Clathrin is required for DNA damage repair

Genetic screening in Arabidopsis reveals that clathrin, a well-known regulator of endocytosis, is required for homologous recombination, a precise mechanism for repairing DNA double- strand breaks. Notably, CLATHRIN LIGHT CHAIN 2 localizes in the nucleus, suggesting that clathrin has non-canonical functions in the nucleus. Maintenance of genome stability is crucial for the survival and reproduction of all organisms. However, various exogenous and endogenous factors frequently induce DNA damage, threatening genome stability. Therefore, all organisms have evolved complex and sophisticated DNA damage response (DDR) mechanisms including transcription reprogramming, cell cycle arrest, DNA repair, and cell death (Ciccia and Elledge, 2010). Compared with the research in mammals and yeasts, the DDR mechanisms in plants are far less well-understood (Herbst et al., 2024). To identify new regulators of DDR in plants, we performed a forward genetic screening in Arabidopsis for DNA Damage Response Mutants (DDRMs) using bleomycin (BLM), which causes double-strand breaks (DSBs), the most toxic forms of DNA damage. The wild-type (WT) Arabidopsis seeds were mutagenized with ethyl methanesulfonate (EMS), and the M2 seeds were grown vertically on medium containing BLM for 8 d. The plants with shorter or longer roots than WT were considered as ddrms. We have previously reported ddrm1, ddrm2, ddrm4, and ddrm6 (Li et al., 2021, 2023; Wang et al., 2022; Yu et al., 2023). In this study, we report ddrm23. Compared with WT, the root length of the ddrm23 mutant was similar under the control condition, but was significantly shorter under BLM treatment (Figure 1A, B), suggesting that DDRM23 is required for DDR. Clathrin is required for DNA damage repair (A, B, E–H, M, and N) The plants were grown vertically on a control medium or medium containing 4 µmol/L bleomycin (BLM) for 8 d. Photographs (A, E, G, and M) and the relative root lengths of plants (B, F, H, and N) are shown. Scale bars, 1 cm. The relative root length data are represented as means ± SD (n > 10) relative to the values obtained under the control condition. The statistical significance was determined using two-way analysis of variance. ***P < 0.001; ****P < 0.0001. (C) Map-based cloning of DAMAGE RESPONSE MUTANT 23 (DDRM23). In total, 200 individual plants were used and the numbers of recombinants at each marker are shown. (D) The genomic structure of Clathrin Heavy Chain 1 (CHC1). Black boxes indicate exons and lines indicate introns. Gray box indicates 5' untranslated region. ATG and TGA indicate the start and stop codons, respectively. The mutation site of ddrm23, the T-DNA insertion site of chc1-1 and chc1-5, and the sites of primers are indicated. (I) Schematic representation of the homologous recombination (HR) reporter system. The reporter line harbors an I-SceI restriction site located between two nonfunctional β-glucuronidase (GUS) fragments and a nearby donor sequence (U). When the single double-strand break (DSB) is repaired through HR, the functional GUS is restored. (J) Representative GUS staining images of cotyledons. The reporter line and trigger line in either chc1-5 or wild-type (WT) background were crossed and the resulting F1 seedlings were used for GUS staining. Scale bars, 1 mm. (K) The relative HR efficiency. The HR efficiency in WT was set to 1.0. Data represent mean ± SD of 60 plants in each genetic background. The statistical significance was determined using Student's t-test, ****P < 0.0001. (L) Subcellular localization of CHC1 and CLC2. CHC1-GFP (green fluorescent protein) or CLC2-GFP was co-expressed with nuclear localization signal (NLS)-mCherry in Arabidopsis protoplasts treated with BLM. The pictures were captured using confocal microscopy. Scale bars, 10 μm. To clone the DDRM23 gene, we first used a map-based cloning approach (Lukowitz et al., 2000). The ddrm23 mutant was crossed to Landsberg erecta (Ler), and plants with shorter roots in the F2 population were selected for mapping. The DDRM23 locus was mapped to the region between the markers T7M13 and T19F11 on Chromosome 3 (Figure 1C). Then, we employed MutMap strategy (Abe et al., 2012). The ddrm23 mutant was backcrossed to WT. In the F2 population, the plants with shorter roots were pooled for DNA extraction, followed by whole-genome sequencing using the next-generation sequencing technology. Data analysis revealed that the conserved splice donor site in the 21st intron of AT3G11130 was mutated from g to a (Figure 1D). Polymerase chain reaction (PCR) analysis suggested that the splicing of the 21st intron was indeed affected in ddrm23 (Figure S1A). Moreover, reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the transcription level of AT3G11130 in ddrm23 was dramatically reduced (Figure S1B). AT3G11130 encodes Clathrin Heavy Chain 1 (CHC1). To confirm that CHC1 is DDRM23, the CHC1-GFP (green fluorescent protein) fusion driven by the CaMV 35S promoter was transformed into the ddrm23 mutant. The root lengths of the resulting transgenic lines were similar to that of WT in the presence of BLM (Figure 1E, F), indicating that CHC1 complements the ddrm23 mutant. Furthermore, we tested the BLM sensitivity of chc1-1 and chc1-5, two T-DNA insertion mutants of CHC1. As expected, both chc1-1 and chc1-5 were more sensitive to BLM than WT (Figure S2), mimicking ddrm23. Additionally, we crossed ddrm23 with chc1-5 and found that all the resulting F1 seedlings were hypersensitive to BLM (Figure 1G, H), suggesting that DDRM23 and CHC1 are allelic. All together, these results demonstrated that CHC1 is required for the resistance to BLM. The hypersensitivity of chc1 mutants to BLM suggested that CHC1 may be involved in DSB repair. One of the important DSB repair pathways is homologous recombination (HR). To test whether CHC1 is involved in HR, we examined the HR efficiency of chc1-5 using the HR reporting system including a reporter line and a trigger line (Roth et al., 2012). The reporter line harbors an I-SceI restriction site within the two nonfunctional β-glucuronidase (GUS) fragments and a nearby donor sequence (U) in direct orientation. The trigger line expresses the endonuclease I-SceI. When the reporter line is crossed with the trigger line, I-SceI induces DSB, which can be repaired through HR and thus produces a functional GUS gene, resulting in blue sectors after GUS staining (Figure 1I). We found that the HR efficiency in chc1-5 was significantly reduced, about 30% of that in WT (Figure 1J, K), suggesting that CHC1 is required for HR. CHC1 is a subunit of clathrin, which is an evolutionarily conserved vesicle coat protein complex. It is known to localize at the plasm membrane and trans-Golgi network/early endosome, where it regulates endocytosis (Wang et al., 2023). Given that DSB occurs in the nucleus, the involvement of CHC1 in HR suggested that CHC1 may go into the nucleus after BLM treatment. Therefore, we transiently expressed 35S:CHC1-GFP in Arabidopsis protoplasts. However, no CHC1-GFP fluorescence was observed in the nucleus (Figure 1L). Clathrin is composed of three CHC and three Clathrin Light Chains (CLC). Arabidopsis encodes two CHC genes and three CLC genes. The absence of CHC1 in the nucleus prompted us to test whether CLCs go into the nucleus. Among three CLCs, we found that CLC2-GFP was able to localize in the nucleus (Figure 1L). This interesting result led us to test whether CLC2 also regulates DSB repair. Indeed, the clc2-1 mutant exhibited hypersensitivity to BLM compared with WT (Figure 1M, N), indicating that CLC2 is required for DSB repair. In summary, through genetic screening, we identified CHC1 as a novel regulator of DSB repair. The HR efficiency was dramatically reduced in the chc1 mutant. Further study revealed that CLC2, a CHC1 partner, is also required for DSB repair. These genetic data strongly suggested that clathrin is required for DSB repair in plants. Given that clathrin is highly conserved in eukaryotes, it is possible that clathrin regulates DNA repair in other eukaryotes. However, the underlying mechanism remains to be further studied. It is of note that CLC2 localizes in the nucleus (Figure 1L), raising the possibility that clathrin may have other functions in addition to its canonical function in endocytosis, which is worthwhile for future exploration. We are grateful to Dr. Chao Wang for providing clc2-1 seeds. This work was supported by the National Natural Science Foundation of China (32270306 and 32070312), HZAU-AGIS Cooperation Fund (SZYJY2022004), and Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (2662024PY019). The authors declare no conflict of interest. S.Y. designed the experiments; T.Y., X.L., and L.D. performed the experiments. S.Y., T.Y., and L.W. wrote the manuscript with input from all other authors. All authors read and approved the contents of this paper. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13910/suppinfo Figure S1. The splicing and transcription of Clathrin Heavy Chain 1 (CHC1) are promised in the damage response mutant 23 (ddrm23) Figure S2. The T-DNA insertion mutants of Clathrin Heavy Chain 1 (CHC1) are more sensitive to bleomycin (BLM) than wild-type (WT) Table S1. Primers used in this study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.