PP2ACdc55 is required for multiple events during

Report

Cell Cycle 10:9, 1420-1434; May 1, 2011; © 2011 Landes Bioscience

PP2ACdc55 is required for multiple events during meiosis I Jocelyn K. Nolt,1,†,‡ Lyndi M. Rice,1,‡ Christina Gallo-Ebert,1 Margaret E. Bisher2 and Joseph T. Nickels1,* Venenum Biodesign; A Division of Genesis Biotechnology Group; Hamilton, NJ USA; 2Department of Molecular Biology; Princeton University; Princeton, NJ USA

1

†

Current address: Doctoral Program in Molecular and Cell Biology and Genetics; Drexel University College of Medicine; Philadelphia, PA USA These authors contributed equally to this work.

‡

Key words: meiosis, protein phosphatase 2A, DNA replication, homologous recombination

Protein phosphatase 2A (PP2A) is a heterotrimer consisting of A and B regulatory subunits and a C catalytic subunit. PP2A regulates mitotic cell events that include the cell cycle, nutrient sensing, p53 stability and various mitogenic signals. The role of PP2A during meiosis is less understood. We explored the role of Saccharomyces cerevisiae PP2A during meiosis. We show a PP2ACdc55 containing the human B/55 family B subunit ortholog, Cdc55, is required for progression through meiosis I. Mutant cells lacking Cdc55 remain mononucleated. They harbor meiotic gene expression, premeiotic DNA replication, homologous recombination and spindle pole body (SPB) defects. They initiate but do not complete replication and are defective in performing intergenic homologous recombination. Bypass alleles, which allow cells defective in recombination to finish meiosis, do not suppress the meiosis I defect. cdc55 cells arrest with a single SPB lacking microtubules or duplicated but not separated SBPs containing microtubules. Finally, the premeiotic replication defect is suppressed by loss of Rad9 checkpoint function. We conclude PP2ACdc55 is required for the proper temporal initiation of multiple meiotic events and/or monitors these events to ensure their fidelity.

©201 1L andesBi os c i enc e. Donotdi s t r i but four families ofe B. subunits (B/55, PR72/130, B/56 and striatins) Introduction

The cell cycle and meiosis are distinct developmental processes that are regulated by the genetic composition of the cell along with environmental factors, resulting in divergent developmental pathways or cell fates.1,2 Similar to mitotic cell division, meiosis is executed by all eukaryotes and is a complex process that requires restructuring of the cell cycle.3,4 It shares major events with mitosis including DNA replication and chromosome segregation.5 There are numerous other events unique to meiosis, which include homolog pairing, synaptonemal complex formation, genetic recombination and a reductional division.6,7 Following a single round of premeiotic DNA replication, a diploid cell undergoes two successive rounds of chromosome segregation (meiosis I and meiosis II or MI and MII, respectively) giving rise to four haploid germ cells, all differing in genetic content. A common meiosis model is the yeast Saccharomyces cerevisiae, in which the end result of meiosis is four spores enveloped within an ascus. Phosphorylation/dephosphorylation is a major regulatory mechanism cells use to regulate growth.8 PP2A is one of four highly conserved serine/threonine phosphatases (PP1, PP2A, PP2B and PP2C) and is a heterotrimeric protein made up of an A structural subunit, a C catalytic subunit and a B regulatory subunit that gives the complex substrate specificity.9 In higher eukaryotes, there are two isoforms of A and C subunits, while

exist, each containing multiple members.10,11 The combination of subunits gives rise to a number of PP2A variants, differing in substrate specificity, subcellular localization and tissue specificity. Some of the mitotic events regulated by PP2A include DNA replication,12,13 oncogenic transformation,14 tumor suppression,15 nutrient sensing,16,17 cell cycle progression,18 RNA transcription,19 apoptosis20 and RNA splicing and translation.21 Early replication studies showed loss of PP2A repressed DNA replication in rat primary hepatocytes22 and Xenopus egg extracts.23 Initiation of DNA replication requires PP2A PR48 dephosphorylation of Cdc6.24 PP2A also regulates replication through regulation of Cdc45,25 replication protein A 26 and the DNA polymerase α-primase.27 The major PP2A catalytic activity in S. cerevisiae is encoded by two redundant genes, PPH21 and PPH22.28 Deletion of both PPH21 and PPH22 results in a growth defect and is lethal in combination with loss of the catalytic-like subunit, PPH3.29 Studies using strains lacking all PP2A catalytic activity harbor defects in the mitotic cell cycle,18 DNA replication,18 organization of the actin cytoskeleton18,19,29 and cell morphology.30 A single A regulatory subunit, TPD3,21 and two B regulatory subunits, CDC55,30 and RTS1,31 orthologous to B (B55 or PR55) and B’ (B56) subunit families respectively, complete the remaining PP2A subunits. Mutants lacking TPD3 or RTS1 are temperature

*Correspondence to: Joseph T. Nickels; Email: [email protected] Submitted: 01/25/11; Accepted: 03/15/11 DOI: 10.4161/cc.10.9.15485 1420

Cell Cycle

Volume 10 Issue 9

report

Report

Table 1. Sporulation efficiency of various pp2a mutants Strain

Mononucleateda

Binucleated

CDC55

36

CDC55b

41

pph21/22 sit4 pph3

100

tpd3

100

-

-

-

0

rts1

33

8

59

-

64

b

Tetranucleated

>4

Sporulation

7

57

-

64

3

56

-

59

99

-

2C peak was observed in SK1 and W303 strain backgrounds. We found rts1, sit4 and tap42-11 cells were capable of completing premeiotic DNA replication, but tpd3 and pph3 were defective (not shown). Finally, we observed that W303 pph21 pph22 cells were unable to complete replication (Fig. 4). Based on our results, we conclude PP2ACdc55 is required for premeiotic DNA replication. CDC55 is required for the completion of DNA replication. The >2C peak suggested cdc55 cells initiated but could not complete DNA replication (Fig. 4). To investigate this, we visualized origin firing using 2D agarose gel electrophoresis, which identifies active replication forks (seen as bubble arcs), random termination structures, stalled replication forks and accumulation of recombination intermediates.53,54 We looked at replication initiation at the rDNA ARS (autonomously replicating sequence) over a 7 h period. cdc55 cells initiated replication, as evidenced by the presence of a bubble arc by 2 h (Fig. 5A). Wild-type cells initiated replication by 3 h. cdc55 cells had sustained accumulation of structures possibly indicative of stalled replication forks, random termination intermediates and recombination intermediates, which were not seen in wildtype cells (Fig. 5B; enlargement of 4 h time point). To determine whether replication initiation could occur at other origins of replication, we used the alkaline gel electrophoresis method described by Diffley and colleagues.55 Alkaline gel electrophoresis releases replication intermediates into the gel as small fragments, while parental DNA remains in the well. Wild-type and cdc55 cells were treated with hydroxyurea for 3 h to increase synchrony and transferred to sporulation media. We analyzed premeiotic replication at the early and late origins, ARS305 and ARS603, respectively; we used the highest concentration of hydroxyurea that did not cause growth inhibition (50 mM) (Fig. 6). Upon release from hydroxyurea, wild-type cells accumulated replication intermediates at ARS305 by 3 h (Fig. 6A). cdc55 cells accumulated these intermediates also, but their synthesis was delayed and they further accumulated at 24 h. Qualitatively, these intermediates accumulated to a greater extent than in wild-type

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Figure 4. FACS analysis reveals a role for PP2ACdc55 in premeiotic DNA replication. Various pp2a strains were shifted to sporulation medium and cell aliquots were taken at 0 and 24 h. DNA content was determined using propidium iodide.

These results were corroborated by RT-PCR and LacZ promoter fusion β-galactosidase assays (data not shown). Western analysis of Ime2 and Spo11 supported our expression data (Fig. 3), indicating early and middle sporulation genes are expressed prematurely in SK1 cdc55 cells, while late gene expression is defective. In the W303 strain background, cdc55 cells temporally expressed early meiotic genes like wild-type, but were defective in expressing NDT80, SPS4 (late gene) and DIT1 using northern analysis (Sup. Info. and Sup. Fig. 1); we used northern analysis, as it is more sensitive than slot blot analysis. The temporal expression

www.landesbioscience.com

Cell Cycle

1423

cells. Wild-type origin firing was visualized by 6 h for the late ARS603 and was reduced thereafter (Fig. 6B). Once again, cdc55 cells were delayed in firing by at least 3 h and intermediates accumulated at 24 h. Loss of the Rad9 checkpoint protein suppresses the premeiotic DNA replication defect of cdc55 cells. Our data suggests that PP2ACdc55 is required for completing premeiotic DNA replication, so we next asked if loss of CDC55 activates a DNA replication and/or DNA damage checkpoint. If so, does elimination of the checkpoint allow cells to complete replication and/or meiosis I? The yeast ATM/ATR kinase ortholog Mec1 regulates the DNA damage checkpoint.56 Rad9 is another critical checkpoint signaling factor.57 Rad9 and Mec1 function during meiosis to monitor DNA metabolism.58 We constructed cdc55 mec1 and cdc55 rad9 strains and monitored replication; we used a mec1 diploid strain harboring the sml1 allele required for viability.59 Loss of MEC1 did not suppress the replication defect of cdc55 cells as visualized by FACS analysis (Fig. 7). cdc55 rad9 cells accumulated 4C DNA content, yet cells remained mononucleated. Thus, although loss of Rad9 partially suppresses the premeiotic DNA replication defect, cells remain blocked in meiosis I, possibly a second downstream checkpoint has been activated.60 Since loss of RAD9 remediated the observed meiotic replication defect, we asked if there was any type of genetic interaction between RAD9 and CDC55 with regards to checkpoint function. Loss of RAD9 in cdc55 cells caused a synthetic lethal phenotype in the presence of hydroxyurea concentrations of 50 mM or greater (Fig. 8). cdc55 cells were more resistant to hydroxyurea than cdc55 rad9 cells, while the sensitivity of rad9 cells was similar to wild-type. Thus, there is a synthetic sick phenotype associated with loss of both RAD9 and CDC55 under conditions of DNA damage. Spindle pole body and microtubule dynamics are altered in cdc55 cells. Duplication of the spindle pole body (SPB) is a hallmark event during meiosis I. Cells activated at the pachytene checkpoint arrest prior to SPB duplication.61,62 To determine if the pachytene checkpoint was activated in cdc55 cells, we first determined if loss of Cdc55 caused defects in SPB duplication and/or microtubule dynamics. We integrated a TUB4-GFP (γ tubulin), which associates with the SPB proteins, Spc97 and Spc98,63 or TUB1-GFP (α tubulin) allele and examined SPB and microtubule dynamics using fluorescence microscopy. The majority of cdc55 cells (93%) contained a single Tub4GFP focus after 12 h in sporulation medium, whereas most wildtype cells contained either 2 (18.5%) or 4 foci (58%) (Fig. 9A); this time point was used as wild-type spore formation was not yet observed. Even after 24 h, when most wild-type cells formed spores containing a single Tub4-GFP focus, cdc55 cells remained mononucleated with a single focus representative of either unduplicated or unseparated SPBs. We next examined microtubule dynamics using Tub1-GFP and we found wild-type cells had elongated meiotic spindles at 8 h, with most cells completing meiosis II by 16 h. cdc55 cells arrested with what looked to be short spindles (Fig. 9B). SK1 strains gave similar results. Fluorescence microscopy cannot distinguish between a single SPB and two SPBs that have not separated. To examine if


1424

Figure 5. cdc55 cells initiate premeiotic DNA replication earlier at the rDNA ARS than wild-type cells. (A) 2D-gel electrophoresis analysis of time-dependent DNA replication initiation at the rDNA ARS. (B) magnification of the 4 h time point of cdc55 diploid cells. arrow, foci indicative of stalled replication and recombination intermediates.

cdc55 cells duplicated SPBs but arrested prior SPB separation, we used electron microscopy to examine SPB dynamics at 16 h in W303 cells. At this time point, wild-type cells formed asci containing four spores (Sup. Fig. 2A). cdc55 cells either had two SPBs that failed to separate but had attached microtubules (Sup. Fig. 2B) or a single “horseshoe-like” SPB lacking microtubules (Sup. Fig. 2C). These structures suggest cdc55 cells are arrested at the pachytene checkpoint, but more analysis is needed to make a definitive conclusion.64 CDC55 is not required for the initiation of homologous recombination, but is required for the proper timing of initiation. Since our analysis of SPB duplication pointed to cdc55 cells being blocked at pachytene, we examined several homologous recombination events.65-68 We first determined if PP2ACdc55 was required to form the double strand breaks (DSBs) necessary for initiation of homologous recombination at the YCR048W recombination hotspot; all strains harbored the rad50K181S (rad50S) allele, allowing for DSB accumulation.69 Cells were synchronized in YPA medium, then grown in sporulation medium and DSBs were visualized by Southern analysis. DSBs accumulated 3 h earlier in cdc55 cells (Fig. 10) and were dependent on

Cell Cycle

Volume 10 Issue 9

Figure 6. cdc55 diploid cells initiate premeiotic DNA replication at an early and late ARS sequence. Alkaline agarose gel electrophoresis was used to analyze DNA replication firing at early (ARS305) and late (ARS603) ARS sequences. DNA replication intermediates were visualized using Southern analysis.

Spo11-dependent single strand break initiation (cdc55 spo11). rts1 and tpd3 cells did not accumulate DSBs. sit4 cells accumulated these recombination intermediates to wild-type levels, while pph3 did not (not shown). pph21 pph22 cells were severely delayed in DSB accumulation. Based on these results, we conclude PP2ACdc55 is not required to initiate the DSBs necessary for homologous recombination, but is required for their proper temporal initiation. PP2ACdc55 is required for intergenic recombination. cdc55 cells initiate DSBs but can they complete homologous recombination? To determine whether this was the case, we determined intragenic recombination frequencies at the trp1, arg4 and leu2 loci. We determined trp1 recombination in the W303 strain described by Honigberg and colleagues.70,71 arg4 and leu2 frequencies were determined in SK1 strains kindly provided by the laboratories of Alain Nicolas72 and Rochelle Esposito,73,74 respectively. Heteroalleles of trp1, leu2 or arg4 can be repaired through intragenic recombination, generating wild-type alleles. cdc55 cells completed intragenic recombination at all three loci with frequencies very similar to wild-type (Fig. 11). Recombination at the LEU2 locus was delayed, but eventually did reach wild-type level. Intergenic recombination was severely affected (Fig. 11). Crossover events at the CENII-HIS3/LYS2 and CENIII-MATa/ MATa intervals were absent. In addition, cdc55 cells remained diploid throughout meiosis and were unable to commit to proceed through meiosis I using “return to growth” assays.73,74 Recombination bypass and checkpoint mutations do not suppress the meiosis I defect of cdc55 cells. We next asked whether any recombination bypass mutations were able to suppress the block in meiosis I. Deletions of MEI4, SPO11 or SPO13 allow mutants defective or blocked in recombination (or pachytene) to bypass meiosis I chromosome segregation and undergo a single equational division.75-77 Deletion of recombination genes, REC102 and REC104, can also suppress recombination defects.78 If our replication defect was caused by defects in homologous recombination, these bypass mutations should allow cdc55 cells to complete meiosis I. The loss of MEI4, SPO11, SPO13 (Sup. Fig. 1), REC102 or REC104 (data not shown) did not suppress the meiotic block of cdc55 cells, as evidenced by the continued appearance of a meiotic replication defect and mononucleated cells. These results possibly suggest that the replication defect we observed was not due solely because of defects in homologous recombination. cdc55 cells are not blocked in meiosis I due to activation of the pachytene checkpoint. The pachytene checkpoint monitors meiotic recombination, and in the presence of a defect, becomes activated and arrests cells until DNA repair has been completed.79,80 The fact that bypass mutations were unable to suppress the meiosis I defect of cdc55 cells suggested to us that they were not blocked at pachytene. We further tested this hypothesis. RAD17 and RAD24 are checkpoint components, and their loss



Cell Cycle

1425

inactivates the pachytene checkpoint and permits recombination mutants to complete meiosis I.81,82 cdc55 rad17 and cdc55 rad24 cells remained blocked in meiosis I at a point identical to cdc55 cells (not shown). During pachytene checkpoint activation the downstream target Swe1 becomes hyperphosphorylated and stable, and it inactivates Clb-Cdc28 Cdk activity to arrest cells.80 Deletion of SWE1 permits recombination mutants to complete meiosis.83 PP2ACdc55 negatively regulates Swe1 stability during mitosis.84 We tested if it does the same during meiosis, thus the pachytene checkpoint is constitutively activated in cdc55 cells due to accumulation of Swe1. This may possibly explain why cdc55 rad17 and cdc55 rad24 cells remained blocked. The loss of SWE1 did not suppress the meiotic replication defect of cdc55 cells (Sup. Fig. 1). In fact it altered the ability of cells to enter meiosis. Northern analysis of meiotic gene expression showed cdc55 swe1 cells were incapable of initiating early meiotic gene expression in the W303 strain background (Fig. 12A). swe1 cells expressed these genes albeit with a slight delay compared to wild-type cells.85 swe1 cells formed spores and tetrads were viable. Expression of a dominant allele of CDC28 does not suppress the meiotic block of cdc55 cells. cdc28-4ts mutants initiate meiotic replication and intragenic recombination at high temperature, but arrest at pachytene.80 clb5/6 mutants are replication deficient, suggesting Cdc28-Clb5/6 activity is required for meiotic replication.86,87 These data place the execution point of Cdc28 activity during meiosis around the time cdc55 cells are blocked. We tested the hypothesis that PP2ACdc55 positively regulates Cdc28 during meiosis, possibly through regulating Swe1 degradation, thus cdc55 mutants are blocked because they are unable to sustain Cdc28 activity. We expressed the dominant activated CDC28AF allele and determined its effects on meiosis. hop1 cells defective in recombination expressing this allele have increased nuclear division.83 cdc55 CDC28AF cells were unable to form bior tetranucleated cells or initiate and complete meiotic replication in W303 strains (Sup. Fig. 1). While removing the phosphorylation-dependent inhibition of Cdc28 did not suppress the meiotic replication block, it did have an effect on the meiotic gene expression pattern of cdc55 cells. CDC28AF cells expressed IME1 and IME2 to levels similar to wild-type cells85 (Fig. 12B). Expression patterns of HOP1, NDT80, CLB1, SPS4 and DIT4 were also normal (not shown). cdc55 CDC28AF cells expressed IME1 and IME2 later than wild-type, cdc55 or CDC28AF cells. Once IME1 expression was induced it was sustained and continued to increase. IME2 expression levels were drastically lower. While CDC28AF suppressed the loss of expression of SPS4 and DIT4 in cdc55 cells, expression levels were much lower than wild-type cells, and induction occurred all at once at 24 h. These results suggest phosphorylation/dephosphorylation regulates the temporal expression of meiotic genes, through a Cdc55-Swe1-Cdc28 pathway.88-90 cdc55 null cells are blocked in meiosis I prior to sister chromatid separation. Recent work has shown cells prematurely separate sister chromatids during meiosis in the absence of securin and Cdc55.39 These studies used a cdc55 strain harboring a galactose inducible CDC55 expression system. There is a 10-fold reduction

Figure 7. Loss of RAD9 partially suppresses the premeiotic DNA replication defect of cdc55 cells. Strains were shifted to sporulation medium and cell aliquots were taken at 0 and 24 h. DNA content was determined using propidium iodide.


1426

Figure 8. There is a lethal synthetic interaction between loss of RAD9 and loss of CDC55. Strains were grown to exponential phase and spotted onto YEPD plates containing the indicated mM hydroxyurea concentrations. Cells were grown at 30°C for 48 h.

in Cdc55 level compared to Rts1,91 which would predict only a small amount of Cdc55 is required for PP2ACdc55 function. We believe enough Cdc55 was present during the initial stages of meiosis I to allow cells to proceed to metaphase. To ensure that our cdc55 nulls cells were not blocked due to premature sister chromatid separation, we examined the meiotic dynamics of multiple tandem repeats of an integrated tetO sequence.32 We did not see any premature sister chromatid separation for up to 48 h for both SK1 and W303 cells (not shown). Eventually we did observe greater than 4 foci (96 h) suggesting separation (or fragmentation) had occurred. Discussion PP2ACdc55 regulates several steps in meiosis I. PP2A is a heterotrimeric protein phosphatase that regulates several events during

Cell Cycle

Volume 10 Issue 9

Figure 9. cdc55 diploid cells harbor defects in SBP dynamics. (A) number of SBP-associated Tub4 foci visualized after 12 h in sporulation medium, (B) time-dependent Tub1 dynamics.

the mitotic cell cycle.12,13,18,19 During meiosis Cdc55 regulates sister chromatid separation through shugoshin-dependent separase regulation.32,43 The results presented lead us to conclude PP2ACdc55 regulates other events during meiosis, as well. Meiotic entry, premeiotic DNA replication and DSB formation were accelerated due to loss of CDC55, suggesting a shortening of meiotic S phase. Deletion of the DNA damage-checkpoint Rad9 resulted in only partial suppression of DNA replication defects, as cells remained blocked in meiosis I; the continued meiosis I block may result from defects that activate a second meiotic checkpoint.60 Loss of Rad9 function increases nuclear division in the presence of HU-mediated DNA damage.60 cdc55 mutants harbor “leaky” defects in SPB dynamics that we believe are secondary to disruption of a more upstream event(s). Finally, cdc55 null cells did not show defects in premature sister chromatid separation until after wild-type cells had completed sporulation and formed ascicontaining spores. Why are multiple meiotic events accelerated in SK1 cdc55 cells? cdc55 mutants have a lower steady state level of Cln2 in W303 (McCourt P and Nickels JT, manuscript in preparation); we did not test whether Cln1/3 levels were also reduced. Cln2 is a negative regulator of meiosis through its regulation of IME1 expression and Ime1 localization, and it is degraded prior to entry into meiosis.92 G1 cyclin-deficient cells do not require starvation in order to enter meiosis.92 It is possible that the steady state Cln2 defect, and possibly Cln1/3, may be responsible for accelerating meiotic events in SK1 strains. Ume6 is a repressor of meiotic genes and it does so through its interaction with the Sin3-Rpd3 complex.51,93 Association of Ume6 with Ime1 converts it from a repressor to an activator.94 Deletion of Ume6 results in the vegetative expression of some meiotic genes, while it also results in a delay and/or reduction in meiotic gene expression during meiosis.94 cdc55 mutants express

early meiotic genes but are defective in late gene expression. The Ime1-Ume6 interaction is regulated by the Rim11 and Rim15 kinases.89 Thus, there is a well defined regulatory loop responsible for Ume6 regulation, which may be perturbed in cdc55 cells. This may explain why we observe an altered meiotic gene expression pattern. The expression defects we observed were seen only in the SK1 background, so strain differences exist and this may be due to differences in synchrony. PP2ACdc55 regulates premeiotic DNA replication and homologous recombination. Our replication data show cdc55 mutants accumulate what appear to be altered DNA structures. We see a greater than 2C peak by FACS analysis, and the accumulation of possible replication intermediates using 2D and alkaline gel electrophoresis. We have seen what appears to be recombination intermediates by electron microscopy (data not shown).64 Spo11dependent DSB accumulation occurs earlier in mutant cells and Cdc55 is required for the completion of intergenic (crossover), but not intragenic (gene conversion) recombination. The DSB repair pathway repairs all Spo11-dependent DSBs. Intergenic recombination is initiated by the formation of DSBs followed by formation of a heteroduplex, and rejoining of the ends forming Holiday junctions (HJs). Gene conversion is the result of mismatch repair in the region of heteroduplex formation.95 Gene conversion results from DSB repair or synthesis-dependent strand annealing (SDSA), as heteroduplex formation precedes pathway selection.96 Resolution of meiotic HJs is performed only by DSB repair.97,98 Since gene conversion is intact in cdc55 cells, possibly through SDSA, but intergenic recombination is defective, Cdc55 may be required directly or indirectly for DSB repair. Defective intergenic recombination could also be caused by defects in DNA replication that are due to a loss of Cdc55 and the accumulation of DNA damage. The latter explanation may resolve why recombination bypass mutations such as mei4, spo11, spo13, rec102



Cell Cycle

1427

or rec104 do not allow cdc55 cells to sporulate. cdc55 cells do not commit to undergo meiosis I, so most likely PP2ACdc55 regulates a pathway that is epistatic with premeiotic DNA replication and homologous recombination. Cdc55 cells are not arrested at pachytene. We considered the possibility that the damage due to loss of Cdc55 activated the pachytene checkpoint, as cdc55 cells had SPBs that were duplicated but unseparated, a hallmark of the pachytene stage of the meiotic cell cycle.61,62 Moreover, we presumed there was an accumulation of the pachytene checkpoint downstream target Swe1.84 rad24 and rad17 cells have a delay in DSB repair, and show a decrease in colocalization of Dmc1 and Rad51 and their association with chromosomes.99 Loss of DMC1 results in pachytene arrest, while rad17 dmc1, rad24 dmc1 and mec1 dmc1 cells complete meiosis I/II and sporulation.81 Generating null mutations of RAD17, RAD24 or MEC1 in cdc55 cells did not suppress the meiosis I defect (Sup. Fig. 1), leading us to believe that the defect observed in cdc55 cells lies prior to the pachytene checkpoint. The pachytene checkpoint responds to unrepaired recombination intermediates by arresting cells through NDT80 inhibition;79,100 ndt80 cells arrest at pachytene. When the pachytene checkpoint is activated, NDT80 expression is reduced and any Ndt80 becomes functionally inactive.100 cdc55 cells in the SK1 strain background express NDT80, suggesting the pachytene checkpoint Figure 10. DSBs are initiated earlier in diploid cells lacking PP2ACdc55. Various pp2a is not activated. However, northern analysis of mutants homozygous for rad50K181S (rad50S) were transferred to sporulation media, W303 cdc55 cells showed NDT80 is not expressed, aliquots were isolated and genomic DNA was isolated. DSBs were analyzed by Southern analysis examining DSB initiation at YCR048w locus. suggesting an arrest prior to pachytene (Fig. S2). dmc1 mutants have a block in middle sporulation gene expression, whereas dmc1 rad17 cells express of Rhp1 (Rad51 ortholog) loading onto ssDNA and when topoithese genes normally through their bypass of the pachytene somerase III resolves homologous recombination intermediates.103 checkpoint.101 If a defect in NDT80 expression caused the meiosis In mammals, it regulates the BRCA2-Rad51 interaction.104 The I defect in cdc55 cells, then we would assume deletion of RAD17 expression of the dominant CDC28AF allele in cdc55 cells remediwould restore NDT80 expression, bypass the pachytene check- ates their cs elongated phenotype, so a genetic interaction exists point, and allow cells to complete meiosis I. This was not the between Cdc55 and Cdc28.84,105 We expressed the dominant case (Sup. Figs. 1 and 2) further supporting our hypothesis that CDC28AF allele in an attempt to bypass a potentially activated cdc55 cells arrest prior to the pachytene checkpoint. Additionally, pachytene checkpoint. Presumably, this allele would bypass any mutation of rad17 in SK1 cdc55 cells does not restore the meiosis regulation by Cdc55. cdc55 CDC28AF cells remained blocked in I defect, again demonstrating cells are arrested prior to pachytene meiosis I, so if the block was due to CDK-dependent phosphorylation-dependent misregulation of homologous recombination, (Nolt J and Nickels JT, unpublished data). Phosphorylation/dephosphorylation regulates homologous we would predict cells would progress through meiosis I or be recombination. Homologous recombination can be regulated blocked late in meiosis I subsequent to DNA replication/homoloby cell cycle-dependent phosphorylation. In budding yeast, the gous recombination. cyclin-dependent Cdc28 kinase (CDK) regulates end resecThere are a number of meiotic events, including homologous tion through regulating Rad51-Rpa loading.102 The same kinase recombination, regulated by protein phosphatases. In addition regulates the Mre11/Rad50/Xrs2 complex and its role in end to PP2ACdc55 regulating shugoshin-dependent regulation of separesection. Conservation exists in the cell cycle regulation of rase,39 PP2A Rts1 stabilizes the meiotic cohesin, Rec8 until chromohomologous recombination in fission yeast103 and mammalian some separation is properly initiated;32,106 we found rts1 mutants cells.104 The fission yeast CDK, Cdc2, is important at the time did not accumulate DSBs. Very recently, Falk and Colleagues107


1428

Cell Cycle

Volume 10 Issue 9

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Figure 11. cdc55 diploid cells harbor defects in intergenic homologous recombination. Wild-type and cdc55 cells were shifted to sporulation medium and aliquots of cells were isolated at the indicated times. 10-fold serial dilutions were prepared and cells were plated onto multiple selective medium to determine the number of intergenic and intragenic events. The values were compared to glucose and acetate grown cells.

found that the PP2A catalytic-like subunit, Pph3, regulated meiotic crossover repair through modulating the phosphorylation state of Zip1. In our hands, pph3 RAD50s diploid cells did not accumulate DSBs, which is in contrast to what was reported.107 These contrasting results could be due to the fact that we examined DSB initiation at the YCR048 locus and not the HIS4LEU2 recombination hotspot. The Cdc25 phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells.108 The FEAR pathway, which activates the Cdc14 phosphatase, is required for meiotic chromosome segregation, nucleolus division and spindle disassembly.109,110 Here, we present results that have expanded our knowledge of the meiotic events regulated by PP2ACdc.55 Isolating the factors directly associated with and regulated by PP2A will go a long way in defining the complexity of meiosis-specific PP2A-dependent signaling events. Materials and Methods Strains. All strains are isogenic to SK1,69,79 or W303,71-73 backgrounds. Yeast gene nomenclature is found in Supplemental Table 2.


Sporulation and meiotic time courses. Yeast strains were grown in YEPD (1% yeast extract, 2% Bacto peptone, 2% glucose), YPA (1% yeast extract, 2% Bacto peptone, 1% potassium acetate) or SP medium (2% potassium acetate, supplemented with appropriate nutrients [when necessary]). For sporulation studies, cells were grown to 107 cells/ml at 30°C in YEPD, shifted to YPA to increase synchrony and subsequently transferred to SP medium; these growth conditions were required, since cdc55 cells harbor a severe growth defect when grown in the presence of acetate. Cells were collected for the indicated times. DAPI staining of DNA. Cells were collected at times indicated, gently pelleted and subsequently washed in 1x PBS, followed by fixation in 95% ethanol for minimum of 1 h at 4°C. Wash in 1x PBS. Treat cells with 10 ug/ml 4',6-diamidino-2-phenylindole (DAPI) at 4°C in the dark for 1 h. Finally, cells were washed and resuspended in 1x PBS and nuclei were visualized by fluorescence microscopy using a Leica DRME fluorescence microscope, UV optics and a PlanAPO 100x objective. Double strand break detection assay. All diploid strains used for meiotic DSB analysis were homozygous for rad50K181S (rad50S). Strains harboring this mutation do not resect

Cell Cycle

1429

meiotic DSBs after their formation.111,112 Cells were grown as described above. 5 x 108 cells were collected every hour for 12 h. Cells were washed twice in 50 mM EDTA, pH 7.5 and resuspended in 330 ul of 50 mM EDTA. 200 ul of resuspended cells equilibrated to 40°C were mixed with 330 ul LMP Agarose Mix (1% Low Melting Temperature agarose (NuSieve GTG Agarose), 0.125 M EDTA) and 68 ul Solution 1 (1 M Sorbitol, 0.1 M Sodium Citrate, 0.06 M EDTA, pH 7.5, 5% β-mercaptoethanol, 1 mg/ml Zymolyase 100T) and poured into a disposable plug mold (Bio-Rad) and solidified at 4°C for 20 min. Plugs were incubated at 37°C for 2 h in Solution 2 (0.45 M EDTA, 10 mM Tris-HCl, 7.5% β-mercaptoethanol, 10 μg/ml RNase A) followed by incubation overnight at 50°C in Solution 3 (0.25 M EDTA, 10 mM Tris-HCl, 1% Sodium lauroyl sarcosinate, 1 mg/ml Proteinase K). Plugs were stored at -20°C in a 50 mM EDTA, 50% Glycerol solution or processed by washing twice in 1x TE, pH 7.4 for 30 min at room temperature, then washed twice for 30 min at room temperature with 1x restriction enzyme buffer (NEBuffer 3, New England Biolabs). Plugs were removed from excess buffer, melted at 65°C for 10 minutes and digested for 2 h in 20 units AseI (New England Biolabs), followed by 2 h digestion in 10 additional units of enzyme. Plugs were melted at 65°C for 10 min and loaded into a “dry” 1% agarose gel and cooled for 5 min. The gel was run at 280–360 V-hrs. Neutral Figure 12. Loss of SWE1 or expression of the CDC28AF allele in cdc55 diploid DNA transfer was performed using Hybond N membrane 32 cells alters meiotic gene expression. (A) swe1 and cdc55 swe1 or (B) CDC28AF (GE Lifesciences) and membranes were hybridized with P and CDC28AF cdc55 cells were grown in glucose, shifted to acetate medium labeled probes (Megaprime Labeling Kit, GE Lifesciences) and then transferred to sporulation medium. Cell aliquots were isolated at using the following primers: 5'-GGA TTT GTT GCA AGA the indicated times and total RNA was extracted. mRNA expression of various CGA AG and 5'-TAA TGA TAC TGG GCC CTG AA in meiotic genes was determined by northern analysis. order to analyze DSBs at the YCR048w locus. Radioactivity was detected using a Storm 840 PhosphorImaging System and Storm Scanner Control software. Cells and ghosts are pelleted and 1.05 g/ml CsCl (Sigma) was Origin firing assays/2D-agarose gel electrophoresis. Origin added to the supernatant and dissolved, followed by the addiFiring Assays we performed as described at the website http:// tion of Hoechst 33258 dye. Samples were centrifuged at 55 K fangman-brewer.genetics.washington.edu with minor modifica- for 18 h in a NVT65 (Beckman Coulter) rotor. The appropriate tions. Briefly, cells were grown as described above for SK1 strains. band of DNA was drawn from the tube and washed three times 1 L of mid-log phase cells were isolated at each time indicated. with an equal volume of 5:1 isopropanol:water solution was genCells were killed by 0.1% Sodium Azide and chilled by adding tly added and mixed with DNA. Once phases separated, alcohol culture to frozen mixture of 0.2 M EDTA and 17% glycerol (80 layer was discarded. DNA was precipitated using three volumes ml frozen mixture per 330 ml culture) and shaken vigorously. of ice-cold 70% ethanol and resuspended pellet in TE. 25 μg of Cells were centrifuged, washed in water and resuspended in ice- DNA was digested in NheI (New England Biolabs), to generate a cold Nuclear Isolation Buffer (17% glycerol, 50 mM MOPS, DNA fragment size of 5.7 kb. DNA was treated with 1 M NaCl, 150 mM potassium acetate, 2 mM MgCl2, 0.5 mM Spermidine then enriched for replication intermediates using BND Cellulose (Sigma) and 0.15 mM Spermine (Sigma), pH 7.2). Cells were (Sigma), which was prepared using NET Buffer (1 M NaCl; vortexed with 5 ml acid-washed glass beads (425–600 μm, 1 mM EDTA; 10 mM Tris, pH 8.0). DNA was washed with Sigma) until >90% are broken (ghosts). Supernatant is removed isopropanol, precipitated in 70% ethanol and resuspended in TE. The first dimension was run as a 0.4% agarose gel for and beads were washed twice with Nuclear Isolation Buffer, retaining and pooling the supernatant each time. Centrifugation approximately 470 V-hrs. Second dimension was a 1% agarose was performed to obtain pellet of nuclei, ghosts and unbroken gel and ran for approximately 1,300 V-hrs at 4°C. DNA was cells. Pellets were resuspended in 8 ml of TEN buffer (50 mM transferred under neutral conditions to Hybond N+ membrane Tris, pH 8.0; 50 mM EDTA, pH 8.0; 100 mM NaCl). Sarkosyl (GE Lifesciences). Membranes were hybridized with 32P labeled (Sigma) was added to a final concentration of 1.5%. After gen- probes (MegaPrime DNA Labeling Kit, GE Lifesciences) using tly mixing, Proteinase K was added to a final concentration the following primers: 5'-TCT GAA GAG TTA AGC ACT CC of 600 μg/ml and incubated at 37°C for a minimum of 2 h. and 5'-CTT CCC GAG CGT GAA AGG AT to generate a 720


1430

Cell Cycle

Volume 10 Issue 9

bp fragment in order to analyze the rDNA ARS. Radioactivity was detected using the Pharos FX Molecular Imager (Bio-Rad) and ImageQuant software (GE Lifesciences). Meiotic mRNA expression analysis. Cells were grown as described above. Total RNA was extracted from 1 x 108 cells using acid-washed glass beads, SDS and buffered-saturated hot phenol. Total RNA was denatured in formaldehyde-SSPE (10 mM NaH2PO4 buffer, pH 7.7, containing 0.18 M NaCl and 1 mm EDTA) for 15 min at 65°C. 20 μg of denatured RNA was applied to a nylon membrane using a slot blot apparatus (Bio-Rad). Membranes were washed in SSPE, UV crosslinked and hybridized with radiolabeled PCR products that were obtained using the Megaprime Labeling Kit (GE Lifesciences). The hybridization buffer consisted of SSPE containing Denhardt’s solution. Post-hybridization, membranes were washed several times with SSPE containing 0.1% SDS. Hybridization and all washes were performed at 65°C. The level of U2 mRNA expression was used as a loading control. Gene expression levels were determined by Storm 840 PhosphorImaging system and Storm Scanner Control software. RNA slot blot analysis. Cells were grown as described above for SK1 strains. Total RNA was extracted from 108 cells per sample. RNA was extracted in the presence of glass beads, SDS and phenol saturated by sodium acetate buffer (50 mM Na Acetate, 10 mM EDTA, pH 5.0) alternating vortexing and incubation at 65°C for 10 min. Samples were cooled and centrifuged, followed by ethanol precipitation from the aqueous phase. RNA was dissolved in water and denatured in formaldehyde-SSPE (10 mM NaH2PO4 buffer, pH 7.7; 180 mM NaCl; 1 mm EDTA) 65°C for 15 min. 20 μg of denatured RNA was applied to a non-charged modified blotting nylon membrane (Hybond N, GE Lifesciences) using a slot blot apparatus (Bio-Rad). Membranes were washed in SSPE, UV crosslinked at 1,200 μJ/sec (Stratalinker) and hybridized with radiolabeled PCR products that were obtained using the MegaPrime DNA Labeling Kit (GE Lifesciences). Hybridization was performed at 65°C in Churches Buffer. Posthybridization, membranes were washed several times with SSC containing 0.5% SDS. Washes were performed at room temperature and 65°C for increasing stringency. The level of U2 mRNA expression was used as a loading control. Gene expression levels were determined by Storm 840 PhosphorImaging system and Storm Scanner Control software. Northern analysis. Total RNA was resolved using 6% formaldehyde agarose gels. 20 μg of total RNA in loading buffer (20 mM MOPS, pH 7.0 containing 10 mM sodium acetate, 2 mM EDTA, 45% formamide, 6% formaldehyde, 1% ethidium bromide, 0.003% bromophenol blue, 0.03% xylene cyanol FF, 1.5% Ficoll) was used for each sample. Total RNA was blotted onto Hybond N nitrocellulose (Amersham) overnight at room temperature using 10x SSC (40 mM sodium citrate and 1.8 M NaCl). Churches Buffer (500 ml Sodium-phosphate buffer, 2 ml EDTA, 10 g BSA, 70 g SDS, TV = 1 L in distilled water) was used for all hybridization procedures. Hybridization was performed overnight at 65°C. Gel-purified radiolabeled probes were boiled in 200 μl salmon sperm DNA prior to use. Post-hybridization, blots

were washed twice in 2x SSC at room temperature, twice in 2x SSC/0.5% SDS at 65°C and twice in 0.1x SSC at room temperature. Gene expression was determined by autoradiography using X-OMAT film (Kodak). FACS analysis. W303 and SK1 cells were collected at times indicated, gently pelleted, then washed in 1x PBS followed by fixation and permeabilization by the dropwise addition of 1 ml ice cold 75% ethanol while vortexing. Cells were incubated for a minimum of 1 h at 4°C. Cells were washed and treated with 1 mg/ml RNase A in 1x PBS followed by 10 minutes of incubation at 37°C. Finally, cells were stained for 20 minutes at 4°C in 5 ug/ml Propidium Iodide (Sigma) in 1x PBS followed by appropriate washing in PBS. FACS profile shown is typical of five independent experiments. Hydroxyurea sensitivity spot assay. Yeast SK1 strains were grown to exponential phase in YEPD. 107 cells were spotted as 10-fold serial dilutions onto YEPD plates or YEPD plates containing 0.2 M, 0.1 M, 0.05 M or 0.01 M Hydroxyurea (USB Products, Affymetrix, Inc.,) and incubated at 30°C. Cell growth was examined after 72 h. Western analysis. SK1 strains were sporulated as described above. Total cell protein extract was obtained from 108 cells that were collected at times noted. Briefly, cells were pelleted and washed then resuspended in one milliliter of Solution A (25 mM Glycerol Phosphate, 1 mM Na3VO4 and 100 ug/ml 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride). Samples are then incubated in 150 ul of 2 M NaOH and 8% 2-mercaptoethanol for 10 minutes at 4°C. 150 ul of cold 50% trichloroacetic acid was added and incubated for an additional 10 min while mixing gently. Samples were pelleted and washed twice in ice-cold acetone followed by protein extraction at 95°C for 4 min in Sample Buffer (3% SDS; 100 mM Tris-HCl, pH 6.8; 380 mM 2-mercaptoethanol; 15% glycerol; 4 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride; 0.01% bromphenol blue). Insoluble material was removed by centrifugation and proteins in the supernatant were resuspended in Laemmli buffer, and proteins were resolved using SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. All steps for western analysis were performed at room temperature. Membranes were blocked for 1 h with 5% nonfat dry milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05% Tween-20. Incubations with primary and secondary antibodies were performed at room temperature for 1 h in 1% nonfat dry milk, 10% goat serum, 10 mM Tris-HCl, pH 7.4 and 150 mM NaCl. Membranes were washed five times after antibody incubations with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.05% Tween-20. Primary antibodies used for immunoblot include anti c-myc (Covance) and α-tubulin (a generous gift from Dr. Jane Azizkahn-Clifford at Drexel University College of Medicine). Primary antibodies were detected with polyclonal goat antimouse horseradish peroxidase (Amersham Biosciences). Proteins were detected using ECL chemiluminescence (GE-Amersham). Homologous recombination assays. Recombination assays determining the intragenic recombination frequency at TRP1 and the frequencies of intergenic at CEN3 to MATa/MATα and



Cell Cycle

1431

CEN2 to HIS3/LYS2 intervals were performed using homozygous deletion strains that were constructed using the W303 haploid strains YJN699 (MATa ade2-1 can1::ADE2:CAN1 his311,15 lys2(3' del):HIS3:lys2(5' del) leu2-3,112 trp1-1 ura3-1) and YJN700 (MATα ade2-1 can1::ADE2:CAN1 his3-11,15 leu23,112 trp1-3' del ura3-1) first described by Lee and Honigberg.70,71 The frequencies of intra- and intergenic recombination were determined using prototrophy selection and mating assays as described previously. Strains used to examine intragenic recombination at the LEU2 and the ARG4 loci were grown as described above for SK1 cells and recombination frequencies were determined by prototrophy selection.74 Alkaline gel electrophoresis. Cells were grown as described above for SK1 strains. 1.5 x 108 cells were collected at indicated times and separated from growth media by centrifugation. Cells were resuspended in 2 volumes of lysis buffer (1% SDS; 2% Triton X-100; 100 mM NaCl; 10 mM Tris, pH 8.0; 1 mM EDTA). 1 volume of glass beads (425–600 μm, Sigma) were added to cells followed by 1 volume of Phenol-Chloroform-Isoamyl Alcohol, 25:24:1 (PCIAA, Sigma). Cells were gently disrupted. Cells were then treated with 1 volume TE, inverted to mix, then phases were separated by centrifugation. The aqueous phase was treated with 500 μl PCIAA, inverted to mix until slurry is developed, centrifuged to separate phases and repeated again. The aqueous layer is precipitated in 1 ml of 95% ethanol and 75 μl of 3 M Sodium Acetate. Precipitate was washed with 70% ethanol, dried and resuspended in TE and treated with RNase A. Alkaline agarose gel electrophoresis was carried out as described in reference 55. Briefly, DNA was separated on 0.7% agarose gels. Agarose was melted in 50 mM NaCl and 1 mM EDTA. Cooled gel was equilibrated for 30 minutes in alkaline running buffer (5 mN NaOH; 1 mM EDTA, pH 8.0). DNA concentration was measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific). 35 μg of DNA from each sample was mixed with 0.2 volumes of 6x alkaline loading buffer (300 mN NaOH, 6 mM EDTA, 18% Ficoll T-400, 0.15% bromocresol green, 0.25% xylene cyanol FF) and loaded into wells of the gel. Gel was run for approximately 360 V-hrs at