The Mitotic DNA Damage Checkpoint Proteins Rad17 and Rad24 Are Required for Repair of Double-Strand Breaks During Meiosis in Yeast

Corresponding author: Akira Shinohara, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka, 560-0043 Japan., ashino{at}bio.sci.osaka-u.ac.jp (E-mail)

Communicating editor: M. E. ZOLAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the -ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.

MEIOSIS generates gametes by halving the diploid genome. This process is accomplished by two successive rounds of chromosome segregation, which follow a single round of DNA replication. Reciprocal crossover recombination, together with sister chromatid cohesion, provides physical connections that facilitate proper segregation of homologous chromosomes at the first meiotic division.

In the budding yeast Saccharomyces cerevisiae, the DNA events of meiotic recombination have been defined in some detail. Recombination is initiated by double-strand breaks (DSBs), the ends of which are resected to produce 3'-single-strand tails (ROEDER 1997 ; KEENEY 2001 ). Intermediates are differentiated into two types: those that will ultimately form crossovers and those that will not, i.e., noncrossovers. The crossover/noncrossover differentiation process is thought to occur at a very early stage, as one end of a processed DSB becomes engaged with homologous sequences on a nonsister chromosome in a nascent joint molecule intermediate. Extensive strand-exchange ensues to form a displacement-loop intermediate known as a single-end invasion (SEI; HUNTER and KLECKNER 2001 ). Then, new DNA synthesis and interaction of the second DSB end forms a joint molecule structure called a double-Holliday junction (dHJ; SCHWACHA and KLECKNER 1994 , SCHWACHA and KLECKNER 1995 ). Both SEIs and dHJs are thought to be specific to the crossover pathway (ALLERS and LICHTEN 2001 ; HUNTER and KLECKNER 2001 ). Noncrossovers presumably arise via intermediates that are less readily detectable, e.g., are less stable and/or more transient (ALLERS and LICHTEN 2001 ; HUNTER and KLECKNER 2001 ).

In yeast, meiotic recombination involves many different proteins (ROEDER 1997 ). Two RecA homologs, Rad51 and Dmc1, play a critical role in strand invasion and exchange of single-strand DNA (ssDNA) with the homologous double-strand DNA (dsDNA; BISHOP et al. 1992 ; SHINOHARA et al. 1992 ). Rad51 is necessary for both mitotic and meiotic recombination, but Dmc1 is specific to meiosis. Rad51 and Dmc1 occur together on meiotic chromosomes, and the complex can be seen by immunostaining (BISHOP 1994 ; DRESSER et al. 1997 ; SHINOHARA et al. 2000 ). Rad51 and Dmc1 cooperate both in the formation of crossovers and in the control of recombination (SHINOHARA et al. 2003 ).

In mitosis, checkpoint proteins sense DNA damage and link repair with cell cycle progression (WEINERT 1998 ; ZHOU and ELLEDGE 2000 ). These proteins can detect one or a few DSBs in the genome (SANDELL and ZAKIAN 1993 ; LEE et al. 1998 ). A number of the proteins have been identified in budding yeast, and they are highly conserved from yeast to human. For instance, in yeast, Rad24 forms a complex with RFC2/3/4/5 and recruits a PCNA-like complex, Rad17-Mec3-Ddc1, onto chromatin (ZHOU and ELLEDGE 2000 ). This recruitment activates a key protein kinase, Mec1/Esr1, which binds directly to the site of DNA damage (KONDO et al. 2001 ; MELO et al. 2001 ). Activated Mec1/Esr1 triggers a kinase cascade, which delays the cell cycle and induces a transcriptional response to DNA damage.

In meiosis, the checkpoint proteins are also required. Some checkpoint mutations suppress the meiotic prophase arrest induced by abnormal recombination and chromosome synapsis in mutants such as dmc1, hop2, and zip1 (LYDALL et al. 1996 ; LEU et al. 1998 ; SAN-SEGUNDO and ROEDER 1999 ; HONG and ROEDER 2002 ). These studies have established the concept of the pachytene checkpoint, which is believed to coordinate recombination, and possibly chromosome synapsis, with progression of meiosis (ROEDER and BAILIS 2000 ).

At the pachytene checkpoint, Rad24-RFC and Rad17-Mec3-Ddc1 complexes recognize incomplete recombination and activate the Mec1/Esr1 kinase, as in mitosis (BAILIS and ROEDER 2000 ; HONG and ROEDER 2002 ). This phosphorylates a meiosis-specific kinase, Mek1/Mre4. Activated Mek1/Mre4 in turn promotes the phosphorylation of a meiosis-specific chromosomal protein, Red1 (BAILIS and ROEDER 2000 ; HONG and ROEDER 2002 ). Dephosphorylation of Red1 by Glc7 plays a critical role in the exit from pachytene (BAILIS and ROEDER 2000 ), although the role of Glc7 itself in meiosis is controversial (TACHIKAWA et al. 2001 ). In mitosis, Mec1/Esr1 also phosphorylates the Rad53 kinase, which is homologous to Mek1/Mre4, but Rad53 itself is not required for meiosis (ROEDER and BAILIS 2000 ). Interestingly, in mitosis, a few DSBs are sufficient to delay or arrest the cell cycle. In meiosis >200 DSBs are formed per nucleus, but the checkpoints have not yet been studied in detail.

In meiosis, the DNA damage checkpoint proteins also determine gamete viability. This is inferred because spore viability is reduced in some checkpoint single mutants of budding yeast (LYDALL et al. 1996 ). In fruit flies, a mutant of the mei-41 gene, the homolog of yeast Mec1/Esr1, also reduces crossover frequencies (BAKER and CARPENTER 1972 ; CARPENTER 1979 ). Similarly, the mec1/esr1 mutation decreases the frequencies of crossovers when assayed by forcing the meiotic mutant cells into mitotic growth (KATO and OGAWA 1994 ). Furthermore, some checkpoint single mutants show increased ectopic recombination, which is an exchange between nonallelic sites on nonsister chromosomes (GRUSHCOW et al. 1999 ). The checkpoint mutations also increase meiotic recombination between sister chromosomes (THOMPSON and STAHL 1999 ), but the exact role of these proteins in meiosis remains unclear.

In this report, we show that the rad17 and rad24 checkpoint mutants are defective in the repair of meiotic DSBs. These mutants also affect formation of Rad51 and Dmc1 complexes on chromosomes. We also found a genetic interaction between RAD51 and RAD24 in both meiosis and mitosis. These findings suggest that Rad24 and Rad17 are involved in the strand invasion and exchange steps of meiotic recombination. In addition, we unexpectedly found that in the mutants meiosis proceeds more slowly than usual. The control of the cell cycle in response to DSBs is also discussed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains:
All strains were derivatives of rapidly sporulating yeast SK-1 and are listed in Table 1. The spo11-Y135F mutant strains are generous gifts from Scott Keeney.

Plasmids:
See Table 1. To construct a high-copy RAD51 plasmid, pMS48, a 3.7-kb BamHI fragment containing the RAD51 gene was inserted into the BamHI site of YEplac195 (GIETZ and SUGINO 1988 ). For pMS117, a PstI-EcoRI fragment containing the RAD54 gene was inserted into the PstI and EcoRI sites of YEplac195. pMS181 was constructed by inserting a BamHI-PstI fragment containing the MRE4/MEK1 gene into the BamHI and PstI sites of pRS424 (CHRISTIANSON et al. 1992 ). pMS155 was made by inserting a XhoI-XbaI fragment containing DMC1 into the XhoI and XbaI sites of pRS314. pMS300 was constructed by cloning a PCR-amplified fragment containing TID1/RDH54 (nos. 382564–386130 of chromosome II in the YPD database) into the BamHI and XbaI site of YEplac195. pMS180 was made by inserting a PCR-amplified fragment (nos. 669869–672932 of chromosome XII) containing the RED1 gene into the BamHI and EcoRI sites of pRS424. The sequences of oligonucleotides for PCR amplification are available upon request.

Physical analysis of genomic DNAs:
Meiotic time course experiments were carried out as described (CAO et al. 1990 ; M. SHINOHARA et al. 1997 ). Presporulation culture was carried out in special presporulation medium for 16 hr. The cells were collected, washed, and resuspended in sporulation medium (SPM) to initiate meiosis. Aliquots of cells were withdrawn and genomic DNAs were prepared as described (M. SHINOHARA et al. 1997 ). For DSB analysis, genomic DNAs were digested with PstI and subjected to electrophoresis in a 0.8% agarose gel for 24 hr at 10 V/cm. For crossover analysis, DNAs were cut with XhoI and analyzed on a 0.6% agarose gel for 48 hr. For crossover/noncrossover and heteroduplex analysis, DNAs were digested with XhoI and MluI and with XhoI, BamHI, and MluI, respectively, and analyzed on a 0.6% agarose gel for 48 hr. DNAs were transferred onto a nylon membrane (Hybond N; Amersham, Buckinghamshire, UK) by capillary transfer and analyzed by Southern hybridization. Blots were visualized and quantified using a phosphorimager, BAS2000 (Fuji). Probes were pNKY155 (STORLAZZI et al. 1995 ) for crossover/noncrossover and heteroduplex assays and pNKY291 for DSB and crossover assays. The amounts of crossover and noncrossover heteroduplexes were <0.5% of total DNA, which made it difficult to accurately compare absolute amounts of the products. The ratio of crossover-to-noncrossover heteroduplexes is a more accurate measurement for the comparison, since the amount of total DNA could be ignored.

Cytology:
To determine the frequency and kinetics of meiotic division, cells were fixed with 70% ethanol and frozen at -20°, stained with 4',6-diamidino-2-phenylindole (DAPI), and then observed and counted under a fluorescent microscope. At least 200 nuclei were assessed. Spindles were examined by staining with rat antitubulin (YOL1/57; Sera Lab) as described previously (KAISER et al. 1994 ).

Meiotic chromosome spreads were prepared as described (BISHOP 1994 ; SHINOHARA et al. 2000 ). Meiotic cells were spheroplasted and surface spread on glass slides in the presence of detergent (lipsol) and fixative (4% paraformaldehyde). After drying, nuclei were immunostained as described previously (SHINOHARA et al. 2000 ). The slides were incubated simultaneously with guinea pig anti-Rad51 and rabbit anti-Dmc1 overnight at 4°, followed by incubation with secondary antibodies for 2 hr at 4°. Epifluorescence microscopy was carried out using Olympus BX51 or Zeiss Axiovert 135M. Images were captured with a cooled charge-couple device digital camera (Cool Snap; Photometrix) and processed using IP lab (Solution Systems) and Photoshop (Adobe) software. For triple staining, filter exchange was carried out using an automatic filter wheel (Roper Japan). The absence of offset between each filter was assessed using 500-nm microsphere beads with multiple colors (TetraSpeck microspheres; Molecular Probes, Eugene, OR). There was little offset of images by exchanging filters. Foci were scored, and colocalization frequency was determined as previously described (PADMORE et al. 1991 ; SHINOHARA et al. 2000 ).

Determination of -ray sensitivity:
Three individual transformants were analyzed for their ability to repair -ray damage. The strains were pregrown in synthetic medium lacking tryptophan or uracil (SC-Trp or SC-Ura) overnight. After sixfold dilution, the cells were grown for 3 hr and irradiated with -ray using a SHIMADZU Isotron RTGS-21 (Shimadzu, Tokyo). After serial dilution, cells were plated on SC-Trp or SC-Ura plates and incubated for 4 days. The numbers of colonies on the plates were counted.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Meiotic cell cycle progression of rad17 and rad24 mutants:
In mutants such as dmc1, zip1, and hop2, abnormal meiotic recombination arrests the cell cycle, but mutations in the DNA damage checkpoint genes can suppress this delay (LYDALL et al. 1996 ; LEU and ROEDER 1999 ; BAILIS and ROEDER 2000 ). However, the effect on meiosis of mutations in individual checkpoint genes has not been analyzed in detail. We therefore studied the timing of meiotic divisions I (MI) and II (MII) in mutants such as rad17 and rad24, since the mutants show the worst spore viability among checkpoint mutants (LYDALL et al. 1996 ; LEU et al. 1998 ; SAN-SEGUNDO and ROEDER 1999 ; HONG and ROEDER 2002 ), and compared each of them to wild-type cells. To assess cell cycle progression, cells were stained with DAPI, and DAPI-staining spots were counted. In this study, MI and MII are defined as cells with more than two (two, three, and four) and more than three (three and four) DAPI-staining spots, respectively, and thus score cells after anaphase I and anaphase II. We also studied the spo11-Y135F mutant, which is defective in the formation of meiotic DSBs but does not affect the progression of premeiotic S-phase (CHA et al. 2000 ).

As reported, spo11-Y135F mutant cells enter MI 1 hr earlier than wild type (data not shown). Surprisingly, the rad24 and rad17 mutants show a substantial delay (0.9 and 1.0 hr, respectively; Student's t-test, P < 0.001) in entry into MI (Fig 1A and Fig E). In addition, in the checkpoint mutants, 20–30% of the cells did not enter MII (Fig 1B). The delay in the rad17 mutant was reported previously, although neither mentioned nor analyzed statistically (GRUSHCOW et al. 1999 ). These results are somewhat unexpected, considering the role of RAD17 and RAD24: if checkpoint proteins monitor meiotic DSBs and delay cell cycle as in mitosis, then mutants would be predicted to enter divisions earlier than wild-type cells.

View larger version (30K): In this window In a new window Download PPT slide   Figure 1. Meiotic cell cycle progression of mutants in single checkpoint genes. (A and B) Cells incubated with sporulation medium were collected at different time points and scored for meiotic division by staining with DAPI. Nuclei with more than two and three or four DAPI-staining bodies are plotted in A and B, respectively. More than 200 cells were counted for each time point. Time "0" is the time when cells were transferred into SPM. , wild type (NKY1551); , rad24 (MSY717); , rad17 (MSY587). (C and D) Cells were stained with antitubulin antibody, cells with elongated spindles were counted, and the percentages of the cells were plotted (C). More than 200 cells were counted for each time point. Cumulative curves (D) were calculated from noncumulative curves in C, as described in PADMORE et al. 1991 . , wild type (NKY1551); •, spo11-Y135F (KSY170); , rad24 (MSY717); , rad17 (MSY587). (E) Time of entry into MI (anaphase I), when 50% of cells enter into MI, was determined from several independent experiments of DAPI and antitubulin staining as described above (A and D). Confidence intervals indicate ± values for 95% confidence in the mean values given. One-way Student's t-tests were carried out to obtain P values for each mutant against wild type. Similar results were obtained by a one-way ANOVA test (data not shown).

We confirmed the delay by analyzing the timing of spindle elongation (Fig 1, C–E). Meiotic cells were stained with antitubulin antibody and cells containing short spindles were counted. When cells enter the metaphase of MI, short spindles are formed, and these spindles elongate slightly before the nuclear division. Compared to wild type, in the spo11-Y135F mutant, elongation occurs 0.7 hr earlier, while in the rad17 and rad24 mutants, elongation occurs 1 hr later. These findings confirm that meiotic prophase progression is slower in rad17 and rad24 than in wild type.

The delay of cell cycle progression seen in rad17 and rad24 is not due to a prolonged premeiotic S-phase. Analysis of DNA contents by fluorescence-activated cell sorter indicates little delay (K. SAKAI and A. SHINOHARA, unpublished results). Consistent with this, the checkpoint mutant with the spo11-Y135F mutation shows kinetics of meiosis I and II similar to those of the spo11-Y135F mutant (K. SAKAI and A. SHINOHARA, unpublished results), implying that the defect in the delay is dependent on SPO11 function, and possibly on DSB formation. Furthermore, the meiotic DSBs form at the same time as they do in the wild type (see below), suggesting normal S-phase progression.

Repair of meiotic DSBs is impaired in the rad17 and rad24 mutants:
The spore inviability cannot be explained by the known role of the checkpoint genes in meiosis. We therefore used Southern blotting to analyze the formation and repair of meiotic DSBs at a well-known recombination hot spot, HIS4-LEU2 (CAO et al. 1990 ). After a 3-hr incubation in SPM, wild-type cells gave two bands at this locus, implying the formation of a break. The bands disappeared after a 7-hr incubation (Fig 2B and Fig C), indicating that the break had been turned over. In the rad17 and rad24 mutants, the bands formed at the same time as they did in wild type, but their disappearance was delayed (compare blots at 6 and 7 hr in mutants and wild type). In addition, the bands in the mutants are much more heterogeneous than those in wild type. LYDALL et al. 1996 showed the formation of 3'-OH ssDNA of DSB ends. The defect is common to mutants that are defective in strand exchange, e.g., dmc1, rad51 (BISHOP et al. 1992 ; A. SHINOHARA et al. 1992 , A. SHINOHARA et al. 1997 ; SCHWACHA and KLECKNER 1997 ). Furthermore, rad50S rad24 double mutants accumulate an amount of DSBs similar to that of the rad50S single mutant (A. SHINOHARA and M. SHINOHARA, unpublished results), indicating that the checkpoint mutations do not affect the formation of meiotic DSBs. These results suggest that the rad17 and rad24 mutants are defective in the conversion of meiotic DSBs into the next recombination intermediate.

View larger version (79K): In this window In a new window Download PPT slide   Figure 2. DSB repair and recombinant formation in checkpoint mutants. (A) Schematic drawing of the HIS4-LEU2 recombination hotspot. B, BamHI; M, MluI; P, PstI; X, XhoI. (B and C) DSB and its repair in the checkpoint mutants. Genomic DNAs from cells harvested at different times after the induction of meiosis were analyzed for DSB (B) and quantified (C). (C) , wild type (NKY1551); , rad24 (MSY717); , rad17 (MSY587). (D) The formation of heteroduplex in various mutants. Genomic DNAs from cells harvested at different times after the induction of meiosis were analyzed for heteroduplex. P1 and P2 are parental DNAs. HD1 and HD3 are noncrossover structures while HD2 (contains two bands) is a crossover structure. ER1 and ER2 are products of ectopic recombination. Wild type, NKY1551; rad24, MSY717; rad17, MSY587. (E) The ratios of amounts of noncrossover heteroduplexes (HD1 plus HD3) to that of crossover HD (HD2) at 10 hr were calculated. We show the ratios of the products, which give more accurate measurement than the amounts, since the amount of each product is <0.5% of total DNAs. The average ratios for three independent experiments are shown.

The rad17 and rad24 mutants reduce the formation of crossover and noncrossover intermediates:
Since DSB turnover is delayed, we were interested in looking at the formation of crossover and noncrossover heteroduplexes at the HIS4-LEU2 recombination hotspot (Fig 2A). The analytical method was originally developed by Kleckner and her colleagues (STORLAZZI et al. 1995 ). Parental chromosomes contain a unique restriction site (MluI or BamHI) near the site of the DSB (DSB I). In addition, a polymorphism at XhoI restriction sites can distinguish crossover molecules from parental molecules. Thus, after the digestion of genomic DNAs with BamHI, MluI, and XhoI, DNA molecules containing a heteroduplex migrate more slowly than parental molecules. Furthermore, crossover and noncrossover molecules containing the heteroduplex exhibit a unique mobility.

In wild type, as shown previously, both crossover and noncrossover heteroduplexes were formed simultaneously (Fig 2D). They started to appear at 5 hr and accumulated during further incubation. However, in rad17 and rad24 mutants, crossover and noncrossover heteroduplexes appeared 2 hr later (compare blots at 5 and 7 hr in mutants and wild type). Furthermore, the mutants showed extra bands, which are consistent with intrachromosomal ectopic recombination between LEU2 of the HIS4-LEU2 locus and the leu2::hisG locus (GRUSHCOW et al. 1999 ). The mutants also showed lower amounts of crossover and noncrossover heteroduplexes relative to wild type, with noncrossover heteroduplexes reduced more severely than crossover heteroduplexes. The ratio of noncrossover-to-crossover heteroduplex in wild type, rad17, and rad24 mutants is 1.6, 1.1, and 1.1, respectively (Fig 2E).

In parallel, we analyzed the formation of crossover and noncrossover products by digesting DNAs with MluI and XhoI, which distinguish crossover and noncrossover irrespective of the presence or absence of heteroduplex. The amount of crossover recombinants in the mutants was reduced to 70% in wild type (data not shown). The results were similar to those discussed above. In summary, in the rad17 and rad24 mutants: (1) recombinants with or without heteroduplex form later (GRUSHCOW et al. 1999 ); (2) there are fewer total recombinants (GRUSHCOW et al. 1999 ); (3) the ratio of crossover to noncrossover is altered (Fig 2E) because noncrossovers are preferentially reduced; and (4) there is a concomitant increase in ectopic recombination (GRUSHCOW et al. 1999 ).

Formation of Rad51 and Dmc1 foci and their colocalization in the rad17 and rad24 mutants:
The accumulation of DSBs is reminiscent of the phenotypes of mutants defective in strand exchange, such as rad51. It would therefore be interesting to know whether the checkpoint mutants also affect the assembly and disassembly of Rad51 and Dmc1 complexes. Rad51 and Dmc1 colocalize on meiotic chromosomes as punctate staining called foci, and the colocalization of the two proteins is genetically controlled (BISHOP 1994 ; SHINOHARA et al. 2000 ). We therefore prepared meiotic chromosome spreads, stained them with anti-Rad51 and anti-Dmc1 antibodies simultaneously, and counted foci under an epifluorescence microscope (Fig 3).

View larger version (33K): In this window In a new window Download PPT slide   Figure 3. Colocalization of Rad51 and Dmc1 on meiotic chromosomes in rad17 and rad24 mutants. (A) Immunolocalization: nuclear spreads of wild type (NKY1551), rad17 (MSY587), and rad24 mutants (MSY717) were stained with anti-Rad51 and anti-Dmc1 and then with the secondary antibodies. (i–iv) Wild type; (v–viii) rad24; (ix–xii) rad17. Rad51 (green, ii, vi, and x) and Dmc1 (red, iv, viii, and xii) were pseudocolored. Nuclei with a side-by-side configuration of Rad51 and Dmc1 foci in rad17 (xiii) and rad24 (xiv) mutants are shown. Nuclei with only Rad51 foci in the rad24 are shown in xv. Bar, 2 µm. (B) Noncumulative analysis of Rad51- and Dmc1-focus positive stages. The fractions of nuclei containing more than five Rad51 or more than five Dmc1 foci were counted at each time. At least 100 nuclei were counted at each point. , Rad51-focus positive nuclei; •, Dmc1-focus positive nuclei. (C) Cumulative analysis of Rad51- and Dmc1-focus positive stage, based on the previous figure and analyzed as described in SHINOHARA et al. 2000 . , entry of Rad51-focus positive nuclei; •, exit of Rad51-focus positive nuclei; , entry of Dmc1 focus-positive nuclei; , exit of Dmc1-focus positive nuclei.

First, we analyzed the distribution of Rad51 and Dmc1 separately. We counted nuclei containing Rad51 foci and nuclei with Dmc1 foci, to define the assembly and disassembly phases of each type of molecule (Fig 3B and Fig C). In wild type, as reported previously (SHINOHARA et al. 2000 ), both focus-positive nuclei show very similar kinetics, indicating that both proteins load onto chromosomes at the same time. However, the coordination is compromised in rad17 and rad24. The life span of Dmc1-positive nuclei is slightly longer (0.2–0.4 hr), but the life span of Rad51-positive nuclei is extended by 1.5 hr (Table 2). Consistent with this, we found nuclei with only Rad51 foci (Fig 3A, xv), which are not detected in wild type. Thus, in the checkpoint mutants, Rad51 foci outnumber Dmc1 foci later in meiosis.

Next, we analyzed colocalization of Rad51 and Dmc1 foci after a 3-hr incubation, when most of the rad17 and rad24 mutant cells are in the assembly phase. In wild type, 76% of the foci contained both Rad51 and Dmc1, as previously reported (SHINOHARA et al. 2000 ), whereas in the rad17 and rad24 mutants, only 52 and 43% of the foci contained both molecules (Table 2). Interestingly, the checkpoint mutants show more nuclei with a side-by-side configuration of Rad51-Dmc1 (Fig 3A, xiii and xiv), which is rarely found in wild type. This shows that the rad17 and rad24 mutants are partially defective for the colocalization of Rad51 and Dmc1. In addition, the total number of foci containing either Rad51 or Dmc1 in the mutants is the same or slightly lower than in wild type (Table 2 and Table 2M. SHINOHARA, unpublished results). This suggests that assembly of Rad51 and/or Dmc1 is slightly defective in the mutants, given that the turnover of the foci is delayed in the mutants; e.g., the number of the foci should be higher in the mutants than in wild type, as in the tid1/rdh54 mutant (SHINOHARA et al. 2000 ). Thus, both RAD17 and RAD24 are required for the proper assembly/disassembly of the RecA homologs, particularly Rad51, on meiotic chromosomes.

A high copy of RAD51 and RAD54 partially suppresses spore inviability of the rad24 mutant:
The results described above suggest that Rad24 acts after DSB formation. We therefore studied genetic interactions between RAD24 and various recombination genes, particularly ones engaged in strand invasion and exchange. Extra copies of RAD51, RAD54, and TID1/RDH54 were introduced into the rad24 mutant cells to check whether they could restore spore viability. A substantial increase in spore viability was observed when RAD51 was overexpressed (from 26.2% with vector alone to 40.5%; Table 3). RAD51 also increased the fraction of asci containing four viable spores from 7.3 to 18%. These differences are statistically significant (Mann-Whitney's U-test, P < 0.001). The overexpression of RAD54 also significantly increases spore viabilities of the rad24 and fractions of four-viable spores per tetrad. In addition, a high copy of TID1/RDH54 substantially decreases the spore inviability of the rad24 mutant. We also tested a high copy number of RED1 and MEK1/MRE4, which are downstream targets of Rad24 in the pachytene checkpoint (BAILIS and ROEDER 2000 ; HONG and ROEDER 2002 ), but neither could suppress the inviability of rad24. These results support the idea that RAD24 works with RAD51, RAD54, and TID1/RDH54 during meiotic recombination.

A high copy number of RAD51 and RAD54 substantially suppresses the rad24 mutant's sensitivity to -rays in mitosis:
The above result prompted us to test whether a high copy of RAD51 and RAD54 could suppress the defects of checkpoint mutants in mitosis. Compared to wild-type cells, the rad24 mutant is sensitive to ionizing radiation, presumably because it is unable to repair damage. We therefore created rad24 cells containing various high-copy-number plasmids, irradiated them with various doses of -rays, and measured cell survival. As shown in Fig 4A, a high copy of both RAD51 and RAD54 partially suppresses sensitivity to radiation, while a vector alone has no effect. This indicates that an increased dosage of RAD51 and RAD54 suppresses rad24's effect. The overexpression of RAD51 or RAD54 does not increase the resistance of wild-type haploid cells (Fig 4B). In addition, the overexpression of TID1/RDH54 has an opposite effect: it increases -ray sensitivity of the rad24 mutant significantly and that of the wild type slightly. Thus, the positive effect of TID1/RDH54 overexpression on the rad24 is specific to meiosis, consistent with a much more critical role of the gene in meiosis than in mitosis (M. SHINOHARA et al. 1997 ).

View larger version (18K): In this window In a new window Download PPT slide   Figure 4. Radiation sensitivity of rad24 with increased dosage of various recombination genes. (A) rad24 mutant haploids (MSY966) transformed with various high-copy-number plasmids were irradiated with various doses of -ray and plated on SC plates lacking tryptophan or uracil. After a 4-day incubation, colonies were counted. The data are an average of three independent experiments. Error bars indicate 95% confidence intervals. , RAD24 (a low-copy plasmid); •, vector alone; , RAD51; , RAD54; , TID1/RDH54. (B) Wild-type cells (MSY833) transformed with various high-copy-number plasmids were irradiated with various doses of -rays. , vector alone; , RAD51; , RAD54; , TID1/RDH54.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

RAD17 and RAD24 are required for normal meiotic recombination:
Previous genetic analyses suggest that DNA damage checkpoint proteins identified in mitosis are also involved in meiotic recombination (see Introduction). However, it was not clear which biochemical process these checkpoint proteins perform. Here, we report that mutants of two DNA damage checkpoint genes, RAD17 and RAD24, are partially defective in the repair of meiotic DSBs, as well as in the formation of crossover and noncrossover products. The mutants are also deficient in coordinating Rad51 and Dmc1 complex formation and share some similarity of meiotic phenotypes with mutants of TID1/RDH54 and RAD51, which play a direct role in meiotic recombination, e.g., strand invasion/exchange. The rad51 and tid1/rdh54 mutants accumulate DSBs with more resected ends and form reduced amounts of crossover (A. SHINOHARA et al. 1997 ; M. SHINOHARA et al. 1997 ). The tid1/rdh54 mutant reduces colocalization of Rad51 and Dmc1 (SHINOHARA et al. 2000 ). In addition, similar to checkpoint mutants, the tid1/rdh54 mutant shows increased ectopic recombination (M. SHINOHARA and A. SHINOHARA, unpublished results). Taken together, these results strongly suggest that Rad17 and Rad24 are involved in strand invasion and exchange (see below).

The rad17 and rad24 mutations affect not only the coordination of Rad51 and Dmc1 complexes on DNA, but also the disassembly of Rad51 complexes, suggesting that Rad17 and Rad24 are likely to work together with Rad51 during meiosis. This idea is further strengthened by the fact that an increased dosage of RAD51 and RAD54 substantially improves spore viability of the rad24 mutant. Interestingly, a high copy of the TID1/RDH54 also increases spore viability of the rad24 mutant, but its effect is specific to meiosis. Tid1/Rdh54, which interacts with both Rad51 and Dmc1, plays a more critical role in meiosis than in mitosis (M. SHINOHARA et al. 1997 ). We propose that during meiosis, the Rad24-RFC complex and the PCNA-like complex containing Rad17 collaborate with Rad51 (and Rad54 and Tid1/Rdh54) and, possibly, facilitate Rad51-filament formation to promote proper DSB repair.

The high-copy suppression of low spore viability of the rad24 mutant by the recombination genes instead could be explained by the overexpression of recombination genes activating a second recombination pathway, which is independent of RAD24 function. The overexpression of RAD54 suppresses the defects in the dmc1 null mutant by bypassing to a second recombination pathway, which does not occur in wild type (BISHOP et al. 1999 ).

Although checkpoint proteins are likely to play a direct role in meiotic recombination, we cannot eliminate indirect pathways. For example, a downstream target protein belonging to a checkpoint pathway that depends upon Rad17 and Rad24 appears to modulate meiotic recombination. The downstream target(s) could be proteins involved in the repair of DSBs. Obvious candidates are Rad51 or Rad51-accessory proteins. In mitosis, one of the Rad51-binding proteins, Rad55, is phosphorylated in response to DNA damage (BASHKIROV et al. 2000 ). Rad55 forms a complex with Rad57 and promotes proper assembly of Rad51 filaments on ssDNA (SUNG 1997 ). Rad55 may also be involved in meiosis, as the rad55 mutant partially delays DSB repair, decreases crossover formation, and is defective in the formation of Rad51 foci (SCHWACHA and KLECKNER 1997 ; GASIOR et al. 1998 ). However, Rad55 phosphorylation depends on Rad53 and Dun1 kinases, neither of which is required during meiosis (ROEDER and BAILIS 2000 ).

During meiosis, Rad17 and Rad24 activate a meiosis-specific kinase, Mek1/Mre4, which in turn appears to phosphorylate Red1, a meiosis-specific chromosome component (BAILIS and ROEDER 1998 ; DE LOS SANTOS and HOLLINGSWORTH 1999 ). The Mek1/Mre4-Red1 pathway is proposed to mediate the pachytene checkpoint (ROEDER and BAILIS 2000 ). In the mitotic DNA damage checkpoint, the overexpression of Rad53, a paralogue of Mek1/Mre4 (BAILIS and ROEDER 2000 ; ROEDER and BAILIS 2000 ), can suppress the mitotic defect in mutants of upstream checkpoint genes (SANCHEZ et al. 1996 ; SUGIMOTO et al. 1996 ). On the other hand, a high copy number of Mek1/Mre4 does not suppress meiotic defects in the rad24 mutant (Table 3). This argues against a function for the checkpoint proteins in signaling during meiosis.

The roles of checkpoint proteins in meiotic recombination:
Given the structural analogy between the RFC-PCNA complex and the Rad24-Rad17 complex, Rad17 and Rad24 are likely to bind to a D-loop structure such as SEI formed during meiotic recombination and to stabilize it by preventing the 3'-OH strand from dissociating from the duplex.

Alternatively, although not exclusively, the checkpoint proteins might prevent the invading end in the SEI from being extended by DNA polymerase(s) before the second end interacts with the same dsDNA. In this scenario, checkpoint proteins might monitor recombination events and cause both ends of a DSB to interact with the same dsDNA. This idea is similar to GRUSHCOW et al. 1999 original proposal: in checkpoint mutants, the coordination of DSB ends is disrupted. This is consistent with our observation that the colocalization of Rad51 and Dmc1 is disrupted in the rad17 and rad24 mutants. We previously suggested that Rad51 forms a complex on one end of the DSB, and Dmc1 forms an independent complex on the other end of the DSB (SHINOHARA et al. 2000 ). The checkpoint proteins might coordinate assembly of recombination complexes on the DSB ends.

What couples meiotic recombination with meiotic cell cycle progression?
The pachytene checkpoint is proposed to inhibit meiotic cell cycle progression in response to incomplete meiotic recombination and chromosome synapsis (ROEDER and BAILIS 2000 ). This has been inferred from the analysis of abnormal meiosis induced by a class of mutants, e.g., dmc1, zip1, and hop2. These mutants arrest or delay meiotic prophase, but this arrest is alleviated by mutations in some mitotic DNA damage checkpoint genes, suggesting that the checkpoint genes might also act in meiosis as in mitosis. However, meiosis involves at least 200 intrinsic DSBs and the cell cycle control might be quite different. Here, we suggest that in meiosis DSBs are not monitored solely by the mitosis checkpoint proteins Rad17 and Rad24, as rad17 or rad24 delay the entry into MI relative to wild type. If these genes were involved in the checkpoint control of meiosis, the mutations should abolish the checkpoint and cause either no delay or, possibly, acceleration. Alternatively, in meiosis, the defect in these mutants might trigger some other checkpoint response controlling the cell cycle. Previous analyses of meiotic prophase arrest by rad50S have shown that a second checkpoint response operates to monitor unprocessed DSBs. This arrest requires the Mec1/Esr1 homolog, Tel1, and Rad9 (USUI et al. 2001 ). However, Tel1 is unlikely to play a role in the delay seen in the checkpoint mutants studied here, since the tel1 rad24 double mutant shows meiotic cell cycle progression similar to the rad24 single mutant (A. SHINOHARA and M. SHINOHARA, unpublished results). Furthermore, the Tel1-dependent rad50S checkpoint still requires RAD24 function (USUI et al. 2001 ).

Although meiotic cells certainly monitor DSBs, the DNA damage checkpoint genes identified in mitosis may not be involved. The link between DNA damage and control of timing in meiosis is complex. Mutants such as spo11 and rec104, which are deficient in the formation of meiotic DSBs, enter MI earlier than wild-type cells, arguing for monitoring. On the other hand, the mei4 mutant, which is also deficient in the formation of the DSBs, enters MI at the same time as wild type, which is different from the other early recombination mutants (GALBRAITH et al. 1997 ; JIAO et al. 1999 ). Furthermore, the induction of a single DSB into a rec104 mutant does not delay the first division (JIAO et al. 1999 ), suggesting that the cell cycle is normally slow enough that DSBs can be repaired. Here, we showed that single checkpoint mutants enter MI later than wild type. If the DNA damage checkpoint proteins normally monitor 200 meiotic DSBs, checkpoint mutants should enter into MI earlier than wild type, similar to the spo11 mutant, but clearly this does not happen. In mitosis, checkpoint proteins are usually very sensitive to strand breaks. One irreparable DSB is sufficient to delay the cell cycle, and two irreparable DSBs are sufficient to arrest it (SANDELL and ZAKIAN 1993 ; LEE et al. 1998 ). Thus, these suggest that DNA damage checkpoint genes are not required for the meiotic cell cycle control at least in wild-type meiosis.

In yeasts as well as in fruit flies and nematodes, the combination of a mitotic checkpoint mutant with meiotic recombination-deficient mutants accumulating abnormal recombination intermediates has been used to analyze the checkpoint in meiosis (GHABRIAL and SCHÜPBACH 1999; GARTNER et al. 2000 ; SHIMADA et al. 2002 ). However, as described in this article, the SPO11-dependent delay in entry into meiosis I is independent of mitotic DNA damage checkpoint proteins in wild-type cells. Therefore, the concept of the pachytene checkpoint in wild-type meiosis should be treated cautiously.

	ACKNOWLEDGMENTS

We thank D. Bishop for sharing unpublished results. We are grateful to Neil Hunter and Valentin Boerner for helpful discussion and critical reading of the manuscript. We also thank Doug Bishop, Scott Keeney, Nancy Kleckner, and Ted Weinert for strains and plasmids. This work was supported by grants from the Ministry of Education, Science and Culture of Japan, priority area (to A.S. and T.O.), Hayashi Memorial Foundation (to M.S.), and the Human Frontier Science program (to A.S.).

Manuscript received January 17, 2003; Accepted for publication March 14, 2003.

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