The DREAM complex functions as conserved master regulator of somatic DNA-repair capacities

C. elegans strains

All strains were cultured under standard conditions78 and were always incubated at 20 °C during the experiments. The strains used were N2 (Bristol; WT):

DREAM: MT8839 lin-52(n771) III, MT10430 lin-35(n745) I, MT15107 lin-53(n3368) I/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III), MT8879 dpl-1(n2994) II, MT11147 dpl-1(n3643) II, JJ1549 efl-1(se1) V, BJS634 dpl-1(n2994) II; lin-52(n771) III.

SynMuv B and NuRD: RB951 lin-13(ok838) III, RB1789 met-2(ok2307) III, PFR40 hpl-2(tm1489), MT8189 lin-15(n765) X, MT14390 let-418(n3536) V.

NER: RB1801 csb-1(ok2335) X, FX03886 xpc-1(tm3886) IV, RB864 xpa-1(ok698) I, FX04539 csa-1(tm4539) II, BJS21 xpc-1(tm3886) IV; csb-1(ok2335) X, BJS631 lin-52(n771) III; csb-1 (ok2335) X, BJS629 lin-52(n771) III; xpc-1 (tm3886) IV, BJS630 lin-52(n771) III; xpc-1 (tm3886) IV; csb-1 (ok2335) X, BJS772 xpa-1(ok698) I; lin-52(n771) III, BJS825 csa-1(tm4539) II; lin-52(n771) III.

HRR: DW102 brc-1 (tm1145); brd-1(dw1) III, BJS890 brc-1 (tm1145) III; brd-1(dw1); lin-52(n771) III, BJS868 lin-52(n771) III; brc-1(tm1145) III; brd-1(dw1) III; cku-70(tm1524) III.

NHEJ: FX1524 cku-70(tm1524) III, BJS887 lin-52(n771) III; cku-70(tm1524) III.

MMS: XF132 polh-1(lf31) III, BJS722 polh-1 (lf31) III; lin-52 (n771) III.

UV-irradiation assay during somatic development

The effects of UV-B in worm development were analyzed as previously described79 after bleach synchronization79. UV-B irradiation was performed with a 310-nm PL-L 36W/UV-B UV6 bulb (Waldmann, 451436623-00005077). OP50 Escherichia coli was added to the plates, and worms were incubated for 48 h. Larval stages were determined using a dissecting microscope. For the strain MT15107 lin-53(n3368) I/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III), lin-53(n3368) homozygotes were distinguished using a fluorescence microscope (Leica M165 FC) and assessing the larval stage of worms that did not express green fluorescent protein.

UV-irradiation assay for germline development

Synchronized late-L4 worms were irradiated with different doses of UV-B and allowed to recover for 24 h. Irradiated and mock-irradiated worms were transferred to a fresh seeded NGM plate to lay eggs for 4 h (5 worms per plate). Upon removal of the adults, the plates were incubated for 24 h, after which the number of eggs laid and percentage of eggs that survived and hatched were evaluated.

NHEJ-dependent IR-sensitivity assay

As previously described13,14, L1 worms repair DSBs mainly through NHEJ repair. To analyze worm sensitivity to IR in a NHEJ-dependent way, synchronized L1 worms were irradiated with different doses of IR using an IR-inducing cesium-137 source, and were left for 48 h to allow development. The different larval stages were determined using a dissecting microscope.

HRR-dependent IR-sensitivity assay

Early embryos highly rely on HRR to repair DSBs13,14. To study the capacity of the different strains to tolerate IR-induced DSBs during embryogenesis, day-1 adults were left to lay eggs on seeded NGM plates for no longer than 1.5 h. Upon removal of the adult worms, early eggs were irradiated using an IR-inducing cesium-137 source. After 24 h, the percentage of surviving embryos that hatched was evaluated using a dissecting microscope.

Alkylation-damage induction by using MMS

Synchronized L1 worms were incubated with different concentrations of MMS (Sigma, 129925) diluted in M9 buffer for 1 h. Worms were washed three times with M9 buffer, and plated in seeded NGM plates. After incubation for 48 h, worm development was evaluated with a dissecting microscope.

ICL induction by using cisplatin

Synchronized L1 worms were exposed to different concentrations of cisplatin in dimethylformamide (DMF) diluted in M9 buffer or were mock-treated with DMF (Sigma, 227056)-diluted M9 buffer for 2 h. Worms were washed three times with M9 and incubated for 48 h in NGM plates, and the larval stages were quantified using a dissecting microscope.

Lifespan assay

Synchronized day-1 adult worms were irradiated or mock-irradiated with UV-B light using a 310-nm PL-L 36W/UV-B UV6 bulb (Waldmann, 451436623-00005077) or with IR using an IR-inducing cesium-137 source, and then were placed on fresh OP50-seeded NGM plates. At the beginning of the experiment, the worms were transferred to new plates every other day to avoid progeny overgrowth. Worms presenting internal hatching or protruding or ruptured vulvas were censored and removed from the experiment, and worms were scored as dead when no movement or pumping was observed even upon physical stimulus. Lifespan curves were analyzed with Graphpad Prism 7.03 log-rank test.

DNA-repair capacity assay in L1 worms

The quantification of DNA repair via immunostaining of CPDs and 6-4PPs of DNA samples in a slot blot was performed as has been described, with slight changes79. Bleach-synchronized L1 worms (at least 30,000 per plate) were irradiated with UV-B light and split in two groups, one to be immediately quick-frozen in liquid nitrogen, to serve as controls with unrepaired damage, and the other one was left in seeded plates for 24 h to allow for DNA repair to occur. After this, worms were washed 5 times, incubated for 2 h to permit the removal of intestinal bacteria, washed another 5 times and quick-frozen.

DNA extraction was performed using the Gentra Puregene Tissue Kit (Qiagen, 158667) and the protocol for DNA purification from tissue. The protocol was adapted to increase the volumes, but we still used a 1.5-ml Eppendorf tube to aid the supernatant extraction and pellet formation. That is, instead of the specified amounts, we used 500 µl cell lysis solution, 2.5 µl Puregene Proteinase K, 2.5 µl RNase A solution, 170 µl protein precipitation solution, 500 µl isopropanol and 500 µl 70% ethanol at the respective steps in the protocol. Cell lysis solution was directly added to the thawed sample, and an additional step with Proteinase K was performed. The DNA concentration was measured using the Qubit dsDNA HS Assay Kit (Invitrogen, Q32851). Serial dilutions of the DNA were denatured at 95 °C for 5 minutes (min) and transferred onto a Hybond nylon membrane (Amersham, RPN119B) using a Convertible Filtration Manifold System (Life Technologies, 11055). DNA crosslinking to the membrane was achieved by incubating the membrane at 80 °C for 2 h. The membrane was blocked for 30 min in 3% milk/phosphate-buffered saline (PBS)-T (0.1%) at room temperature. The membrane was incubated overnight at 4 °C with anti-CPD antibodies (Clone TDM-2, 1:10,000, Cosmo Bio, CAC-NM-DND-001) or anti-6-4 PP antibodies (Clone 64M-2, 1:3,000, Cosmo Bio, CAC-NM-DND-002), then washed three times with PBS-T (5 min at room temperature), and blocked for 30 min with 3% milk/PBS-T. The secondary antibody was a goat anti-mouse AffiniPure peroxidase-conjugated secondary antibody (1:10,000, Jackson Immuno Research, 115-035-174). Addition of the secondary antibody was followed by three washes in PBS-T and incubation with ECL Prime (Amersham, RPN2232). The DNA lesions were visualized by using a Hyperfilm ECL (Amersham, 28906836).

In order to quantify the total amount of DNA per sample, the membrane was incubated overnight at 4 °C in PBS with 1:10,000 SYBR Gold Nucleic Acid Stain (Invitrogen, S11494), then washed in PBS at room temperature and imaged using a BIO-RAD Gel Dox XR + Gel Documentation System (BIO-RAD, 1708195).

Adult somatic and germline DNA-repair assay

Synchronized day-1 adult worms were irradiated or mock-irradiated with a 310-nm UV-B light Philips UV6 bulb in a Waldmann UV236B irradiation device. Half of the worms were left in seeded NGM plates for 60 h to allow DNA repair to occur, whereas the others were collected directly after the irradiation.

After irradiation or incubation, worms were picked and placed in a drop of M9 buffer on top of a HistoBond+ Adhesion Microscope Slide (Marienfeld, 0810461). Using a hypodermic needle, we cut the worms close to the head, which also releases one of the germline arms, and then placed a coverslip over the slide and kept it at −80 °C for at least 30 min. After this, the coverslip was removed quickly to perform freeze-cracking80. Worms were fixated in liquid methanol at −20 °C for 10 min, then washed for 5 min in PBS. Seventy microliters of 2 M HCl were added on top of the worms for 30 min at room temperature to denature the DNA. Slides were washed three times with PBS, and blocked with 70 µl of 20% fetal bovine serum (FBS) in PBS for 30 min at 37 °C. The slides were incubated with 70 µl of 1:10,000 anti-CPD antibodies (Clone TDM-2, 1:3,000, Cosmo Bio, CAC-NM-DND-001) in PBS containing 5% FBS, at 4 °C overnight in a humid chamber. After subjecting the slides to three PBS washes, 5 min each, 70 µl of secondary anti-mouse Alexa Fluor 488-conjugated antibody (1:300, Invitrogen, A21202) in 5% FBS PBS was added for 30 min at 37 °C. The slides were washed three times, 5 min each, and mounted using 5 µl of Fluoromount-G with DAPI (Invitrogen, 00495952). Images were obtained using a SP8 Confocal Microscope by Leica using LAS X 3.5.7 software.

Image quantification of CPDs

Image stacks of the heads and germlines of adult worms were analyzed using the analysis software Imaris 9.9 (Oxford Instruments). Nuclei in the area anterior to the pharyngeal-intestinal valve and germlines were determined by using DAPI staining and setting a threshold of size and intensity. False-positive nuclei (due to bacteria in the pharynx) were manually discarded. The CPD signal was quantified using the maximum spherical volume fitting inside each of the nuclei. Owing to variable background signal in the germlines, background intensity was subtracted in these samples.

Cell culture and treatments

U2OS (ATCC, HTB-96) were cultured in DMEM, high-glucose GlutaMAX supplement, pyruvate (Thermo Fisher Scientific, 31966047) with 10% fetal bovine serum (FBS; Biochrom, S0615) and 1% penicillin–streptomycin (Thermo Fisher Scientific, 15140112). Cells were kept at 37 °C in a 5% CO2 incubator (Binder). Cell dissociation was performed with Accutase (Sigma, A6964). To promote quiescence, cells were cultivated in FBS-free medium for 48 h before genotoxic treatment. After 24 h of culture in FBS-free medium, cells were mock treated or received harmine hydrochloride (diluted in water) or INDY (diluted in DMSO) (Sigma, SMB00461 and SML1011) at 10 or 25 µM, respectively. Before the genotoxic treatment, cells were washed with FBS-free medium. For the UV treatment, medium was removed from the plates and cells were irradiated using 254-nm UV-C light Philips UV6 bulbs with 2 mJ/cm2. The MMS treatment was performed by adding MMS at 2 mM for 2 h, followed by 3 washes with FBS-free medium. Then, FBS-free medium was added. Quantification via flow cytometry of cell death and apoptosis was performed 24 h after genotoxic treatment.

Flow cytometry analysis

Collected cells were incubated in annexin V binding buffer (BioLegend, 422201) with Pacific Blue annexin V (BioLegend, 640917) and 7-AAD (Thermo Fisher Scientific, 00699350) at 4 °C for 15 min. Cells were measured using a MACSQuant VYB (Miltenyi Biotec) using MACSQuantify software 2.13.0 and analyzed using FlowJo v10.7.1 (BD). The gating strategy can be found in Supplementary Figure 1.

Animal handling

All animals were maintained in their breeding cages on a 12-h light/dark cycle. Mice were kept on a regular diet and had access to water ad libitum. Body weight was measured weekly. Animals were housed in a temperature- (18–23 °C) and humidity-controlled (40–60%), pathogen-free animal facility at the Institute of Molecular Biology and Biotechnology (IMBB), which operates in compliance with the ‘Animal Welfare Act’ of the Greek government, using the ‘Guide for the Care and Use of Laboratory Animals’ as its standard. All experiments were performed under the Animal license 6ΛΤΑ7ΛΚ-ΚΚΘ, issued by the Veterinary Medicine Directorate of Greek Republic.

Mice experiments

Male and female FVB/nj:C57BL/6j Ercc1–/– and their respective control WT mice81, on the third day after birth (postnatal day P3), were injected intraperitoneally 3 times per week with 10 mg/kg body weight of harmine hydrochloride (SMB00461, Sigma) diluted in 0.9% sodium chloride. Mice were euthanized at postnatal day P15 for retina tissue isolation. Tissues were embedded in optimal cutting temperature (OCT) compound, cryosectioned and stained using the in situ cell death detection kit (TUNEL staining) (11684817910, Roche), according to the manufacturer’s instructions.

For the immunostaining experiments against γH2AX (Millipore, 05–636), retina slices were fixed in 4% formaldehyde in 1× PBS for 10 min at room temperature, permeabilized with 0.5% Triton X-100 in 1× PBS for 10 min, on ice, and blocked with 1% BSA in 1× PBS for 1.5 h at room temperature. After overnight incubation with the primary antibody (1:12,000, in 1% BSA/1× PBS, 4 oC), a secondary fluorescent antibody was added (goat anti-mouse IgG-Alexa Fluor 555, 1:2,000, Invitrogen, A-21422) and DAPI (1:20,000, Thermo Fisher Scientific, 62247) was used for nuclear counterstaining.

Samples were visualized with an SP8 TCS laser scanning confocal microscope (Leica). The detection of nuclei and signal intensity from retinas was performed utilizing Imaris 9.9 (Oxford Instruments).

RNA extraction for RNA-seq and qPCR experiments

For the qPCR and RNA-seq of L1 worms, around 10,000 (qPCR) or 40,000 (RNA-seq) bleach-synchronized L1 worms in triplicates (qPCR) or quadruplicates (RNA-seq) per strain and condition were placed in seeded NGM plates for 3 h. They were mock-treated or UV-B irradiated, and left for 6 h to allow the DNA-damage-related transcriptional changes to take place. Worms were collected and washed three times with M9 buffer, and the pellet was placed in a tube containing 1 ml TRIzol (Invitrogen, 15596018) and 1 mm zirconia/silica beads (Biospec Products, 11079110z).

To extract the RNA, worms were disrupted with a Precellys24 (Bertin Instruments, P000669-PR240-A), and the RNA isolation was performed by using the RNeasy Mini Kit (QIAGEN, 74106) following the manufacturer’s specifications, except we used 1-bromo-3-chloropropane (Sigma, B9673) instead of chloroform. The RNA was quantified using NanoDrop 8000 (Thermo Fisher Scientific, ND-8000-GL).

RNA extraction from U2OS cells was performed after 24 h of harmine or INDY treatment of cells that had been starved for a total of 48 h by using the RNeasy Mini Kit (QIAGEN, 74106), following the manufacturer’s specifications. Cells were disrupted with RLT buffer and homogenized with QIAshredder spin columns (QIAGEN, 79656).

qPCR

Reverse transcription to form complementary DNA (cDNA) was performed using Superscript III (Invitrogen, 18080044). The obtained cDNA was used to perform qPCR by using SYBR Green I (Sigma, S9460) and Platinum Taq polymerase (Invitrogen, 10966034) in a BIO-RAD CFX96 real-time PCR machine (BIO-RAD, 1855196). The analysis of the results was performed by using the comparative CT method82.

All C. elegans qPCR experiments were done in biological triplicates, and the data were normalized to three housekeeping genes. qPCR CT values were obtained using Bio-Rad CFX Manager 3.0.

C. elegans qPCR primers

Primers used for PCR were as follows:

Housekeeping genes:

Forward primer

Reverse primer

Y45F10D.4

5′-CGAGAACCCGCGAAATGTCGGA-3′

5′-CGGTTGCCAGGGAAGATGAGGC-3′

eif-3.C

5′-ACACTTGACGAGCCCACCGAC-3′

5′-TGCCGCTCGTTCCTTCCTGG-3′

vha-6

5′-CTGCTATGTCAATCTCGG-3′

5′-CGGTTACAAATTTCAACTCC-3′

Genes of interest:

   

parp-1

5′-AGCGAATGAAGAAACAATCCGA-3′

5′-ACTAGGCGTTCGATTACTTGTG-3′

polh-1

5′-AGAAATATCGCGACGCTAGC-3′

5′-GTAGGTAATAGCAGCCTGCA-3′

polk-1

5′-GAGATACTGATGGAGAATCTTGAG-3′

5′-AGTAGTTGGATGTGCTCAGC-3′

mus-101

5′-TCGAAAGCCATATACGATGAACC-3′

5′-ACAAGAACGGGAGTACTAGAGAC-3′

exo-3

5′-GGAGGAGACGTTTAAGAACTACAC-3′

5′-TAGATCACTGGCTTCTTCTCGT-3′

lig-1

5′-TGATCAAGGCTGTTGCTAAAGC-3′

5′-AGCCTCAATTCCTTGACATGC-3′

atm-1

5′-GCGAAGTTCTTACACCTCGAC-3′

5′-AGTTCGACACATTCTTCAGCA-3′

RNA-seq

A triplicate of RNA samples from lin-52 mutant and WT L1 worms were rRNA-depleted using Ribo-Zero Plus rRNA Depletion Kit (Illumina, 20037135) and sequenced using a Hiseq4000 (Illumina) with PE75 read length. For RNA quality control, the RNA integrity number was ≥9.4 for all samples. RNA-seq data were processed through the QuickNGS pipeline83, Ensembl version 85. Reads were mapped to the C. elegans genome using Tophat84 (version 2.0.10) and abundance estimation was done using with Cufflinks85 (Version 2.1.1). DESeq2 (ref. 76) was used for differential gene expression analysis.

The human RNA-seq data were processed with Salmon-1.1 (ref. 86) against a decoy-aware transcriptome (gencode.v37 transcripts and the GRCh38.primary_assembly genome) with the following parameters: –validateMappings –gcBias –seqBias. The output was imported and summarized to the gene-level with tximport (1.14.2)87, and differential gene analysis was done with edgeR (3.28.1)88.

Proteomics

WT and lin-52(n771) L1 worms were plated in OP50-containing NGM plates and left to feed for 9 h. Worms were collected and washed 5 times with M9 buffer to remove the OP50, and 8 M urea buffer mixed in 50 mM TEAB with 1× Protease Inhibitor cocktail (Roche) was added to the sample before quick freezing.

Chromatin was degraded using a Bioruptor (Diagenode) for 10 min with cycles of 30/30 seconds. Upon centrifugation, the concentration of protein in the supernatant was calculated using Qubit Protein Assay Kit (Thermo Fisher Scientific). Twenty-five micrograms of protein per sample were transferred to a new tube, dithiothreitol was added to a concentration of 5 mM followed by vortexing and incubation at 25 °C for 1 h. Chloroacetamide was added to a final concentration of 40 mM, and the samples were incubated at room temperature for 30 min. Protein digestion with lysyl endopeptidase was done at an enzyme:substrate ratio of 1:75 and incubated at 25 °C for 4 h. Samples were diluted with 50 mM TEAB to reach a urea concentration of 2 M, and trypsin protein digestion was performed by adding trypsin at an enzyme:substrate ratio of 1:75; the samples were kept at 25 °C overnight.

After protein digestion, SDB RP StageTip purification was performed89. The protein samples were then analyzed utilizing liquid chromatography–mass spectrometry by the CECAD Proteomics Facility on a Q Exactive mass spectrometer that was coupled to an EASY nLC 1000 (Thermo Fisher Scientific). The differential protein levels were obtained by CECAD’s Proteomics Facility. Briefly, a predicted spectrum library was generated using the Prosit webserver90, and data were processed using DIA-NN 1.7.16 (ref. 91) and imported into Perseus 1.5.5.0 (ref. 91) for analysis.

Four replicates per strain and condition were used, each containing around 20,000 worms.

Datasets

The list of 211 genes belonging to the GO term ‘Cellular response to DNA damage stimulus’ was obtained by using data from the GO Consortium92,93 (database released on 8 October 2019) (Supplementary Table 1). GO analysis was performed using the PANTHER 15.0 over-representation test and Venn Diagrams were created using Venn Diagram Plotter 1.5 and GIMP 2.10.12.

Gene IDs from previously published datasets were updated to current databases. Duplicated or dead IDs were eliminated accordingly. Overlap analysis were done by using Fisher’s exact test in R v3.6.3 (ref. 94). Gene set enrichment analysis (GSEA) was done in R v3.6.3 (ref. 94) with the GSEA function of clusterProfiler v3.14.3 (ref. 95) and the parameter settings minGSSize = 3, maxGSSize = 5000, and nPerm = 20000. To calculate the adjusted P values for the GSEA results, statsmodels96 v0.11.1 multipletests methods with the parameter method = ’fdr_bh’ or method = ’bonferroni’ in Python 3.6 (ref. 97) was used.

Promoter analysis: C. elegans

The set of 211 DDR genes was used as input for the findMotifs function of HOMER-4.11-2 (ref. 98) with the parameters -len 8,10 -start −1000 -end 0. Wormbase IDs were converted to the sequence name with WormBase’s SimpleMine99. These identifiers were searched in the ‘worm.description’ file of HOMER to gain the corresponding RefSeq IDs. The P values were calculated with the hypergeometric tests function in scipy(1.5.1). HOMER’s seq2profile function98 was used to convert the previously reported CDE + CHR DREAM complex motif35 with one mismatch and three random base pairs in between to a motif file usable by HOMER with the following parameter: seq2profile.pl BSSSSSNNNTTYRAA 1 (ref. 35). The constructed motif was searched with the findMotifs.pl -find function for the 211 DDR genes with the parameters -start −1000 -end 0. The background enrichment of the motif was calculated for all 20,174 protein-coding genes with a RefSeq ID included in the worm.description file of HOMER. The P values were calculated with the hypergeometric tests function in scipy (1.5.1)100.

Promoter analysis:human

Homer’s seq2profile function was used to convert previously reported DREAM complex motifs56, with no allowed mismatch, with the following parameters:

  • seq2profile.pl TTTSSCGS 0

  • seq2profile.pl VVCGGAAGNB 0

  • seq2profile.pl BNBVNTGACGY 0

  • seq2profile.pl CWCGYG 0

The motifs were searched with the findMotifs.pl -find function with the parameters -start −1000 -end 0 for the up and downregulated genes, after harmine and INDY treatments, respectively, with an FDR cutoff of 0.01.

The background enrichment of the motif was calculated for all protein-coding genes included in the homer.description file of HOMER.

The P values were calculated with the hypergeometric test function in scipy(1.5.1)100 and Python’s statsmodels (0.11.1)96 was used to calculate the Benjamini–Hochberg FDR.

Statistics and reproducibility

In C. elegans, development growth assays and egg-laying assays were performed a minimum of 3 times, each of which included 3 biological replicates per condition with an average of around 40–50 individuals per sample. Slot blots were performed at least three times. Flow cytometry assays were done at least three times, each having three biological replicates. For experiments from which data were obtained from single individuals, such as mice experiments, worm imaging studies, and lifespan assays of worms, the sample sizes are indicated. All attempts at replication were successful.

The sample sizes have been well established in similar experiments in other scientific publications (refs. 9,13,39,79, among others). The statistical analysis performed in each experiment can be found in the figure legend. Two-tailed t-tests were done in Microsoft Excel 2019 and GraphPad Prism 7.03. log-rank, Mann–Whitney and two-way ANOVA tests for the germline image quantification tests and unpaired t-tests with Welch’s correction were done in GraphPad Prism 7.03. Two-way ANOVA for the quantification of worm heads was done with Python’s pingouin v0.3.6. Two-tailed Fisher’s exact tests were done in RStudio 1.2.5019. C. elegans experiments were not randomized was not applied because the group allocation was guided on the basis of the genotype of the respective mutant worms. Worms of a given genotype were nevertheless randomly selected from large strain populations for each experiment without any preconditioning. In mice experiments, allocation was random.

Blinding was generally not applied, as the experiments were carried out under highly standardized and predefined conditions to avoid investigator-induced bias. Developmental assays upon DNA damage with small observed effects were performed blinded to exclude any bias. This affects the developmental growth upon IR, MMS and cisplatin treatment.

Starvation assay

A pool of synchronized starving lin-52 mutant and WT L1 worms was maintained in M9 buffer rolling at 20 °C. From this pool, around 30 worms were transferred to seeded NGM plates over consecutive days, and the number of L1 worms per plate was counted. After 48–72 h in the seeded plates, the number of worms that recovered and survived the ongoing starvation was evaluated. A biological triplicate for each strain was used for each timepoint. The experiment was discontinued after 14 days because all worms from days 13 and 14 had died. Each plate per condition, replicate and day had an average of 30 worms.

Total-egg-hatching assay

Single synchronized day 1 adult worms were transferred to seeded NGM plates and left to lay eggs for 24 h. Each day, the worms were transferred to a new seeded NGM plate, until egg laying stopped for all individuals. The total number of eggs laid and hatching/surviving eggs were evaluated 24 h after the removal of the adult from the plate. Twelve adult WT and lin-52 mutant worms were used. Internal hatching or exploding worms were excluded from the day the event occurred onwards.

Motility assay

Synchronized day 1 adult lin-52 mutant or WT worms were UV-irradiated or mock-treated and incubated at 20 °C for 72 h. Next, 30 worms were transferred to unseeded small NGM plates and left for 30 min to avoid worms accumulating in areas with food, promote movement and facilitate image analysis. To obtain video footage, the plates containing the worms were left under the microscope light without the lid for 30 seconds to allow the worms to get used to the conditions. Thirty-second videos were taken by using a Zeiss Axio Zoom V.16 and Zeiss ZEN 2.3 pro software. Worm footage was analyzed using the plugin wrMTrck in ImageJ 1.53q.

EdU-incorporation assay in L1 and adult worms

Thymidine-deficient E. coli (strain MG1693) were grown in M9 containing 1 % glucose, 1 mM MgSO4, 1.25 µg/ml vitamin B1, 0.5 µM thymidine and 20 µM 5-ethynyl-2′-deoxyuridine (EdU) at 37 °C overnight in darkness. These bacteria were used to seed M9-agar plates (M9 with 1.2% agar and 0.6% agarose) and were left to incubate overnight at room temperature.

Synchronized L1 or adult lin-52 mutant and WT worms were UV-irradiated or mock-treated and transferred to the plates containing the EdU-labeled MG1693 E. coli. Worms were collected after 6 h, 12 h and 24 h for L1 worms, and after 24 h for adults, washed three times in M9 buffer and transferred to fixing buffer (1× egg buffer with 0.1% Tween and 3% PFA). Fixed worms were placed on top of a HistoBond+ Adhesion Microscope Slides (Marienfeld, 0810461), adult worms were cut open, and a coverslip was placed above the worms and slight pressure was applied. Next, the slides were placed on dry ice to allow freeze-cracking80. We used the Click-iT EdU imaging kit (Invitrogen, C10337), following the manufacturer’s instructions, for the preparation of the Click-iT reaction cocktail. Upon washing the worms 3 times for 5 min in PBS, 50 µl of reaction cocktail was added to the slides, followed by incubation for 30 min at room temperature in darkness. Slides were washed once in3 % BSA in PBS and mounted using 5 µl of Fluoromount-G with DAPI (Invitrogen, 00495952). Images were obtained using a SP8 Confocal Microscope by Leica using LAS X 3.5.7 software. Positive EdU nuclei were counted manually from the obtained image stacks.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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