Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle

Abstract

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.

FIG. 1.

Rad53 phosphorylation in response to chemically induced DSBs during meiosis I. spo11Δ and spo11Δ rad9Δ diploid cells expressing Mek1-HA3 from the MEK1 promoter, as well as spo11Δ mek1Δ diploid cells, were grown to stationary phase in YPA medium and then resuspended in SPM medium at time zero. At 210 min after transfer to SPM, half of each cell culture was incubated for 30 min in the presence of 5 μg/ml of phleomycin. Cell samples were collected at the indicated time points after transfer to SPM to analyze DNA content by fluorescence-activated cell sorting analysis (A); the phosphorylation pattern of Mek1 (B, top) and Rad53 (B, bottom) by Western blot analysis with anti-HA and anti Rad53 antibodies, respectively; and the percentages of binucleate (completed meiosis I [M I]) and tetranucleate (completed meiosis II [M II]) cells (C) by fluorescence microscope analysis of propidium iodide-stained cells. In all Western analysis, the same quantity of total protein extracts was loaded in each lane according to Coomassie blue staining.

FIG. 2.

Rad53 phosphorylation in response to programmed DSBs during meiosis I. Wild-type (wt) and dmc1Δ diploid cells expressing Mek1-HA3 from the MEK1 promoter and mek1Δ diploid cells were grown to stationary phase in YPA medium and then resuspended in SPM at time zero. Cell samples were collected at the indicated time points after transfer to SPM to analyze phosphorylation of Mek1 (A, top) and Rad53 (A, bottom) as in Fig. 1B and meiotic DSB formation by Southern blot analysis (B). Southern blotting was performed on EcoRI-digested genomic DNA run on a native agarose gel, and the filter was hybridized with a probe complementary to the 5′ noncoding region of the THR4 gene. This probe reveals an intact parental EcoRI fragment (P) of 7.9 kb and two bands of 5.7 and 7.1 kb corresponding to the two prominent meiotic DSB sites (DSB I and DSB II).

FIG. 3.

Targeting Rad53 to Mec1 results in Rad53 activation in response to meiotic DSB formation. Wild-type (wt) and dmc1Δ diploid cells, carrying the pRS316 DDC2-RAD53-3FLAG plasmid (DDC2-RAD53) or the pRS316 DDC2-rad53K227A D339A-3FLAG plasmid (DDC2-rad53kd), were grown to stationary phase in YPA and then resuspended in SPM at time zero. Cell samples were collected at the indicated time points after transfer to SPM to analyze DNA content by fluorescence-activated cell sorting analysis (A) and DSB formation (C) by Southern blot analysis on EcoRI-digested genomic DNA as described for Fig. 2B. Total protein extracts were prepared from the indicated strains and subjected to Western blot analysis with anti-FLAG and anti-Rad53 antibodies (B) and to ISA (D).

FIG. 4.

Rad53 phosphorylation in sae2Δ meiotic cells requires homologous chromosome segregation. Wild-type (wt), sae2Δ, sae2Δ spo11Δ, ndt80Δ, and ndt80Δ sae2Δ diploid cells, all expressing Mek1-HA3 from the MEK1 promoter, were grown to stationary phase in YPA and then resuspended in SPM at time zero. Cell samples were collected at the indicated time points after transfer to SPM to analyze DNA content (A); phosphorylation of Rad53 (B, top) and Mek1 (B, bottom); the percentages of binucleate (M I) and tetranucleate cells (M II) (C), as described for Fig. 1C; and DSB formation (D) as described for Fig. 2B.

FIG. 5.

Rad53 phosphorylation after segregation of homologous chromosomes carrying unrepaired meiotic DSBs. Wild-type (wt), dmc1Δ, dmc1Δ mek1Δ, dmc1Δ rad54Δ, and dmc1Δ mek1Δ rad54Δ diploid cells were grown to stationary phase in YPA and then resuspended in SPM at time zero. Cell samples were collected at the indicated time points after transfer to SPM to analyze Rad53 phosphorylation (A) and the percentages of binucleate (M I) and tetranucleate cells (M II) (B) as described for Fig. 1C.

FIG. 6.

Progression through meiosis in sae2Δ cells lacking Rad9. (A) Wild-type (wt), sae2Δ, and dmc1Δ diploid cells were grown to stationary phase in YPA and then resuspended in SPM at time zero. Total protein extracts were prepared from the indicated strains and subjected to Western blot analysis using anti-Rad9 antibodies. The asterisk points out hyperphosphorylated Rad9. (B to D) Wild-type, sae2Δ, rad9Δ, and sae2Δ rad9Δ diploid cells were grown to stationary phase in YPA and then resuspended in SPM at time zero. Samples were taken at the indicated time points for fluorescence-activated cell sorting analysis of DNA content (B), Western blot analysis of protein extracts with anti-Rad53 antibodies (C), and determination of the percentages of binucleate (M I) and tetranucleate (M II) cells (D).

FIG. 7.

Rad53 phosphorylation in meiotic sae2Δ cells requires the checkpoint kinases Mec1 and Tel1. (A and B) Wild-type (wt), sae2Δ, sae2Δ tel1Δ, and sae2Δ mec1Δ diploid cells, all expressing Mek1-HA3 from the MEK1 promoter, were grown to stationary phase in YPA and then resuspended in SPM at time zero. Cell samples were collected at the indicated time points after transfer to SPM to analyze phosphorylation of Mek1 (A, left) and Rad53 (A, right) as described for Fig. 1B and DSB formation (B) as described for Fig. 2B. (C and D) Diploid cells carrying the sae2Δ or dmc1Δ allele were grown to stationary phase in YPA and then resuspended in SPM at time zero. (C) 5′-to-3′ resection eliminates EcoRV sites located 0.8 kb centromere-distal from DSB II and 1.8 kb centromere-distal from DSB I, producing larger EcoRV fragments (rDSB II and rDSB I) of 3 kb and 3.7 kb, respectively, detected by the probe. (D) Genomic DNA prepared from aliquots taken at the indicated times after transfer in SPM was digested with EcoRV and separated on an alkaline agarose gel. Gel blots were hybridized with a single-stranded RNA probe specific for the 5′ noncoding region of the THR4 gene, which reveals Spo11-cut and uncut fragments of 1.8 kb and 2.2 kb, respectively.

FIG. 8.

Detection of meiotic DSBs by the checkpoint machineries. Homologs are indicated in black (paternal) and gray (maternal). Zigzag lines represent the meiosis-specific chromosome structure(s). In wild-type cells, DSB repair is accomplished via interhomolog recombination (A). In dmc1Δ cells, the inability to repair meiotic DSBs leads to Mek1 phosphorylation and a meiosis I arrest (B). Unprocessed meiotic DSBs in sae2Δ cells lead to a Mek1-dependent slowing down of meiosis I (D). When homologous chromosomes with unrepaired meiotic DSBs segregate from each other, these DSBs elicit a Rad53-dependent checkpoint that delays meiosis II (C and D).