 40 (G2 foci and anaphase bridges), 80 (lagging chromosomes) or 100 cells (G1), are shown as dots. ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 7 The RPA and SELF, but not the RAD51, binding domains of RAD52 are essential for suppressing TMEJ.

a, Diagram of GFP–RAD52 WT and mutant proteins (left panel). Tables show amino acid positions of the deleted RAD52 domains and the molecular weights of the resulting GFP–RAD52 mutant proteins (right panel). b, (i) Expression levels of GFP–RAD52 mutant proteins were analysed by immunoblotting in two independent clones for each mutant (#1 and 2). (ii) Depletion of endogenous RAD52 with the single siRNA was confirmed by immunoblotting in the control cell line (GFP only). Vinculin was used as loading control. c, γH2AX and pRPA foci in G2 HeLa cells carrying different GFP–RAD52 constructs (clone #2 of each mutant). Cells were transfected with siRNAs, labelled with EdU, irradiated with 2 Gy X-rays and EdUGFP + G2 cells were analysed (n = 3 independent experiments). All data show the mean ± s.e.m. Individual experiments, each derived from 40 cells, are shown as dots. *: P < 0.05; **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.

Source data

Extended Data Fig. 8 RAD52 and BRCA2 inhibit the polymerase function of POLθ via their ssDNA binding modes.

a, Gel images of purified human BRCA2, RAD52, RAD51, RPA and POLθ. 200 ng BRCA2, 2 µg RAD52, 1 µg RAD51, 2 µg RPA and 500 ng POLθ were loaded. The visible contaminants for POLθ include HSP70, HSP90 and tubulin. The gels shown are single experiments. b, Titration of POLθ and E. coli PolI in the primer-extension assay using the 80 nucleotide 5’-overhang substrate. c, Titration of human RAD52 in the primer-extension assay using the 80 nucleotide 5’-overhang substrate. d, Titration of BRCA2 in the primer-extension assay using the 80 nucleotide 5’-overhang substrate. e, Effect of human RAD52 (hRAD52) and S. cerevisiae Rad52 (yRad52) on POLθ-mediated DNA synthesis in the presence or absence of 4 nM human RPA and 100 µM ATP. A titration of hRAD52 in the primer-extension assay using the 80 nucleotide 5’-overhang substrate was performed. A single concentration for yRad52 was also tested. f, Effect of BRCA2 on POLθ-mediated DNA synthesis in the presence or absence of 4 nM human RPA (hRPA) or S. cerevisiae RPA (yRPA) and 100 µM ATP. A titration of BRCA2 in the primer-extension assay using the 80 nucleotide 5’-overhang substrate was performed. g, Titration of hRAD52 and yRad52 in the primer-extension assay using the 80 nucleotide 5’-overhang. The schemes of the reactions are shown in the diagrams at the top. Protein concentrations are indicated. DNAs were analysed on 10% non-denaturing gels. Extension values are normalized as described in the Methods section. For (e,f): All the percentages of extended products are normalized to POLθ-only reaction (±ATP). Representative gels are shown for the quantitation in Fig. 6b, which reports mean ± s.e.m. (n = 3 independent experiments). For (b): Result from a single experiment is shown. The result was consistent with results from two additional experiments performed under slightly varying conditions. For (c,d,g): Results from single experiments are shown that were performed once under the condition shown, and once with slightly varying conditions with consistent results. Source data are available online.

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Extended Data Fig. 9 RPA and RAD51 don’t inhibit the polymerase function of POLθ.

a, Titration of ssDNA binding proteins (human RPA: hRPA, S. cerevisiae RPA: yRPA and the phage ssDNA binding protein: T4 gp32) in the primer-extension assay using the 80 nucleotide 5’-overhang substrate. The ratio between protein and DNA is indicated as molar ratio. Human or yeast RPA fully covers the available ssDNA at the 2:1 ratio, and gp32 fully covers all ssDNA at the 20:1 ratio. b, Titration of human RAD51 in the primer-extension assay using the 80 nucleotide 5’-overhang substrate with the 5’-Cy5-labelled 20-mer. RAD51 fully covers the available ssDNA at the 1 RAD51 per 3 nucleotide ratio (1:3). The schemes of the reactions are shown in the diagrams at the top. Protein concentrations are indicated. DNAs were analysed on 10% non-denaturing gels for (a) and on 15% denaturing gels for (b). Extension values and unutilized primer values are normalized as described in the Methods section. Results from single experiments are shown. The result in (b) was consistent with the result from one additional experiment performed under slightly varying conditions. Source data are available online.

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Extended Data Fig. 10 Immunoblotting and qPCR analyses confirming siRNA efficiencies.

a, Depletion of RAD52 and BRCA2 in HeLa cells, as well as in 82-6 hTERT (WT) and HSC-62 hTERT (BRCA2 mutant: BRCA2*) fibroblasts, was confirmed by immunoblotting. GAPDH, KU70 and KU80 were used as loading controls (data shown represent 3 independent experiments for HeLa and WT, and 2 independent experiments for BRCA2*). b, Depletion of POLθ was confirmed by qPCR, due to the lack of reliable antibodies for immunoblotting. The standard siRNA used for most experiments (siPOLθ_1; referred to as siPOLθ within the manuscript), as well as an independent siRNA (siPOLθ_6) were tested (n = 3 independent experiments, except siCTRL/siPOLθ_1 in fibroblasts: n = 4 independent experiments). All data show the mean ± s.e.m. Individual experiments are shown as dots. **: P < 0.01; ***: P < 0.001 (One-way ANOVA was used to compare data from siPOLθ_1, while an unpaired two-tailed t-test was used for comparing samples from siPOLθ_6). The exact P values are provided as source data. c,d, Depletion of RAD51, RAD52, BRCA2 and LIG4 in HeLa cells was confirmed by immunoblotting. Vinculin, GAPDH and KU80 were used as loading controls. e, Depletion of BRCA2, RAD54, RAD52 and RAD51 (left), as well as PALB2 (right) in HeLa GFP–RAD52 cells was confirmed by immunoblotting. KU80 and GAPDH were used as loading controls. Source data are available online. Data shown represent 2 independent experiments in c and e, and 3 independent experiments in d.

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Supplementary information

Reporting Summary

Supplementary Table 1

List of oligonucleotides used in this study.

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Llorens-Agost, M., Ensminger, M., Le, H.P. et al. POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis. Nat Cell Biol 23, 1095–1104 (2021). https://doi.org/10.1038/s41556-021-00764-0

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