Rad9, a 53BP1 Ortholog of Budding Yeast, Is Insensitive to Spo11-Induced Double-Strand Breaks During Meiosis

Abstract

Exogenous double-strand breaks (DSBs) induce a DNA damage response during mitosis as well as meiosis. The DNA damage response is mediated by a cascade involving Mec1/Tel1 (ATR/ATM) and Rad53 (Chk2) kinases. Meiotic cells are programmed to form DSBs for the initiation of meiotic recombination. In budding yeast, Spo11-mediated meiotic DSBs activate Mec1/Tel1, but not Rad53; however, the mechanism underlying the insensitivity of Rad53 to meiotic DSBs remains largely unknown. In this study, we found that meiotic cells activate Rad53 in response to exogenous DSBs and that this activation is dependent on an epigenetic marker, Dot1-dependent histone H3K79 methylation, which becomes a scaffold of an Rad53 mediator, Rad9, an ortholog of 53BP1. In contrast, Rad9 is insensitive to meiotic programmed DSBs. This insensitiveness of Rad9 derives from its inability to bind to the DSBs. Indeed, artificial tethering of Rad9 to the meiotic DSBs activated Rad53. The artificial activation of Rad53 kinase in meiosis decreases the repair of meiotic DSBs. These results suggest that the suppression of Rad53 activation is a key event in initiating a meiotic program that repairs programmed DSBs.

Keywords: DDR (DNA damage response); Rad53; Rad9/53BP1; checkpoint; meiosis; recombination.

FIGURE 1

H3K79me is required for Rad9-dependent Rad53 activation in meiosis after phleomycin treatment. (A) TCA-precipitated cell extracts were prepared from wild-type (USY543/544), rad9 (USY524/525), dot1 (USY526/527), and dot1 rad9 (USY522/523) diploid strains, and were analyzed by western blotting using anti-Rad9, anti-Flag (for Rad53), anti-H3K79-3me, and anti-tubulin antibodies. “³²P-Rad53” represented ³²P-incorporation to Rad53 in the ISA assay. Band intensities were quantified and are shown as arbitrary units without normalization (fourth panels). Cell extracts derived from 4 × 10⁶ cells were loaded for the ISA assay. (A,C) Cell extracts were prepared from 3.5-h meiotic cells before (labeled as “–”) and after 30-min treatment with 5 μg/mL of phleomycin (labeled as “+”). (B) Diploid cells of the indicated strains were arrested at G2/M by nocodazole and treated with 5 μg/mL of phleomycin for 30 min, followed by TCA precipitation. Wild-type (USY543/544), rad9 (USY524/525), dot1 (USY526/527), and dot1 rad9 (USY522/523) were used in the experiment. Results of an independent experiment are shown in Supplementary Figure 1A. (C) Rad53 activation was examined in various strains in meiosis as shown in (A). Wild-type (USY543/544), rad9 (USY524/525), dot1 (USY526/527), h3-K79R (USY693/677), and rad9-Y798A (USY758/759) diploid strains were used. Results of an independent experiment are shown in Supplementary Figure 1B.

FIGURE 2

Ddc2-Rad9 fusions activate Rad53 in meiosis. (A) TCA-precipitated meiotic cell extracts of the indicated strains were analyzed by western blotting using anti-HA, anti-Flag, anti-H3K79-3me (tri-methylation), anti-Dmc1, and anti–α-tubulin antibodies.³²P-Rad53 represented ³²P-incorporation to Rad53 in the ISA assay, and was quantified as shown Figure 1A (fifth panels). Cell extracts derived from 4 × 10⁶ cells were loaded for the ISA assay. Wild-type (USY543/544), DDC2-RAD9 (USY544/661), DDC2-RAD9 spo11 (USY783/414), and DDC2-RAD9 rad53-KD (USY545/720) were examined. (B) Cell extracts were prepared from the strains expressing C-terminal 3x HA-tagged Ddc2 (USY83/84; DDC2-HA) and N-terminal 3x HA-tagged Rad9 (USY20/35; HA-RAD9) from their native promoters; Ddc2-Rad9 (USY544/671; DDC2-RAD9) and 3x HA-tagged Rad9 (USY544/661; OE-HA-RAD9) from the DMC1 promoter; no HA-tagged wild-type strain (USY543/544); wild type. Rad53 was not tagged with Flag in DDC2-HA and HA-RAD9 strains. Flag-Rad53 was expressed from the native promoter. (C) Rad53 activation was examined in various strains as in (A). Wild-type (USY543/544), DDC2-RAD9 (USY544/671), DDC2-rad9-Y798A (USY544/776), DDC2-RAD9 dot1 (USY768/526), DDC2-RAD9 h3-K79R (USY770/693), and DDC2-rad9-K1088M (USY544/797) were examined. Results of an independent experiment are shown in Supplementary Figure 2C. (D) Rad53 activation was examined in various strains as in (A). Wild-type (USY543/544), DDC2-RAD9 (USY544/671), DDC2-RAD9 mec1 (USY495/785), DDC2-rad9-7A (USY544/667), and DDC2-RAD9 rad53-KD (USY4520) were examined. Results of an independent experiment are shown in Supplementary Figure 2D.

FIGURE 3

Ddc2-Rad9 localizes to meiotic DSBs. (A) The representative images of anti-HA and anti-Rad51 double immunostaining of spread nuclei prepared from DDC2-RAD9 (USY544/671), DDC2-RAD9 dmc1 RAD53 (USY580/591), DDC2-RAD9 dmc1 rad53-KD (USY582/623), HA-RAD9-OE dmc1 rad53-KD (USY582/674), and DDC2-RAD9 dmc1 spo11 rad53-KD (USY559/666) strains are shown at 4.5 h in meiosis, except for DDC2-RAD9 dmc1 RAD53, which is shown at 6 h in meiosis. Typical DNA images stained with DAPI and merged images by HA- and Rad51-staining are presented. Bar equals 2 μm. (B) The percentage of dmc1Δ rad53-KD DDC2-RAD9 nuclei that had more than 5 HA or Rad51 staining foci is presented at the indicated time points. At least 50 nuclei were counted at each time point. Error bars represent standard deviations obtained from 3 independent time courses.

FIGURE 4

Rad9 is insensitive to meiotic DSBs. Rad9 binding to chromatin adjacent to mitotic HO-induced DSBs (A) and to meiotic programmed DSBs at the HIS4-LEU2 locus (B) was tested by ChIP. Formaldehyde-fixed chromatin extracts were prepared at 0 and 1 h after HO DSB induction in G2/M-arrested cells, and at 0 and 4 h after meiotic induction, followed by anti-HA and anti-Rad51 ChIP. Anti-HA- or anti-Rad51-bound DNAs were quantified by real-time PCR. Error bars represent standard deviations obtained from 3 independent cultures. The non-tagged wild-type and HA-RAD9 strains examined were USY99 and USY100 in (A) and NKY1551 and USY2035 in (B), respectively.

FIGURE 5

DDC2-RAD9 impedes cell cycle progression and meiotic recombination. (A) Meiotic nuclear divisions were monitored in wild-type (USY543/544) and DDC2-RAD9 (USY544/671) strains by DAPI staining. The y-axis represents the percentage of cells that completed meiosis I and II (Post-MI). At least 200 cells were counted at each time point. (B) Schematic presentation of the HIS4-LEU2 DSB site. (C,E) Genomic DNAs were prepared from the indicated strains during the meiotic time course. Meiotic DSBs (DSB-I and DSB-II) (C) or crossover recombinants (CO1 and CO2) (E) were detected by Southern blot using the probes pNK291 or pNKY155 after genomic DSBs were digested with PstI or XhoI, respectively. (D,F) Quantitative data of DSB-I (C) and CO1 + CO2 (E) obtained from Southern blot are plotted. (G–L) DDC2-RAD9 influences localization of Rad51 and Zip1 on meiotic chromosome. (G) The representative images of Rad51 immunostaining nuclear foci in spread nuclei prepared from the indicated strains at 4 and 6 h in meiosis are shown. (H) The percentage of the indicated cells’ nuclei that had more than 5 Rad51 foci was plotted at the indicated time points. (I) Distribution of nuclei according to Rad51 foci number per nucleus at the indicated time is presented using the same data samples as in (H). Only nuclei that had more than 5 Rad51 foci were plotted. The size of the bubbles reflects the percentage of nuclei that had a certain foci number of Rad51 among all nuclei examined. Bars represent the average numbers of Rad51 foci. (J) Representative images of Zip1 staining classification are shown. (K) Classifications of Zip1 staining at the indicated time in the indicated strains are shown. The line represents the Zip1 polycomplex (PC). At least 100 nuclei were examined at each time point. (L) Representative images of Zip1 staining in DDC2-RAD9 at 4 h and 6 h are shown. Zip1 PCs are indicated by arrowheads. Experiments were performed at least twice, and representative data are shown. Bar equals 5 μm.

FIGURE 6

Rad9 is incompetent to meiotic DSBs. Exogenous DSBs often occur in nucleosome-rich gene body where Dot1-mediated histone H3K79 methylation marks are enriched (left pathway). The DSBs activates Mec1/Tel1, which in turn phosphorylates histone H2AS129. The two epigenetics marks, H3K79 methylation and H129S phosphorylation, provides multiple binding sites to Rad9. Rad9 is phosphorylated by Mec1/Tel1 and activates Rad53 kinase. At recombination hotspots is located in nucleosome-free region, which shows less H3K79 methylation compared to gene bodies right pathway. This chromatin features make the region insensitive to Rad9 binding.