Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity
- Graduate School of Biostudies, Kyoto University, Yoshidakonoe, Sakyo, Japan
- Department of Molecular and Cell Biology, University of California, United States
- Howard Hughes Medical Institute, United States
- California Institute for Quantitative Biosciences, United States
- Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, United States
- Department of Science, Kyoto University, Japan
- Institute of Evolutionary Biology, Ashworth Laboratories, School of Biological Sciences, University of Edinburgh, United Kingdom
- Radiation Biology Center, Kyoto University, Japan
- Institute for Integrated Cell‐Material Sciences (iCeMS), Kyoto University, Japan
Figures
Figure 1 with 2 supplements
DSB-1 is phosphoregulated in a PPH-4.1PP4- and ATL-1ATR -dependent manner and ATL-1ATR kinase antagonizes PPH-4.1PP4 phosphatase.
(A) Left: Western blot of GFP-fused DSB-1 probed with α-GFP. GFP-DSB-1 detected in extracts from wild-type and gfp-dsb-1 worms (24 hr post-L4 stage) with either control RNAi or pph-4.1 RNAi …
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Figure 1—source data 1
Intensity of phos-GFP-DSB-1 and non-phos-GFP band in Figure 1A.
Related to Figure 1A.
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Figure 1—source data 2
Western blotting raw images in Figure 1A.
Related to Figure 1A.
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Figure 1—source data 3
Western blotting raw images in Figure 1B.
Related to Figure 1B.
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Figure 1—source data 4
Western blotting raw images in Figure 1C.
Related to Figure 1C.
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Figure 1—source data 5
Western blotting raw images in Figure 1D.
Related to Figure 1D.
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Figure 1—source data 6
Western blotting raw images in Figure 1E.
Related to Figure 1E.
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Figure 1—source data 7
RAD-51 foci numbers graphed in Figure 1H.
Related to Figure 1H.
- https://cdn.elifesciences.org/articles/77956/elife-77956-fig1-data7-v2.xlsx
Figure 1—figure supplement 1
Validation of effectiveness of pph-4 RNAi and western blots of DSB-1.
(A) Images of DAPI-stained diakinesis nuclei of control (N2) or gfp-dsb-1 treated with pph-4 RNAi. The control nuclei contained six bivalents while the majority of nuclei contained univalents in gfp-…
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Figure 1—figure supplement 1—source data 1
Western blotting raw images in Figure 1—figure supplement 1B.
Related to Figure 1—figure supplement 1B.
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Figure 1—figure supplement 1—source data 2
Western blotting raw images in Figure 1—figure supplement 1C.
Related to Figure 1—figure supplement 1C.
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Figure 1—figure supplement 1—source data 3
Western blotting raw images in Figure 1—figure supplement 1D.
Related to Figure 1—figure supplement 1D.
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Figure 1—figure supplement 2
Double-strand break (DSB) formation in atl-1 and atm-1 mutants.
(A) Maximum-intensity projection of an entire gonad arm from an atl-1(tm853) mutant. Top left panel shows DAPI staining in magenta and RAD-51 staining in green. Bottom left panel shows RAD-51 foci …
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Figure 1—figure supplement 2—source data 1
RAD-51 foci numbers graphed in Figure 1—figure supplement 2C.
Related to Figure 1—figure supplement 2C.
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Figure 2 with 2 supplements
The dsb-1(5A) mutation rescues double-strand break (DSB) defect and viability loss of pph-4.1 mutants.
(A) A schematic diagram of the DSB-1 protein sequence. Green regions indicate intrinsically disordered regions from the D2P2 database (Oates et al., 2013). Five serines which were mutated into …
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Figure 2—source data 1
RAD-51 foci numbers graphed in Figure 2C.
Related to Figure 2C.
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Figure 2—source data 2
Brood size and number of viable progeny in the genotypes indicated in Figure 2D.
Related to Figure 2D.
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Figure 2—figure supplement 1
Sequence alignment of DSB-1 orthologs.
Multiple sequence alignment (MSA) of DSB-1 orthologs in 11 Caenorhabditis. [ST]Q sites are depicted in magenta. FXXP sites are boxed in red. The region of all proteins scored as intrinsically …
Figure 2—figure supplement 2
Double-strand break (DSB) formation in dsb-2 non-phosphorylatable mutant.
(A) A maximum-intensity projection of an entire gonad from wild type with the DAPI staining on the top and RAD-51 staining at the bottom in grayscale. Scale bar, 50 μm. Boxed inset on the right …
Figure 3 with 1 supplement
Chiasma formation and homologous pairing defects are partially rescued by the dsb-1(5A) allele in pph-4.1 mutants.
(A) FISH images show paired 5S rDNA sites in wild type, dsb-1(5A) and pph-4.1(tm1598); dsb-1(5A) worms (arrows indicate paired foci), and unpaired sites in pph-4.1(tm1598) mutants (arrowheads …
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Figure 3—source data 1
Number of paired and unpaired nuclei in the genotypes indicated in Figure 3B.
Related to Figure 3B.
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Figure 3—source data 2
Number of nuclei with indicated DAPI body numbers of each genotype in Figure 3C.
Related to Figure 3C.
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Figure 3—source data 3
Number of nuclei with indicated DAPI body numbers of each genotype in Figure 3D.
Related to Figure 3D.
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Figure 3—figure supplement 1
Synapsis in pph-4.1; dsb-1(5A) and pph-4.1; atl-1/nT1 mutants.
Immunofluorescence images of wild-type, pph-4.1 (tm1598), dsb-1(5A), pph-4.1(tm1598); dsb-1(5A), atl-1/nT1 and pph-4.1(tm1598); atl-1/nT1 mutants. Left panel shows the maximum-intensity projection …
Figure 4
The dsb-1(5A) mutation rescues double-strand break (DSB) and crossover formation in dsb-2 mutants.
(A) Immunofluorescence images of RAD-51 foci in mid-pachytene nuclei of the indicated genotypes. Scale bar, 5 μm. (B) Quantification of RAD-51 foci in the gonads of the genotypes indicated in (A). …
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Figure 4—source data 1
RAD-51 foci numbers graphed in Figure 4B.
Related to Figure 4B.
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Figure 4—source data 2
Number of nuclei with indicated DAPI body numbers of each genotype in Figure 4C.
Related to Figure 4C.
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Figure 5 with 2 supplements
Alanine substitution of serine 186 in DSB-1 suffices to rescue the dsb-2 mutation.
(A) Diagram depicting a series of dsb-1 phospho mutants: dsb-1(1A) is dsb-1(S186A); dsb-1(2A) is dsb-1(S137A; S143A); dsb-1(3A) is dsb-1(S137A; S143A; S186A) and dsb-1(5A) is dsb-1(S137A; S143A; …
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Figure 5—source data 1
Number of daily laid viable progeny of the indicated genotypes in Figure 5B.
Related to Figure 5B.
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Figure 5—source data 2
Intensity of phos-GFP-DSB-1 and non-phos-GFP band in Figure 5C.
Related to Figure 5C.
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Figure 5—source data 3
Western blotting raw images in Figure 5C.
Related to Figure 5C.
- https://cdn.elifesciences.org/articles/77956/elife-77956-fig5-data3-v2.zip
Figure 5—figure supplement 1
Double-strand break (DSB) formation in a series of dsb-1 non-phosphorylatable mutants.
Quantification of RAD-51 foci in the gonads of the indicated genotypes (data of wild type and dsb-1(5A) were duplicated from Figure 4B). Data are presented as mean ± SEM, three gonads were scored …
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Figure 5—figure supplement 1—source data 1
RAD-51 foci numbers graphed in Figure 5—figure supplement 1.
Related to Figure 5—figure supplement 1.
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Figure 5—figure supplement 2
Double-strand break (DSB) formation and synapsis in pph-4.1; dsb-1(1A) and dsb-2; dsb-1(1A) mutants.
(A) Immunofluorescence images of wild type, dsb-1(1A), dsb-2(me96), dsb-2(me96); dsb-1(1A), pph-4.1(tm1598), and pph-4.1(tm1598); dsb-1(1A) mutants. Maximum-intensity projections of nuclei at …
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Figure 5—figure supplement 2—source data 1
RAD-51 foci numbers graphed in Figure 5—figure supplement 2B.
Related to Figure 5—figure supplement 2B.
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Figure 6 with 1 supplement
Structural prediction of double-strand break (DSB) factors and their interaction.
(A) A representative structure of the DSB-1, DSB-2, and DSB-3 heterotrimer predicted by the AlphaFold structure prediction pipeline (Jumper et al., 2021; Mirdita et al., 2021) is shown at top. Green …
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Figure 6—source data 1
AlphaFold prediction files and settings (ZIP).
Related to Figure 6A.
- https://cdn.elifesciences.org/articles/77956/elife-77956-fig6-data1-v2.zip
Figure 6—figure supplement 1
Phylogenetic prediction of double-strand break (DSB) factors.
Maximum likelihood gene tree of DSB-1/2 homologs in the genus Caenorhabditis.
Tables
Table 1
Embryonic viability, male progeny percentage indicating the rate of X chromosome nondisjunction, and total number of scored embryos is shown for hermaphrodite self-progeny of the indicated genotypes (Table 1—source data 1).
| Genotype | Embryonic viability (%) | Male percentage (%) | Total # eggs scored |
|---|---|---|---|
| WT | 99.28 | 0.04 | 1990 |
| gfp-dsb-1 | 99.10 | 0.19 | 2578 |
| dsb-1(1A) | 98.47 | 0.15 | 3427 |
| dsb-1(2A) | 98.25 | 0.00 | 1347 |
| dsb-1(3A) | 99.29 | 0.19 | 2056 |
| dsb-1(5A) | 99.22 | 0.00 | 2546 |
| pph-4.1(tm1598) | 2.00 | 45.60 | 1424 |
| dsb-2(me96) | 39.55 | 13.85 | 3413 |
| dsb-2(me96); dsb-1(1A) | 93.96 | 1.40 | 3370 |
| dsb-2(me96); dsb-1(2A) | 83.12 | 3.31 | 2413 |
| dsb-2(me96); dsb-1(3A) | 97.56 | 0.64 | 2395 |
| dsb-2(me96); dsb-1(5A) | 98.64 | 0.04 | 1596 |
| pph-4.1(tm1598); dsb-1(1A) | 7.39 | 32.21 | 496 |
| pph-4.1(tm1598); dsb-1(5A) | 40.89 | 7.89 | 1273 |
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Table 1—source data 1
Brood size and number of viable progeny of the genotypes indicated in Table 1.
Related to Table 1.
- https://cdn.elifesciences.org/articles/77956/elife-77956-table1-data1-v2.xlsx
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Caenorhabditis elegans) | dsb-1 | WormBase/Stamper et al., 2013 PMID:23990794 | WormBase ID:WBGene00008580 | |
| Gene (Caenorhabditis elegans) | pph-4.1 | WormBase/Sato-Carlton et al., 2014 PMID:25340746 | WormBase ID:WBGene00004085 | |
| Gene (Caenorhabditis elegans) | atl-1 | WormBase | WormBase ID:WBGene00000226 | |
| Gene (Caenorhabditis elegans) | atm-1 | WormBase | WormBase ID:WBGene00000227 | |
| Gene (Caenorhabditis elegans) | dsb-2 | WormBase/Rosu et al., 2013 PMID:23950729 | WormBase ID:WBGene00194892 | |
| Strain, strain background (Escherichia coli) | pph-4.1 RNAi-L4440 in HT115 | Sato-Carlton et al., 2014 PMID:25340746 | ||
| Strain, strain background (Escherichia coli) | L4440 in HT115 | Ahringer Lab RNAi library (Source Biosciences) | ||
| Strain, strain background (Caenorhabditis elegans) | For C. elegans allele and strain information, see Supplementary file 1 | This paper | N/A | See Supplementary file 1 |
| Genetic reagent (Caenorhabditis elegans) | For CRISPR/Cas9 reagents, see sequence-based reagent and peptide, recombinant protein. | This paper | N/A | Purchased from IDT |
| Antibody | Anti-GFP (mouse monoclonal) | Santa Cruz | Cat#sc-9996 | WB (1:1000) |
| Antibody | Anti-Actin (rabbit polyclonal) | Santa Cruz | Cat#sc-1615 | WB (1:3000) |
| Antibody | Anti-RAD-51(rabbit polyclonal) | SDIX/Novus Biologicals | Cat#29480002 lot#G3048-009A02 | IF (1:10,000) |
| Antibody | Anti-DSB-1 (guinea pig polyclonal) | Stamper et al., 2013 PMID:23990794 | N/A | WB (1:75) |
| Antibody | Anti-ZIM-3 (rabbit polyclonal) | Phillips and Dernburg, 2006 PMID:17141157 | N/A | IF (1:2000) |
| Antibody | Anti-SYP-1 (guinea pig polyclonal) | Sato-Carlton et al., 2020 PMID:33175901 | N/A | IF (1:100) |
| Antibody | Anti-SYP-2 (rat polyclonal) | Sato-Carlton et al., 2020 PMID:33175901 | N/A | IF (1:200) |
| Antibody | Anti-HTP-3 (guinea pig polyclonal) | MacQueen et al., 2005 PMID:16360034 | N/A | IF (1:500) |
| Antibody | Alexa488-anti-rabbit (donkey polyclonal) | Jackson ImmunoResearch | Cat#711-545-152, lot#109880 | IF (1:500) |
| Antibody | DyLight649-anti-guinea pig (donkey polyclonal) | Jackson ImmunoResearch | Cat#706-495-148, lot#95544 | IF (1:500) |
| Antibody | DyLight594-anti-guinea pig (donkey polyclonal) | Jackson ImmunoResearch | Cat#706-515-148, lot#94259 | IF (1:500) |
| Antibody | DyLight649-anti-rat (donkey polyclonal) | Jackson ImmunoResearch | Cat#712-495-153, lot#94218 | IF (1:500) |
| Antibody | HRP-conjugated anti-mouse (sheep polyclonal) | GE Healthcare BioSciences | Cat#NIF825 | WB (1:10,000) |
| Antibody | HRP-conjugated anti-rabbit (goat polyclonal) | Abcam | Cat#ab97051 | WB (1:10,000) |
| Antibody | HRP-conjugated anti-guinea pig (goat polyclonal) | Beckman Coulter | Cat#732868 | WB (1:10,000) |
| Sequence-based reagent | FISH probe to the right arm of Chromosome V (5S rDNA) | Dernburg et al., 1998 PMID:9708740 | N/A | |
| Sequence-based reagent | Alt-R CRISPR-Cas9 tracrRNA | IDT | Cat# 1072532 | |
| Sequence-based reagent | dpy-10 crRNA: 5’-GCTACCATAGGCACCACGAG-3’ | https://www.ncbi.nlm.nih.gov/pubmed/25161212 | N/A | |
| Sequence-based reagent | dpy-10(cn64) homology template for CRISPR 5'-cacttgaacttcaatacggcaagatgagaatgactggaaaccgta ccgcATgCggtgcctatggtagcggagcttcacatggcttcagaccaacagcct-3’ | https://www.ncbi.nlm.nih.gov/pubmed/25161212 | N/A | |
| Sequence-based reagent | For crRNAs, repair templates and genotyping primers, see Supplementary file 2 | This paper | N/A | Purchased from IDT |
| Peptide, recombinant protein | Recombinant Cas9 protein | UC Berkeley QB3 Macrolab | https://macrolab.qb3.berkeley.edu/cas9-nls-purified-protein/ | |
| Commercial assay or kit | Pierce BCA Protein Assay Kit | ThermoFisher Scientific | Cat#23227 | |
| Chemical compound, drug | DAPI (4',6-Diamidino-2-phenylindole dihydrochloride) | Nacalai Inc | Cat#11034–56 | |
| Chemical compound, drug | Nuclease-free Duplex Buffer | IDT | Cat#11-01-03-01 | |
| Chemical compound, drug | GFP-Trap magnetic beads | ChromoTek | Cat#gtma-20 | |
| Chemical compound, drug | Lambda Protein Phosphatase | BioLabs | Cat#P0753S | |
| Chemical compound, drug | Chemilumi-one super | Nacalai Inc | Cat#02230–30 | |
| Chemical compound, drug | Chemilumi-one ultra | Nacalai Inc | Cat#11644–24 | |
| Chemical compound, drug | skim milk | Nacalai Inc | Cat#31149–75 | |
| Chemical compound, drug | SuperSep (TM) Ace, 5%–12%, 13well | Wako | Cat#199–15191 | |
| Chemical compound, drug | NuPAGE 4% to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well | Invitrogen | Cat#NP0322BOX | |
| Software, algorithm | softWoRx suite | Applied Precision/GE Healthcare | N/A | |
| Software, algorithm | OMERO | Burel et al., 2015 PMID:26223880 | https://www.openmicroscopy.org/omero/ | |
| Software, algorithm | Priism | Chen et al., 1996 PMID:8742723 | https://msg.ucsf.edu | |
| Software, algorithm | Prism6 | GraphPad | https://www.graphpad.dom/scientific-software/prism | |
| Software, algorithm | Mafft v7.487 | Katoh and Standley, 2013 PMID:23329690 | https://mafft.cbrc.jp/alignment/software/ | |
| Software, algorithm | Fiji | Schindelin et al., 2012 PMID:22743772 | https://fiji.sc | |
| Software, algorithm | AGAT v0.4.0 | Dainat et al., 2020 doi:10.5281/ZENODO.3552717 | https://github.com/NBISweden/AGAT | |
| Software, algorithm | OrthoFinder v2.5.4 | Emms and Kelly, 2019, Emms, 2022 PMID:31727128 | https://github.com/davidemms/OrthoFinder | |
| Software, algorithm | FSA v1.15.9 | Bradley et al., 2009 PMID:19478997 | http://fsa.sourceforge.net/ | |
| Software, algorithm | IQ-TREE v2.2.0-beta | Nguyen et al., 2015 PMID:25371430 | http://www.iqtree.org/ | |
| Software, algorithm | ETE3 Python module | Huerta-Cepas et al., 2016 PMID:26921390 | http://etetoolkit.org/ |
Additional files
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Supplementary file 1
Table listing strains used in this study.
- https://cdn.elifesciences.org/articles/77956/elife-77956-supp1-v2.docx
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Supplementary file 2
Table listing crRNAs, repair templates, and genotyping primers for transgenes generated in this study.
- https://cdn.elifesciences.org/articles/77956/elife-77956-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/77956/elife-77956-transrepform1-v2.docx
