Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae

Abstract

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80.

FIG. 1.

Cells of a dmc1/dmc1 rad17/rad17 strain and cells of a dmc1/dmc1 strain overexpressing NDT80 enter the meiotic divisions and express middle sporulation-specific genes. (A) Comparison of the efficiency of entry into the meiotic divisions by wild-type cells, dmc1/dmc1 rad17/rad17 cells, dmc1/dmc1 cells containing the high-copy plasmid pNDT80-426 (see Materials and Methods), and dmc1/dmc1 cells containing the vector pRS426. Cells that had been incubated in sporulation medium for the indicated times were fixed, stained with DAPI, and examined by fluorescence microscopy. Cells that appeared binucleate, trinucleate, or tetranucleate were scored as having completed meiosis I (MI). The data presented are representative of at least two trials per strain. (B) Sporulation-specific gene expression in wild-type cells (lanes 1 to 4), dmc1/dmc1 cells containing the pRS426 vector (lanes 5 to 8), dmc1/dmc1 cells containing the high-copy pNDT80-426 plasmid (lanes 9 to 12), and dmc1/dmc1 rad17/rad17 cells (lanes 13 to 16). This Northern filter contained RNA extracted from the indicated strains during vegetative growth (0 h), or 6, 8, or 10 h after transfer to sporulation medium, as noted above the top panel. The filter was hybridized sequentially with radioactively labeled probes specific for NDT80, SMK1, CLB1, SPS1, SPS4, SPS100, and the loading control, pC4 (see Materials and Methods).

FIG. 2.

Deletion of SUM1 in dmc1/dmc1 cells leads to efficient entry into the meiotic divisions and a high level of NDT80-dependent expression of premiddle and middle sporulation-specific genes. (A) Comparison of the efficiency of entry into the meiotic divisions in wild-type cells, dmc1/dmc1 cells, dmc1/dmc1 sum1/sum1 cells, and dmc1/dmc1 sum1/sum1 ndt80/ndt80 cells. Cells were taken during growth (0 h) or at the indicated times after transfer to sporulation medium, fixed, stained with DAPI, and examined by fluorescence microscopy. Completion of the meiosis I (MI) division was assessed as described in the legend for Fig. 1A. The data for wild-type cells and dmc1/dmc1 rad17/rad17 cells are the same as those presented in Fig. 1A. All data presented are representative of at least two trials per strain. (B to H) Sporulation-specific gene expression in wild-type cells (lanes 1 to 4), dmc1/dmc1 cells (lanes 5 to 8), dmc1/dmc1 rad17/rad17 cells (lanes 9 to 12), dmc1/dmc1 sum1/sum1 cells containing the pRS426 vector (lanes 13 to 16), dmc1/dmc1 sum1/sum1 cells containing the high-copy pNDT80-426 plasmid (lanes 17 to 20), and dmc1/dmc1 sum1/sum1 ndt80/ndt80 cells (lanes 21 to 24). A Northern filter was prepared with RNA purified from these cells harvested during vegetative growth (0 h) or at 6, 8, or 10 h after transfer to sporulation medium, as noted above the top panel (see Materials and Methods). The RNA used in the wild-type and dmc1/dmc1 rad17/rad17 lanes was from the same preparations as those used for the experiment shown in Fig. 1B. The filter was hybridized sequentially with radioactively labeled probes specific for NDT80 (B), SMK1 (C), CLB1 (D), SPS1 (E), SPS4 (F), SPS100 (G), and the loading control, pC4 (H) (see Materials and Methods).

FIG. 3.

Deletion of SUM1 allows dmc1/dmc1 cells to form mature spores. Cells were fixed and prepared for examination by electron microscopy 24 h after transfer to sporulation medium (see Materials and Methods). (A) A representative field of wild-type cells showing mature asci. (B) A higher-magnification micrograph of two asci containing three mature spores within the plane of the section. Due to the tetrahedral arrangement of mature spores, a fourth spore may be above or below the plane of this section. The spores are enclosed within mature spore walls, and the ascal wall is closely juxtaposed to the spores (see text for details). (C) A representative field of dmc1/dmc1 cells; no spores are present. (D) A representative field of dmc1/dmc1 rad17/rad17 cells showing asci containing spores at various stagesof maturity; no mature asci are present. (E) A higher magnification of a dmc1/dmc1 rad17/rad17 ascus containing one relatively mature spore (bottom) and two immature spores (top). The ascal wall has not condensed around these spores. (F) A representative field of dmc1/dmc1 sum1/sum1 cells showing asci at various stages of maturity. (G) A higher magnification of two dmc1/dmc1 sum1/sum1 asci. The ascus on the left is indistinguishable from a mature wild-type ascus (see panel B); the spores have mature spore walls and the ascal wall is closely juxtaposed to the spores. The ascus on the upper right contains three spores at various stages of maturity.

FIG. 4.

Examination of the effect of deletion of SWE1 on dmc1-induced arrest. (A) Comparison of the efficiency of entry into the meiotic divisions in wild-type cells, dmc1/dmc1 cells, dmc1/dmc1 swe1/swe1 cells containing the pRS426 vector, dmc1/dmc1 swe1/swe1 cells containing the high-copy pNDT80-426 plasmid, and dmc1/dmc1 swe1/swe1 ndt80/ndt80 cells. Cells were taken during growth (0 h) or at the indicated times after transfer to sporulation medium, fixed, stained with DAPI, and examined by fluorescence microscopy. Completion of the meiosis I (MI) division was assessed as described in the legend for Fig. 1A. The data for wild-type cells, dmc1/dmc1 cells, and dmc1/dmc1 rad17/rad17 cells are the same as those presented in Fig. 2A. All data presented are representative of at least two trials per strain. (B to H) Deletion of SWE1 does not restore a wild-type level of middle gene expression to dmc1/dmc1 cells. A Northern filter was prepared with RNA purified from wild-type cells (lanes 1 to 4), dmc1/dmc1 cells (lanes 5 to 8), dmc1/dmc1 rad17/rad17 cells (lanes 9 to 12), dmc1/dmc1 swe1/swe1 cells containing the vector plasmid pRS426 (lanes 13 to 16) or the high-copy plasmid pNDT80-426 (lanes 17 to 20), and dmc1/dmc1 swe1/swe1 ndt80/ndt80 cells (lanes 21 to 24) harvested during vegetative growth (0 h) or at 6, 8, or 10 h after transfer to sporulation medium, as noted above the top panel (see Materials and Methods). The RNA used in the wild-type, dmc1/dmc1, and dmc1/dmc1 rad17/rad17 lanes was from the same preparations as those used for the experiment shown in Fig. 2B to H. The filter was hybridized sequentially with radioactively labeled probes specific for NDT80 (B), SMK1 (C), CLB1 (D), SPS1 (E), SPS4 (F), SPS100 (G), and the loading control, pC4 (H) (see Materials and Methods). (I to K) Initiation of spore morphogenesis in dmc1/dmc1 swe1/swe1 cells is dependent on NDT80. Cells of a dmc1/dmc1 swe1/swe1 strain (I and J) and of a dmc1/dmc1 swe1/swe1 ndt80/ndt80 strain (K) were fixed and prepared for examination by electron microscopy 24 h after transfer to sporulation medium (see Materials and Methods). (I) A representative field of dmc1/dmc1 swe1/swe1 cells shows no mature asci; some cells contain prospore-like compartments. (J) A higher-magnification micrograph showing a dmc1/dmc1 swe1/swe1 cell that contains immature and aberrant spore-like compartments. (K) A representative field of dmc1/dmc1 swe1/swe1 ndt80/ndt80 cells shows no asci or any cells that have initiated spore morphogenesis.

FIG. 5.

Model for the roles of NDT80, SUM1, and SWE1 as targets of the meiotic recombination checkpoint. See text for details. The top horizontal line denotes the timing of landmark events during sporulation. Lines terminating in arrowheads indicate that the expression of a gene or the function of a protein has been activated, whereas lines terminating in bars indicate that the expression of a gene or the function of a protein has been inhibited. Early during sporulation in wild-type cells, Sum1 represses expression of NDT80 and middle genes. (A) Towards the end of prophase in wild-type cells, Sum1 is destabilized, which allows expression of NDT80. Ndt80 then activates expression of middle sporulation-specific genes, including CLB genes. This leads to an increase in Clb-Cdc28 activity, which drives cells out of pachytene and into the meiotic divisions. The gray arrow refers to aputative feedback mechanism that upregulates Ndt80 in cells that have exited pachytene. (B) In dmc1/dmc1 cells, activation of the checkpoint results in inactivation of Ndt80 and stabilization of Sum1, which prevent expression of middle sporulation-specific genes, including CLB genes. Concomitant stabilization of Swe1 ensures that Clb-Cdc28 is inactive and that cells arrest at pachytene. (C) The absence of Sum1 in dmc1/dmc1 sum1/sum1 cells allows expression of NDT80 prior to activation of the checkpoint signal. Additionally, sufficient Ndt80 accumulates such that the ability of the checkpoint to inhibit the activity of Ndt80 is at least in part titrated. Middle sporulation-specific genes, including CLB genes, are expressed. Increased Clb levels promote entry into the meiotic divisions despite the presence of active Swe1; the high level of expression of middle sporulation-specific genes promotes spore morphogenesis. (D) In dmc1/dmc1 swe1/swe1 cells, inhibition of Clb-Cdc28 is relieved, but inactivation of Ndt80 and stabilization of Sum1 are initially maintained. In some cells there is sufficient Clb-Cdc28 activity to promote exit from pachytene. We speculate that these cells do not progress into the meiotic divisions until a putative feedback mechanism attenuates the checkpoint signal, allowing increased Ndt80 activity. The subsequent increase in the level of Clb's drives the cells into the meiotic divisions.