Cryptochromes (CRYs) act as blue-light photoreceptors that regulate development and circadian rhythms in plants and animals and as navigating magnetoreceptors in migratory birds. DNA double-strand breaks (DSBs) are the most serious type of DNA damage and threaten genome stability in all organisms. Although CRYs have been shown to respond to DNA damage, whether and how they participate in DSB repair is not well understood. Here we report that Arabidopsis CRYs promote the repair of DSBs through direct interactions with ADA2b and SMC5 in a blue-light-dependent manner to enhance their interaction. Mutations in CRYs and in ADA2b lead to similar enhanced DNA damage accumulation. In response to DNA damage, CRYs are localized at DSBs, and the recruitment of SMC5 to DSBs is dependent on CRYs. These results suggest that CRY-enhanced ADA2b–SMC5 interaction promotes ADA2b-mediated recruitment of SMC5 to DSBs, leading to DSB repair.
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All data generated or analysed during this study are included in this article and its supplementary files. The sequence data from this article can be found in the EMBL/GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) or the Arabidopsis Genome Initiative database (https://www.arabidopsis.org/) under the following accession numbers: CRY1 (AT4G08920), CRY2 (AT1G04400), ADA2a (AT3G07740), ADA2b (AT4G16420) and SMC5 (AT5G15920). Source data are provided with this paper.
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We thank C. Lin, H. Liu, C. Yang and Y. Fang for assistance with materials. This work was supported by the National Natural Science Foundation of China grant no. 32000183 to T.G.
The authors declare no competing interests.
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a-c, Yeast two-hybrid assays showing the interactions of CRY1 (a, b) and CRY2 (c) with ADA2a. Yeast cells co-expressing the indicated combinations of constructs were grown on SD-Trp-Leu and selective medium SD-Trp-Leu-His with 25 mM 3-AT (a) or SD-Trp-Leu-His-Ade (b, c) in continuous darkness (Dark) or blue light (BL, 30 μmol/m2/s).
a, b, Pull-down assays showing the interactions of CRY1 (a) and CRY2 (b) with ADA2a. GST-ADA2a served as bait. His-TF, His-TF-CRY1, His-TF-CNT1, His-TF-CRY2, and His-TF-CNT2 served as preys. c, d, Pull-down assays showing the interactions of CRY1 (c) and CRY2 (d) with ADA2a, ADA2b, and SMC5. GST-ADA2a, GST-ADA2b, GST-SMC5 and GST served as baits, His-TF-CRY1 and His-TF-CRY2 served as preys. Input images show CBB staining. Prey proteins pulled down by bait were detected with anti-His antibody.
a, Protein co-localization assays showing that CRY1 and CRY2 were co-localized with ADA2a in the same foci of tobacco leaf cell nuclei. Scale bar, 5 μm. b, c, Split-LUC assays indicating the interactions of CRY1 (b) and CRY2 (c) with ADA2a in tobacco.
Extended Data Fig. 4 Disruptions of CRY1 and CRY2 do not affect DNA damage accumulation in red light.
a, b, WT and cry1 cry2 mutant seedlings showing similar root development under red light. The seedlings were grown in red light (30 μmol/m2/s) for 7 d before they were photographed (a) and their root lengths were measured (b). Scale bar, 5 mm. The data are mean ± SD (n = 30). c, d, WT and cry1 cry2 mutant roots showing similar root apical meristem regions under red light. The seedlings were grown in red light (30 μmol/m2/s) for 7 d before they were stained with propidium iodide (PI). The root apical meristem regions of the seedlings were indicated between the white arrowheads (c), and the number of cells within the root apical meristem was measured (d). Scale bars, 50 μm. The data are mean ± SD (n = 15 seedlings per each genotype). e, f, Comet assays (e) and statistical analyses (f) showing no difference in accumulation of damaged DNA in cry1 cry2 mutant and WT under red light. The leaves of WT and cry1 cry2 seedlings grown under red light (30 μmol/m2/s) for 2 weeks were subjected to comet assays. Scale bars, 50 μm. The statistical data for DNA in tail are mean ± SD (n = 15). Statistical significance was analyzed by t test. ns, no statistical significance (b, d, f).
a, Measurements of relative plant fresh weights showing that ada2b and cry1 cry2 mutants are hypersensitive to UV-C treatments in blue light. Seven-day-old red light (100 μmol/m2/s)-grown plants were irradiated with 1.5, 3, 4.5 and 6 kJ/m2 UV-C once a day for 3 d, respectively, and then grown under blue light (30 μmol/m2/s) for 5 d. Plants were randomly collected into a pool of 15 individuals for relative fresh weight measurements. b, Measurements of relative plant fresh weights showing hypersensitivity of cry mutants to UV-C treatment under blue light. UV-C-treated seedlings grown in 5, 30 and 50 μmol/m2/s blue light (BL5, 30 and 50) for 5 d in Fig. 2h were randomly collected into a pool of 15 individuals for fresh weight measurements. The curves above the columns depict the trends of relative fresh weight loss. c, d, Plant growth assays (c) and relative fresh weight measurements (d) showing that cry mutants and WT display similar sensitivity to UV-C treatment under red light. Plants were randomly collected into a pool of 15 individuals for relative fresh weight measurements. Relative fresh weight was calculated by comparing with untreated WT and mutants, respectively. Data are presented as mean ± SD (n = 3) (a, b, d). Statistical significance was analyzed by two-way ANOVA (a, b) or one-way ANOVA (d) followed by Tukey’s multiple comparisons test. ns, no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (a, b, d).
Extended Data Fig. 6 Overexpression of CRY1 or CRY2 leads to enhanced tolerance to DNA damage induced by UV-C irradiation in blue light.
a-d, Plant growth assays (a, c) and relative plant fresh weight analyses (b, d) showing that overexpression of CRY1 or CRY2 results in enhanced tolerance to UV-C treatment under blue light (a, b), but not under red light (c, d). Seven-day-old red light (100 μmol/m2/s) -grown seedlings were irradiated with UV-C (4.5 kJ/m2) once a day for 3 d and then grown in blue light (30 μmol/m2/s) or red light (30 μmol/m2/s) for 5 d before they were photographed (a, c) and their relative plant fresh weights were analyzed (b, d). Plants were randomly collected into a pool of 15 individuals for plant fresh weight measurement. Relative weight was calculated by comparing with untreated WT and mutants respectively. The relative fresh weight is presented as mean ± SD (n = 4) (b) or (n = 3) (d). ns, no statistical significance, **P < 0.01, ****P < 0.0001 (One-way ANOVA, Tukey’s multiple comparisons test).
a, b, Plant growth assays (a) and relative fresh weight measurements (b) showing enhanced sensitivity of cry1 cry2 and ada2b mutants to Zeocin under blue light. Five-day-old blue light (30 μmol/m2/s)-grown seedlings were transferred onto MS medium supplemented with Zeocin (75 μg/mL) and then grown in blue light for 21 d before they were photographed, and their relative plant fresh weights were analyzed. Relative plant weight was calculated by comparing with untreated WT and mutants, respectively. Data are presented as mean ± SD (n = 4). ****P < 0.0001 (One-way ANOVA, Tukey’s multiple comparisons test). c-f, Plant growth assays (c, e) and relative fresh weight measurements (d, f) showing that cry1 cry2 mutant and WT display similar sensitivity to MMS (c, d) and Zeocin (e, f) under red light. Five-day-old red light (30 μmol/m2/s)-grown seedlings were transferred onto MS medium supplemented with MMS (100 ppm) (d) or Zeocin (75 μg/mL) (f) and then grown in blue light for 14 d before they were photographed, and their relative plant fresh weights were analyzed. Plants were randomly collected into a pool of 15 individuals for plant fresh weight measurement (b, d, f). Relative plant weight was calculated by comparing with untreated WT and mutants, respectively. Data are presented as mean ± SD (n = 6)(d, f). ns, no statistical significance (t test).
Extended Data Fig. 8 CRY1 and CRY2 are co-localized with γ-H2AX during DNA damage under blue light, but not in red light.
a, Another duplicate of immunofluorescence staining assays related to Fig. 3a,b showing that the endogenous CRY1 and GFP-CRY2 are co-localized with γ-H2AX under blue light upon MMS treatment. b, Immunofluorescence staining assays showing that the endogenous CRY1 and GFP-CRY2 are not co-localized with γ-H2AX under red light upon MMS treatment. Etiolated WT or GFP-CRY2-OX/cry1 cry2 seedlings were incubated in liquid half MS with or without MMS (125 ppm) for 30 h, and then exposed to blue light (30 μmol/m2/s) (a) or red light (30 μmol/m2/s) (b) for 15 min before they were fixed. The nuclei isolated from these seedlings were immunostained with anti-CCT1 and/or -γ-H2AX antibodies. The DAPI, γ-H2AX (Alexa Fluor 555), and CRY1 (Alexa Fluor 594) or GFP-CRY2 signals were detected by confocal microscopy. Scale bars, 5 μm.
Extended Data Fig. 9 The recruitment of ADA2b and SMC5 to DSBs is independent of and dependent on CRYs, respectively.
a, Immunoblot assays showing similar expression levels of YFP-ADA2b and SMC5-YFP fusion proteins in WT and cry1 cry2 backgrounds, respectively. Total proteins were extracted from YFP-ADA2b-OX, YFP-ADA2b-OX/cry1 cry2, SMC5-YFP-OX, SMC5-YFP-OX/cry1 cry2 seedlings grown in white light for 7 d, followed by immunoblot analyses with anti-GFP and -HSP82 antibodies, respectively. b-f, Confocal microscopy assays showing that the recruitment of ADA2b (b-d) and SMC5 (e, f) to DSBs is independent of and dependent on blue light and CRYs, respectively. Before subjected to confocal microscopy, YFP-ADA2b-OX and SMC5-YFP-OX/cry1 cry2 seedlings treated with MMS or without MMS (control) were kept in the dark (b, e), YFP-ADA2b-OX/cry1 cry2 seedlings without MMS treatment (control) were illuminated with blue light (30 μmol/m2/s) for 25 h (c), and YFP-ADA2b-OX and SMC5-YFP-OX seedlings treated with MMS were illuminated with red light (30 μmol/m2/s) for 25 h or 30 h, respectively (d, f). Scale bars, 5 μm. g, h, Statistic analyses of the nuclei with different numbers of foci isolated from the seedlings corresponding to b, c, e and Fig. 3c,d. The numbers of foci in 10 pools of 30 nuclei from five independent seedlings were counted for each sample and used to generate the diagrams. Data are presented as mean ± SD (n = 10). Statistical significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. ns, no statistical significance, ****P < 0.0001.
Extended Data Fig. 10 CRY1 and CRY2 interact with SMC5 in tobacco cells and enhance the interaction of ADA2b with SMC5 in blue light.
a, b, Split-LUC assays indicating the interactions of CRY1 (a) and CRY2 (b) with SMC5 in tobacco leaf cells. c, Protein co-localization assays showing co-localization of CRY1 and CRY2 with SMC5 in the same nuclear foci in tobacco leaf cells, respectively. Scale bars, 5 μm. d, e Pull-down assays showing that blue light-activated CRY1 (d) or CRY2 (e) promotes the association of ADA2b with SMC5. GST-SMC5 served as bait, and MBP-ADA2b served as prey. The protein extracts from Myc-CRY1-OX or Myc-CRY2-OX seedlings adapted in the dark and then exposed to red light (50 μmol/m2/s) or blue light (50 μmol/m2/s) for 15 min or the protein extracts from cry1 cry2 seedlings adapted in the dark and then exposed to blue light (50 μmol/m2/s) for 15 min served as effectors. The bait protein (GST-SMC5) and the prey protein (MBP-ADA2b) pulled down by GST-SMC5 was detected with anti-GST and -MBP antibodies, respectively. f, g, Co-IP assays showing that blue light-excited Myc-CRY1 (f) or Myc-CRY2 (g) enhances the interaction of ADA2b with SMC5. Protein extracts from white light-grown SMC5-Flag-OX/cry1 cry2 or YFP-ADA2b-OX/cry1 cry2 seedlings served as bait (SMC5-Flag) and prey (YFP-ADA2b), respectively. The effector was the same as described in d, e. h, Co-IP assays showing that blue light-excited endogenous CRY1/CRY2 enhance the interaction of ADA2b with SMC5. White light-grown seedlings were adapted in the dark for 3 day and treated with MG132, and then exposed to blue light (50 μmol/m2/s) for 15 min. Protein extracts from SMC5-Flag-OX or SMC5-Flag-OX/cry1 cry2 and YFP-ADA2b-OX or YFP-ADA2b-OX/cry1 cry2 seedlings served as bait and prey, respectively. The IP (SMC5) and co-IP signals (ADA2b) were detected in immunoblots probed with anti-Flag and -GFP antibodies, respectively (f, g, h).
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Guo, T., Liu, M., Chen, L. et al. Photoexcited cryptochromes interact with ADA2b and SMC5 to promote the repair of DNA double-strand breaks in Arabidopsis. Nat. Plants 9, 1280–1290 (2023). https://doi.org/10.1038/s41477-023-01461-6
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