PMC

Synaptonemal Complex Disassembly in syp-1 Alleles
(A) Representative images of diplotene region and oocites −4 to −1 from the indicated strains’ fixed germlines immunostained with synaptonemal complex protein SYP-1 and SYP-2 antibodies and counterstained with DAPI. Scale bar, 2 μm.
(B) Representative images of the diakinesis region from the indicated strains’ fixed germlines stained with DAPI.
(C) Quantification of the number of DAPI-stained bodies in the diakinetic oocyte. Data are represented as average ± SD (n, number of oocytes assayed).

Embryonic Lethality of syp-1 Phosphorylation Alleles in a brc-1 Background
(A) Representative images of the mitotic region from N2(WT) fixed germlines immunostained with anti-BRC-1 and anti-SYP-1 antibodies and counterstained with DAPI.
(B) Percentage of embryos of the indicated genotypes that failed to complete embryogenesis. Data are represented as average percentage ± SD.
(C) Proposed model. During meiosis, SPO-11 DSBs are repaired by homologous recombination (HR) using the homolog chromatid as template (top). In a context where excessive DSBs are produced, the DNA damage checkpoint is activated and triggers phosphorylation of SC component SYP-1 to bias repair through the sister chromatid as template (bottom). For simplicity, SC is represented only with SYP-1.

Synaptonemal Complex Assembly in syp-1 Alleles
(A) Representative images of transition zone and pachytene region from the indicated strains’ fixed germlines immunostained with synaptonemal complex protein SYP-1 and SYP-2 antibodies and counterstained with DAPI. Scale bar, 5 μm.
(B) Quantitation of pairing for chromosome X shown as the percentage of nuclei with paired signals in each zone shown in (C). Pairing of the X chromosome was visualized by immunofluorescence against HIM-8, which binds to the left end of the X chromosome at the cis-acting pairing center (PC). At least 15 gonads were scored for each genotype.
(C) Diagram of a hermaphrodite gonad, indicating the zones in which the pairing of HIM-8 signal (one foci versus two foci) was scored. 1, mitotic; 2, leptotene and zygotene; 3, early pachytene; 4 and 5, mid-pachytene; 6, late pachytene; 7, diplotene and diakineis.

ATM-ATR-Dependent Phosphorylation in Response to DNA Damage
(A) Representative images of the meiotic region from N2(WT) fixed germlines immunostained with anti-P^S/T-Q antibody and counterstained with DAPI without DNA damage (left) and 1 h after 75 Gy (right). Scale bar, 5 μm.
(B) Representative images of the meiotic region from N2(WT) fixed germlines immunostained with anti-P^S/T-Q and SYP-1 antibodies and counterstained with DAPI 1 h after 75 Gy, previously incubated with buffer (top), phosphatase (middle), or with the animals previously grown in the presence of 20 mM caffeine for 4 h (bottom). Scale bar, 5 μm.
(C) Representative images of the meiotic region from the indicated strains’ fixed germlines immunostained with anti-P^S/T-Q and synaptonemal complex protein SYP-1 antibodies and counterstained with DAPI 1 h after 75 Gy. Scale bar, 5 μm.
(D) Quantification of P^S/T-Q in the indicated strains in normal conditions (gray bars) or 20 h after 75 Gy (black bars). Graph shows intensity signal (arbitrary units, not normalized) determined by ImageJ software. 20–30 nuclei/germline from mid-pachytene were analyzed.

Meiotic Phosphorylation in Response to DNA Damage
(A) Representative images of whole N2(WT) fixed germlines immunostained with anti-P^S/T-Q antibody and counterstained with DAPI 1 h after 75 Gy. Scale bar, 10 μm.
(B) Western blot using SYP-1, SYP-2, and BRC-1 antibodies of the mock purification and CeBCD complex following tandem immunoaffinity purification (S, soluble and C, chromatin bound, before and after IR treatment). Samples were treated or not with phosphatase.
(C) In vitro phosphorylation of the SYP-1 peptide array by N2(WT) extracts without DNA damage (top) and with N2(WT) extracts after 75 Gy (bottom). Each of the 127 spots represents an 18-mer peptide fragment juxtaposed by three amino acids (aa) scanning the complete SYP-1 protein. Each peptide has a 15-amino acid overlap with the previous peptide and is numbered sequentially from the start codon. Positive serial spots (detected by autoradiography) corresponding to the specific DNA damage-phosphorylated region are boxed. The peptide sequences with specific DNA damage phosphorylation are shown with the possible phosphorylation residues highlighted in red. Scheme shows the phosphorylation site established by the peptide array data.

Defects in DNA Damage Response in the syp-1 Phosphorylation Alleles
(A) Sensitivity of L4-stage worms from the indicated strains to different doses of IR. Relative survival of offspring is shown. Data are represented as average percentage ± SD from at least four experiments with 15 worms each. ^∗p = 0.02, ^∗∗p = 0.0015, ^∗∗∗p = 0.0006, ^∗∗∗∗p < 0.0001; p values for paired t test.
(B and C) Quantification of recombination marker RAD-51 foci in the indicated strains in normal conditions (B) or 20 h after 75 Gy (C). At least 15 gonads were analyzed in each condition and ten nuclei were scored in each zone (mitotic region, 1; transition zone, 2; early-mid-late pachytene regions, 3-4-5; and diplotene-diakinesis regions, 6) for at least three independent experiments.
(D) Germ cell apoptosis was measured by differential interference contrast (DIC) microscopy in animals of the indicated strains at the indicated time points after IR treatment. Data are represented as average ± SD from at least ten worms for each time point of three independent experiments. ^∗∗∗∗p < 0.0001, p value for paired t test.