Copyright 8 1995 by the Genetics Society of America
The Yeast MER2 Gene is Required for Chromosome Synapsis
and the Initiation of Meiotic Recombination
Beth Rockmill,* JoAnne Engebrecht,*”Hany Scherthan,+Josef Loidlf and G. Shirleen Roeder*
*Department of Biology, Yale University, New Haven, Connecticut 06520-8103, tDepartment of Human Biology and Human Genetics,
University of Kaiserslautm, 0-67653 Kaiserslautm, Germany, and fDepartment of Cytology and Genetics,
Institute of Botany, University of Vienna, A-1030 Vienna, Austria
Manuscript received March 8, 1995
Accepted for publication May 30, 1995
ABSTRACT
Mutation of the MER2 gene of Saccharomycescerewisiae confers meiotic lethality. To gain insight into
the function of the Mer2 protein, we have carried out adetailed characterization of the mer2 null mutant.
Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion
and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the
ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that
the mer2 mutation prevents the formation of meiosis-specific,double-strand breaks, indicating that the
Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis
reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex,
but homologous chromosomes fail to synapse. Fluorescence
in situ hybridization of chromosome-specific
DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly
reduced in the w 2 mutant. Although the
gene is transcribed during vegetativegrowth, deletion
or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage
repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic
exchange.
M
EIOSIS is a special type of cell division that produces haploid gametes from diploid parental cells
through two successive rounds of chromosome segregation. At the meiosis I division, homologous chromosomes move to opposite poles, while sister chromatids
remain associated. In most organisms, this reductional
segregation depends on genetic recombination between homologous chromosomes and the intimate association of homologs in the context of synaptonemal
complex (SC) .
Double-strand breaks (DSBs) initiate many, if not all,
meiotic recombination events in Saccharomyces cerevisiae
(GAME1992; ZENVIRTH et al. 1992; Wu and LICHTEN
1994). Meiosis-specific DSBs have been detected at several recombination hotspots (SUNet al. 1989; CAO et al.
1990; GOLDWAY
et al. 1993) and at a number of preferred sites on every chromosome assayed (GAME1992;
ZENVIRTHet al. 1992; WU and LIGHTEN1994). DSBs are
processed to expose single-stranded tails with 3‘termini
(SUNet al. 1991; BISHOPet al. 1992). According to the
DSB repair model of recombination (SZOSTAK
et al.
1983; SUNet al. 1991), these single-stranded tails invade
an homologous DNA duplex to generate a region of
hybrid DNA flanked by Holliday junctions. Repair of
Corresponding author: G. Shirleen Roeder, Department of Biology,
Yale University, P. 0.Box 208103, New Haven, CT 06520-8103.
E-mail: shirleen_roeder@quickmail.yale.edu
‘Present address: Department of Pharmacology, State University of
New York, Stony Brook, NY 117948651,
Genetics 141: 49-59 (September, 1995)
mismatched base pairs present in hybrid DNA results
in gene conversion. Resolution of Holliday junctions
can lead to crossing over between flanking markers.
During meiotic prophase, homologous chromosomes synapse with each other to form the SC, which
is a tripartite structure consisting of two parallel lateral
elements separated by a central region (vON WETTSTEIN
et al. 1984). Each lateral element represents the protein
backbone of one pair of condensed sister chromatids
and is called an axial element before its incorporation
into SC. Most chromatin is located outside the complex
and is folded into a series of loops, each attached at
its base to a lateral element. In some organisms, the
formation of tripartite SC has been shown to be preceded by the side-by-side alignment of homologous
chromosomes at a distance that exceeds the width of
the SC (LOIDL 1990). In yeast, this presynaptic alignment has been visualized by fluorescence in situ hybridization (FISH) using composite, chromosome-specific
DNA probes ( SCHERTHAN
et al. 1992). Throughout this
paper, synapsis is defined as the formation of mature
SC, while pairing refers more generally to the alignment of homologous chromosomes, both before and
during SC formation (GIROUX1988; ALANI et al. 1990;
SYM et al. 1993).
Studies of meiotic mutants are beginning to identify
some of the gene products required for
meiotic recombination and chromosome synapsis.Analysisofyeast
mutants has been facilitated by the spol3 mutation,
50
B. Rockmill et al.
which causesdiploid cells to undergo a single round of
chromosome segregation to produce two-sporedasci
containing diploid spores (KLAPHOLZ and ESPOSITO
1980). Recombination is not required for the production ofviable spores in a $1013 strain (MALONE and
ESPOSITO1981), because chromosomes can segregate
either reductionally or equationally (HUGERAT
and
SIMCHEN
1993). Sporulation-proficient, meiotic-lethal
mutants have been classified into two groups, based on
their behavior in a spol3 strain background (for review,
see PETESet al. 1991). Class 1 mutants produce viable
spores in conjunction with spol3, whereas class 2 mutants produce deadspores. Furthermore, aclass 1 mutation restores spore viability to a spo13 strain carrying a
class 2 mutation. Based on these observations, class 1
mutants are generally assumed to be defective at an
early step in the recombination pathway (e.g., initiation), whereas class 2 mutants are thought
to be blocked
at latersteps (e.g., resolution of recombination intermediates).
The MER2 gene was identified in two different
searches for meiotic genes. MALONE et al. (1991) identified MER2 in a selection for mutations that restore
spore viability to a class 2 mutant carrying a spol3 mutation. ENCEBRECHTet al. (1990) recovered the MER2
gene in a screen for multicopy suppressors of the gene
conversion defect of a meiotic mutant called merl. Preliminary studies (ENGEBRECHT
et al. 1990) of a mer2 null
mutant and more
extensive characterization (COOLand
MALONE 1992) of a mer2 allele induced by ultraviolet
light (W)indicate that the Mer2 protein is required
for most or all meiotic recombination events.Only
1% of the spores derived from a mer2 mutant diploid
are viable.
The MER2 gene displays a unique pattern of regulation. Many yeast genes have been identified that function specifically in meiotic cells. With the exception of
MER2, all of these genes are regulated at the level of
transcription; there is little or no transcription during
vegetative growth and transcription is strongly induced
during sporulation (MITCHELL1994). In contrast, the
MER2 gene is transcribed constitutively. The primary
transcript of the MER2 gene contains an intron that
must be removed byRNA splicing for the transcript
to encodea full-length, functional protein. Efficient
splicing of MER2 pre-mRNA requires the productof the
MER1 gene, which is transcribed only during meiosis
(ENGEBRECHT
et al. 1991). In vegetative cells that lack
Merl, only -10% of MER2 pre-mRNA is spliced; in
meiotic cells containing Merl, the efficiency ofsplicing
is increased dramatically. Recently, Merl has been
shown to be an RNA-binding protein withspecificity
for MER2 pre-mRNA (NANDABALAN
and ROEDER
1995).
To gain further insight into the molecular function
of the MER2 gene product, we have used a variety of
genetic, molecular and cytological methods to characterize the mer2 null mutant. The results presented con-
-
firm the hypothesis that the mer2 mutation blocks meiotic recombination at avery early step and demonstrate
that the MER2 gene product is required for chromosome synapsis and for stable pairing between homologous chromosomes.
MATERIALSAND METHODS
Yeast strains and genetic manipulations: Yeast media were
prepared andgenetic methods were carried outas described
by ROSEet al. (1990). YEPAD is YEPD medium supplemented
with adenine. Yeast strains were transformed usingthe lithium
acetate procedure of ITO et al. (1983).
The genotypes of yeast strains used in this study are presented in Table 1. Isogenic derivatives ofY20 and 5114 were
obtained by transforming the disomic haploids. S2701 is derived from S1570 by MER2 disruption; thus, URA3 is inserted
into the ribosomal DNA (rDNA) array at the same location
in both strains. Isogenic derivatives of 5360 and BR2495 were
obtained by transforming the haploid parents and then matingthe
transformants.Strain
S1570 was obtained from
THOMASMENEES.
Strain U469, obtained from RODNEY ROTHSTEIN, contains the 1.1-kb Hind111 fragment of URA3 inserted
at the Hind111 site immediately centromere-proximal to the
wild-type SlJF'4 gene. U469 was crossed to JE193-2B (ENGEBRECHT et al. 1990) transformed withpAM506 (SMITHand
MITCHELL1989) and pME58 (ENGEBRECHT
et al. 1990) to
generate S2703.
BR2920 was constructed using two strains, (21017-6D and
G911-10D, generously provided byJoHN GAME.Both strains
are congenic withSKI(FAST 1973); G911-10D containsa
circular derivative of chromosome ZZZ. G911-10D was crossed
to a lys2 derivative of SKI to obtain a lys2 rad5@Kl81::UBI3
segregant carrying a circular chromosome ZZZ. This segregant
(PC404) was mated to G1017-6D to obtain BR2920. G10176D and PC404 were transformed with pR1400 to introduce
the mer2::LYS2allele and transformants were mated to generate BR2921.
Strains carrying an intronless version of the MER2 gene
were constructed by two-step transplacement(ROTHSTEIN
1991)using pR1106.Derivatives that had lost the plasmid
were analyzed by Southern blot hybridization to identify those
in which the intronless version of MER2 remained on the
chromosome.
Plasmids: Plasmid R1400, constructed by LAURA PRICE, was
used to generate yeast strains carrying the merZ::LYS2 allele.
To make pR1400, the 4.8-kb XbaI fragment containing the
LYS2 gene from pDP6 (FLEIGet al. 1986) was inserted at the
XbaI site in the MER2 gene in pR1040. Plasmid R1040 is a
derivative of pBR322 in which the 3.Gkb EcoRI-BamHI fragment containing MER2 (ENGEBRECHT
et al. 1990) has been
inserted between the EcoRI and BamHI sites. Before transformation into yeast, pR1400 was digested with EcoRI and SphI.
Plasmid ME58, carrying the mer2::ADE2 mutation(ENGEBRECHT et al. 1990), and pME302, carrying the spoll::ADE2
mutation (ENGEBRECHT
and ROEDER1989), have been described previously.
Plasmid R1106 was used to construct strains carrying an
intronless version of the MER2 gene. To construct pR1106,
the EcoRI-BamHI fragment containing the intronless MER2
et al. 1991) was inserted
gene from pME268 (ENGEBRECHT
between the EcoRI and BamHI sites of YIp5. Before transformation into yeast, pR1106 was cleaved with BglII.
Plasmid R1307, obtained from BRADLEY OZENBERGER,
was
used to insert the URA3 gene into the rDNA array. A 3.841
PuuII fragment ofrDNA containing the 3' end of the 25s
rRNA gene and a portion of the nontranscribed spacer was
Yeast Meiotic Recombination Gene
51
TABLE 1
Yeast strains
Strain
Genotype
5360
MATa his4-260 W C l O leu2-27 trpl-289 CYHlO lys2 ura3-l
MATa HIS4cdcl0 leu2-27
trpl-1 cyhlO LYS2 ura3-1
spol3::URA3 arg4-9 THRl ade2-1
spol3::URA3 arg4-8 thrl ade2-1
5352
same as 5360but homozygous for m2::ALlE2
Y20
MATa CRY1 leu2-112 his4-260,39
ura3 trpl-H3 spol3::TRPI ade2-l lys2-1 qhlO
MATa q 1 leu2-3 his4280
S1791
S1570
S2701
same as Y20 but mer2::ADE2
same as Y20 but RLlNI::URA3
same as Y20 but RDNl::URA3 mer2::LYS2
J114
his4
MATa CDCIO leu2
ura3 trpl canl cyh2 a&2-1 $013-I sap3 lys2-99
MATa cdcl0 LEU2::pNH18-1 HIS4
S1793
same as J114 but mer2::ADE2
BR2920
MATa leu2-Al linear III lys2 canl ura3 hom3 hisl-7 trp5-arad5O-K181::URA3ade2-4"
MATa leu2-29 circular I I I G CANl ura3 HOM3 hisl-1 trp5-g rad5O-K181::URA3 ADE2
BR2921
same as BR2920 but homozygous for mer2::LYS2
BR2495
MATa leu2-27 his4-280 ura3-I trpl-289 CYHlO
are-8 thrl-1 ade2-1
~
MATa leu2-3,112 his4-260 ura3-1 trpl-1 cyhlO ARG4 thrl-4 ade2-l
S2500
S1590
S2888
S2702
S2947
same as BR2495 but
same as BR2495 but
same as BR2495 but
same as BR2495 but
same as BR2495 but
S2703
MATa leu2-27 his4-260 ~trpl-289 lys2 CANl ura3-I arg4-9 HIS3 ade2-1
MATa leu2-3,112 HIS4 trpl-lLYS2canl
ura3-1 ARG4 his3-11,15 ade2-l
imel-1::TRpl mer2::ADE2
SUP4
Ii?IEl
MER2
SuP4::URA3
heterozygous for ADE2
homozygous for mer2::ADE2
homozygous for spoll::ADE2
homozygous for MEm-c
homozygous for MERZ-c and heterozygous for ADE2
The following strains have been described previously: Y20 (MENEESand ROEDER
(1989),5114 (ENCEBRECHT
and ROEDER
1989),5360 and 5352 (ENGEBRECHT
et al. 1990), BR2495 (ROCKMILLand ROEDER1990) and S2500
(MENEESet al. 1992). The rad5GK181 allele present in BR2920 and BR2921 is referred to as rad50Selsewhere
in the text.
used to replace the polylinker in pUC18. The URA3 gene was
inserted as a 1.1-kb Hind111 fragment into the unique BglII
site after filling in the endsof both fragments with the Klenow
fragment of DNA polymerase I . Before transformation into
yeast, pR1307 was digested with FuuII.
Determination of recombinationfrequencies: Spontaneous mitotic and meiotic frequencies of intragenic recombination were determined as described previously (ENGEBRECHT
and ROEDER1989). Median frequencies were calculated by
the method of LEAand COULSON
(1949).
Recombination induced by U V was measured as follows.
Cells were grown to saturation in YEPAD and then appropriate dilutions were plated on synthetic complete medium
or medium lacking histidine or threonine and exposed to a
germicidal lamp (Sylvania G15T8) at a distance of 12 inches
for 30 sec. After treatment with UV, plates were incubated in
the dark at 30" for 3 days.
Recombination induced by methyl methane sulfonate (MMS)
was determined as follows. Cells grownto saturation in YEPAD
were diluted 1 in 20 into YEPAD and MMSwas added to a
final concentration of 0.01%. After incubation at 30" for 6 hr
with shaking, an equal volume of 10% sodium thiosulfate was
added and cells were pelleted by centrifugation. Appropriate
dilutions were then plated on synthetic complete medium
and medium lacking histidine or threonine.
DSB assay: Fresh overnight cultures of strains BR2920 and
BR2921 in YEPAD were diluted 100-fold into YPA (CAO et al.
1990) and grown at 30" for 12 hr. Cellswere then washed
and diluted twofold into 2% potassium acetate and incubated
with vigorous shaking at 30".Aliquots of sporulating cells were
harvested at the times indicated and chromosome plugs were
prepared according to SHERMAN and
WAKEM(1991) using
InCert agarose (FMC BioProducts, Rockland, ME). Yeast
chromosomes were separated on a crossed field gelapparatus
et al. 1981) by electrophoresis at 150 V with a 25(SOUTHERN
sec pulse for 20 hr. The gel was blotted and probed with a
et
radiolabeled 2.2-kb EcoRV fragment of pSG315 (GOLDWAY
al. 1993), derived from the THR4 locus on chromosome ZII.
Cytology: Chromosome spreads were prepared from d i p
loid BR2495 and isogenic derivatives. Cellsweregrown
to
saturation at 30" in YEPAD medium (ROSE et al. 1990) and
then diluted 7.5-fold into 2% KAc and incubated at 30" for
52
B. Rockmill et al.
TABLE 2
TABLE 3
Meiotic gene conversion in a mer2 null mutant
Meiotic crossing over in a mer2 null mutant
Strain, relevant
genotype
His prototrophs
Mitotic
Meiotic
Fold decrease
Leu protrophs
Mitotic
Meiotic
Fold decrease
Y20,
MER2
2.9 X 1 0 - ~
1.2 x
1x
5.2 X
1.4 X 1 0 - ~
1x
S1791,
rner2::ADEZ
1.6 X 1 0 - ~
2.4 X
50X
4.8 X
4.3 x 10-6
326X
His and Leu prototrophs were selected in diploids carrying
heteroalleles at the HIS4 and LEU2 loci. Mitotic and meiotic
recombination frequencies represent the mean values obtained
from two independent cultures. Folddecrease is the mean
meiotic frequency for wild type divided by the mean meiotic
frequency for the mutant. S1791 is a transformant of Y20.
15 hr. For electron microscopy, cells werespread and stained
with silvernitrate as described by DRESSER
and GIROUX
(1988)
and ENCEBRECHT
and ROEDER
(1990), except that cells were
spread onto clean glass slides that had not been precoated
with plastic. Spreads were subsequently overlaid with plastic
and lifted onto copper mesh grids (LOIDLet al. 1991). For
FISH, cells werespread as for electron microscopy with modifications noted by NAG et al. (1995).
FISHwith chromosome-specific, composite DNA probes
(chromosome painting) was carried out as described by
et al. (1992). Chromosome I and ZII probes were
SCHERTHAN
generated and detected as described by LOIDLet al. (1994).
The chromosome Z probe spanned 60 kb of this 230-kbchromosome and was detected as a red signal (tetramethylrhodamine isothiocyanate fluorescence). The chromosome III
probe spanned 185 kb of this 340-kb chromosome and was
detected as a green signal (fluorescein isothiocyanate fluorescence). The frequency of associations between homologous chromosomes was corrected for accidental associations
as determined from the frequency of associations between
nonhomologous chromosomes (LOIDLet al. 1994). The corrected frequencies for chromosomes I and ZII were averaged
in each experiment. To compare chromosome condensation
or homolog pairing in strains of different genotype, the results of all experiments for each of two strains were analyzed
using the Wilcoxon two-sample rank-sum statistic.
RESULTS
Meioticinterchromosomalrecombination
is abolished in a mer2 null mutank The effect of a mer2 null
mutation on intragenic recombination was examined
in haploid spol3 strains that are disomic for chromosome III and carry heteroalleles at the HIS4 and LEU2
loci. Hisand Leu prototrophic recombinants result primarily from gene conversion. As shown in Table 2, meiotic prototroph formation in the mer2 mutant isreduced dramatically relative to wild type. The frequency
of recombinants in the mutant is not increased above
the mitotic background level, demonstrating that the
mer2 mutation confers an absolute defect in meiotic
gene conversion.
Strain, relevant genotype
Spore viability
2-spore viable dyads analyzed
Distance HIS4-WClO (ZIZ)
Distance WClO-MAT (IIZ)
Distance CYHl O-LYS2 (II)
Distance ARM-THRl (WII)
Aberrant segregation ZI
Reductional segregation IZ
Aberrant segregation III
Reductional segregation III
5360,
MER2
75%
101
29cM
12 cM
49 cM
10 cM
0%
9%
2%
3%
5352,
rner2::ADE2
94%
152
<0.7
<0.7
<0.7
<0.7
cM
cM
cM
cM
0%
0%
0%
0%
The map distances for 5360 are taken from ENGEBRECHT
et
al. (1990). Map distances were calculated as described preet al.
viously (ENGEBRECHT
and ROEDER1989; ENGEBRECHT
1990; ROCKMILL
and ROEDER1990). The predominant pattern of chromosome segregation was equational. Reductional
and aberrant segregations of chromosomes II and ZII were
identified as described previously (ENGEBRECHT
et al. 1990).
The pattern of segregation of chromosome WII could not be
determined due to the absence of a tightly centromere-linked
marker. 5360 and 5352 are isogenic.
The effect of the mer2 deletion on meiotic crossing
over was measured by dissection and analysis of dyads
from isogenic mer2 spol3 and MER2 spol3 diploids. Map
distances were measured in four different intervals:
HIS4-OCIO and CDCIO-MAT on chromosome III,
CYHIGLYS2 on chromosome II and ARM-THRI on
chromosome WII,As shown inTable 3, meiotic crossing
over is completely eliminated by the mer2null mutation.
mer2 eliminates meiotic intrachromosomal recombination: The effect of a mer2 mutation on meiotic intrachromosomal recombination was measured in spol3
haploid strains disomic for chromosome III, using the
assay developed by HOLLINCSWORTH
and BYERS(1989).
An 11.4kb segment of DNA between HIS4 and LEU2
is duplicated on one copy of chromosome IIJ inserted
between the repeats is the wild-type CYH2 gene. Recombinants that havelost the CYH2 markerdueto
exchange between the repeats can be selected on medium
containing cycloheximide, due to a recessive mutation
conferring cycloheximide-resistanceat the CYH2 locus
on chromosome V I . In this assay, a mer2::ADE2 strain
displays a level of recombination that is20-foldless
than the wild-type level and no higher than the mitotic
background level (Table 4).
Previous studies have provided evidence that meiotic
intrachromosomal recombination in the naturally OCcurring rDNA array is regulated differently from recombination in artificial duplications (GOTTLIEBet d .
1989). To determine
whether MER2 is required formeiotic recombination in the rDNA, spol3 haploids carrying a URA3 insertion in the rDNA array were examined by dyad dissection (Table 5). In the MER2 strain,
meiotic recombination resulting in lossof the URA3
Yeast Meiotic Recombination Gene
TABLE 4
rad5OS
7 10.5
0 3
Meiotic intrachromosomal recombination
in a mer2 null mutant
Strain, relevant
genotype
mer2 rad5OS
0 3 7 10.5
J114,
MER2
mer2::ADE2
linear I l l +
linearized
circle
111")
othercut
products?
8.5 X 1 0 - ~
1.7 X
7.7 x lo-%
1x
22%
66%
12%
3.8 X 1 0 - ~
3.3 X 1 0 - ~
3.8 X 1 0 - ~
20 x
87%
0%
13%
FIGURE
1.-Double-strand break assay in wild type and mer2
LYS2. Chromosomes extracted from BR2920 ( M E R 2 rud50S)
and BR2921 (mer2::LYS2 rud50S) cells after 0, 3, 7 and 10.5
hr in sporulation medium were separated by pulsed-field gel
electrophoresis, blotted to a filter and then hybridized with
a probe for chromosome IIZ.
S1793,
CyhRfrequency
Mitotic
Meiotic
Corrected meiotic
Fold decrease
Equational segregation
Reductional segregation
Aberrant segregation
53
Mitotic and meiotic frequencies of CyhRrecombinants are
the median and mean frequencies, respectively, obtained
from three independent cultures. CyhR recombinants were
selected on medium lacking histidine and leucine as deand BYERS
(1989). The corrected
scribed by HOLLINGSWORTH
meiotic frequency was obtained by dividing the meiotic frequency by the fraction of dyads displaying
equational segregation because recombinants can be detected only when chroand
mosome IZZ segregates equationally (HOLLINGSWORTH
BYERS
1989). Fold decrease is the correctedmeiotic frequency
for the wild type divided by the corrected meiotic frequency
for the mutant. Aberrant, reductional and equational segregations of chromosome ZIIwere identified as described by ENGEBRECHT and ROEDER (1989). SI793 is a transformant of J114.
gene was observed in 7.1% ofthe dyads examined. Meiotic recombination occurred in <0.1% of dyads from
the mer2 mutant.
mer2 eliminatesreductionalsegregation
in spol3
strains: $013 diploids undergo a mixed meiotic division, inwhich some chromosomes segregate equationally, whileothers undergo eitherreductional or aberrant segregation (KLAPHoLz and ESPOSITO 1980;
HUCERAT
and SIMCHEN
1993). Crossing over promotes
reductional segregation and increases the frequency of
missegregation. In a spol3 strain background, the mer2
mutation abolishes reductional segregation and improves spore viability (Tables 3 and 4), as shown preTABLE 5
Meiotic recombmation in the rDNA array
in a mer2 null mutant
Strain, relevant genotype
Twespore viable dyads analyzed
Ura+:Ura+dyads
138
Ura+:Ura-dyads
Ura-:Ura-dyads
Meiotic recombination frequency
S2701,
S1570,
MER2
128
104
8
16
7.1%
mer2::ADE2
152
0
14
<0.7%
Nonrecombinant dyads contain two Ura+ spores; recombinant dyads contain one Ura' and one Ura- spore. Dyads
containing two Ura-spores are assumed to result from mitotic
recombination. The percent meiotic recombination was calculated as the number of Ura+:Ura-dyads multiplied by 100
divided by the total of Ura+:Ura+dyads and Ura+:Ura-dyads.
viously for other mutants with defects in recombination
(MALONE1983; MALONE and ESPOSITO 1981; ENCEBRECHT and ROEDER1989; HOLLINGSWORTH
and BYERS
1989; MENEES and ROEDER 1989; ROCKMILLand
ROEDER1990; BHARGAVA
et al. 1991).
mer2 fails tomake meiotic DSBs: Because mer2 strains
display no induction of meiotic recombination, it was
of interest to determine whether the
w2mutation prevents the formation of meiotically induced DSBs. To
do so, the physical assaydeveloped by GAMEet al. (1989)
was employed. The diploid strain used for these experiments carries one linear copy of chromosome ZZZand
one circular variant of this chromosome. The circular
chromosome IZZdoes not enter a pulsed-field gel; however, a meiotic DSB at any location on the circular c h r e
mosome generates a linear molecule that does enter
the gel and migrates with a faster mobility than its homolog. The strain used also carries the rads0S allele.
rad50S mutants fail to process meiotic DSBs, leading to
an accumulation of this otherwise transient intermediate (AIANI et al. 1990).
Figure 1 shows the analysis of DNA extracted from
wild-type and mer2 cells before meiotic induction and
at various times after the introduction into sporulation
medium. In wild type, only the original linear chrome
some IIZis evident in DNA from premeiotic cells. After
a few hours of meiotic induction, the linear version of
the circular chromosome is also detected, as are a number of fragments of faster mobility, which presumably
are derived from both chromosome ZZZhomologs. Consistent with previous studies (GAME1992; ZENVIRTHet
al. 1992), distinct bands were observed, implying that
many of the breaks occur at specific sites. In the mer2
mutant, no DSB products were detected, even after p r e
longed incubation in sporulation medium. Thus, the
mer2 null mutation eliminates the formation of meiotic
DSBs.
mer2 is defective in SC assembly: To examine the
effect of the mer2 mutation on chromosome synapsis,
meiotic chromosomes were surface spread, stained with
silver nitrate and examined in the electron microscope.
At the 13-and 15hr time points examined, a significant
fraction of wild-type cells were in the pachytene stage
R. Rockmill
54
us
AE
MI
MII
MI
MII
50
15 hours
us
AE
sc
FIGURE%-Distribution of cells in meiotic nuclear spreads
at IS and I5 hr of sporulation. Solid bars represent wild-type
nuclei (S2500) and hatchedbars represent mpf2::ADE2 nuclei
(S1590). The categories of nuclei include US (unstructured),
AE (axial elements), SC, MI (meiosis I ) and MI1 (meiosis 11).
US nuclei show uniform staining and no evidence of SC or
axial elements. AE nuclei contain axial element$. SC nuclei
contain fully synapsed chromosomes. In most of the US, AE
and SC nuclei, duplicated but unseparated spindle polebodies are apparent. MI nuclei contain two separate spindle pole
bodies, whereas MI1 nuclei contain four separate spindle pole
bodies o r two pairs of duplicated, but unseparated spindle
pole bodies. MI and MI1 nuclei do not contain any SC or
axial element!. S2500 and SI590 are isogenic.
of meiosis(Figure 2). An example of awild-type nucleus
containing fully synapsed chromosomes is shown inFigure 3A. Within most SCs, two parallel lateral elements,
separated by a less densely stained central region, are
evident. Unsynapsed axial elements were not observed
in spreads from wild-type cells. In contrast, in the mer2
mutant, nuclei containing multiple axial elements (Figure 3B) were observed at a frequency similar to the
frequency of pachytene nuclei in wild type (Figure 2).
In some of these nuclei, the axial elements were significantly shorter than expected for full-length chromosomes; in other nuclei, the elements appeared to be
fully developed. SC was never observed in spreads from
the mer2 mutant. Cells undergoing meiosis I or I1 were
observed at approximately wild-typelevels, indicating
that the mer2 mutant is proficient in nuclear division.
w 2 strains undergo a low levelof homologous chromosomepairing To assay homolog pairing, meiotic
chromosomes were surface spread and painted with
composite probes for chromosomes I and III. Only nuclei containing clear and compact hybridization signals
el
al.
for both chromosomes were scored (see LOIDLet al.
1994). Signal compaction is a reflection of chromatin
condensation, which is maximal at pachytene (DRESSER
and GIROUX1988; LOIDLet al. 1994). Homologs were
classified as paired if they were so close together that
their FISH signals had fused into asingle spot or if their
signals were touching each other.
In the wild-type cellsharvested after 15 hr in sporulation medium, almost all FISH signals were paired (Table 6). In contrast, in the mer2 mutant, less than onethird of the homologs were associated. For comparison,
homolog pairing was measured in an isogenic spoll
mutant (Table6); homolog pairing in the spoll diploid
was significantly lower than in mer2 ( P < 0.005).
The percent of nuclei that display compact FISH signals represents the fraction of cells in whichchromatin
is condensed; thus, the FISH procedure provides an
assessment ofchromatin condensation. In the
mer2 and
s t 0 1 I mutants, the fraction of nuclei containing condensed chromosomes is not significantly different from
wild type (Table 6).
w 2 does not affect spontaneous
or induced mitotic
recombination: To explore thepossibility that theMer2
protein plays a role during vegetative growth, the mer2
null mutant was examined in assays ofspontaneous and
induced mitotic recombination. In addition, mitotic recombination was measured in diploids homozygous for
the intronless versionof the MER2 gene (MEM-c),
which produce about 10 times as much spliced MER2
RNA as wild type (ENCEBRECHT
et al. 1991) and therefore presumably 10 times as much Mer2 protein. The
wild type, mer2 and MER2-c diploids showed similar levels of recombination spontaneously and after exposure
to UV or MMS (Table 7). Furthermore, the levelof
survival of mer2 and MER< strains treated with U V or
MMSwas similar to wild type (Table 7).
MER2 is on the right arm of chromosome Xi The
MER2gene was localized to chromosomeXby Southern
blot analysis ofelectrophoretically separated yeast chromosomes (CHUet al. 1986) (data not shown). Tetrad
analysis positioned MER2 on the right arm of the chromosome between IMEl and SUP4 (Table 8).
DISCUSSION
MER2 is essential for the initiation
of meiotic recombination: Our studies demonstrate that theMER2 gene
is absolutely required for meiotic interchromosomal recombination. Neither gene conversion (Table 2) nor
crossing over (Table 3) is induced above the mitotic
background level whenthe mer2 null mutant undergoes
sporulation. When COOLand MALONE (1992) analyzed
a $013 diploid homozygous for an UV-induced MER2
allele, they observed a low frequency of recombinants
among dissected dyads and random spores. In contrast,
we observed no crossovers inmeiotic products from the
mer2 null mutant (Table 3). This comparison suggests
Yeast Meiotic Recombination
55
Gene
A
FIGURE3.-Electron micrographs of meiotic nuclei from MER2 and mer2 strains. (A) Pachytene nucleus from the wild-type
strain, BR2495, showing fully synapsed chromosomes. (B) Meiotic nucleus from the mer2::ADE2 strain, S1590, displaying unsynapsed axial elements. The darkly staining body in each micrograph is the nucleolus. Bar, 1 pm.
that the MER2 allele analyzed by COOLand MALONE
(1992) is not a null mutation. Our data indicate that
w 2 is similar to the$01 1 (KIMHOLZ et al. 1985), rad50
(GAMEet al. 1980; MALONE and ESPOSITO
1981), mrell
(AJIMURA
et al. 1993;JOHZUKA and OGAWA
1995), m2
(IVANOV
et al. 1992), mei4 (MENEESand ROEDER
1989),
red02 (BHARGAVA
et al. 1991; COOLand MALONE 1992),
reclO4 (GALBRAITH
and MALONE 1992) and red14 (PI?TMAN et al. 1993) null mutations, which completely abolish meiotically induced recombination.
h4ER2 is also required for meiotic intrachromosomal
recombination. Previous studies have provided evidence fortwo distinct pathways ofmeiotic intrachromosoma1 exchange in yeast.The Rad50 protein is required
for recombination within artificial duplications of sequences thatnormally exist ina single copy, but not for
recombination in the naturally occurring rDNA array
(GOITLIEBet al. 1989). In addition, the rDNAis the
only segment of yeast nuclear DNA that fails to engage
in SC formation (DRESSER
and GIROUX
1988). Our data
(Tables 4 and 5) indicate that the Mer2 protein, like
Spol 1 (WAGSTAFF
et al. 1985), is required for meiotically
induced intrachromosomal recombination both in an
artificial duplication and in the rDNA. Thus, Rad50
remains the only known protein that acts differentially
in the two pathways.
To determine whether Mer2 acts at the initiation of
meiotic recombination, we monitored DSB formation
in the mer;! null mutant. DSBs are often assayed at specific recombination hotspots, using Southern blot analysis to detectrestriction fragments truncated at one end
( . g . , SUNet al. 1989; G\o et al. 1990). Because of the
limited sensitivity ofsuch assays, mutations that strongly
reduce DSBs are difficult to distinguish from those that
completely abolish breaks. Furthermore, if a meiotic
mutation alters the distribution of DSB sites, then DSB
TABLE 6
Homolog pairing and chromosome condensation in wild type, mer2 and spoil
Strain, relevant genotype
Percent
Percent
Percent
Percent
of homologous FISH signals paired
of wild-type homolog pairing
of nuclei with condensed chromosomes
of wild-type chromosome condensation
BR2495, S2888,
MER2 SPOI I
94.4 2 1.6
100
25.9 2 9.7
100
S 1590,
w2::ADE2
spo1I::ADEZ
28.2 2 7.2
29.9
16.7 2 8.4
64.5
12.2 2 1.6
12.9
21.6 2 10.1
83.4
The percentages given are the averages, together with standard deviations, obtained in seven (BR2495), six (S1590) or five
(S2888) experiments for each strain. Six of the seven experiments for strain BR2495 are reported this issue of Genetics by NAG
et al. (1995). In every experiment, approximately 100 nuclei from each strain were scored for condensation and 200-300 FISH
signal pairs were scored for pairing.
56
B. Rockmill et al.
TABLE 7
Effect of mer2 on spontaneous and induced mitotic recombination
Strain,
relevant
genotype
S2500, MER2
Spontaneous His+
Spontaneous Thr'
UV-induced His+
UV-induced Thr'
Survival after W
MMSinduced His+
MMSinduced Thr+
Survival after MMS
5.6 X
1.8 X 1 0 - ~
9.7 X 1 0 - ~
1.8 X 1 0 - ~
24%
2.2 X 1 0 - ~
2.3 X
11%
S1590, mer2::ADE2
5.5 X
2.2 X
7.3 X
2.6 X
S2702 or S2947, MER2-c
10-5
10-7
10-3
5.4 x 10-5
2.0 X 1 0 - ~
7.1 X 1 0 - ~
10-~
1.6 X
16%
1.8 X 10-3
2.7 X 10-3
12%
24%
1.8 X 1 0 - ~
2.8 X 1 0 - ~
14%
His+ andThr+ recombinants were selectedfrom diploids heteroallelic atHIS4 and THRl. The spontaneous
from five independent cultures.
Each UV-induced
prototroph frequencies are the median frequencies obtained
frequency is the average of four independent culturesand each MMSinduced frequencyis the average of two
independent cultures.For MER2-c, spontaneous and induced frequencies were derived
from strain S2702 and
W-induced frequenciesfrom strainS2947. Survival after UV and MMS indicates the percent
of cells remaining
viable after exposure to UV or MMS as described in MATERIALS AND METHODS.
formation at a specific locus may not provide a good
indication of the overall level of DSBs. To overcome
these objections, we used a sensitive assay developed by
GAMEet al. (1989) that can detect DSBs anywhere on
chromosome ZZZ. The assay employs a circular chromosome 111 and pulsed-field gel electrophoresis of undigested, chromosomal DNA. The circular chromosome
fails to enter the
gel, whereas linear derivatives resulting
from cleavage at any site on the chromosome enter the
gel and migrate with a unique mobility. The rad50S
mutation, which prevents DSB processing (ALANI et al.
1990),was included in the strains used for this analysis
so that any DSBs induced would persist. Using
this assay,
we detected no DSBs in the mer2 mutant, even at late
time points when DSB formation in wildtype had
reached its maximum level (Figure 1).These datademonstrate that the Mer2 protein acts at or before the
initiation of meiotic recombination events. Four other
class 1 mutations, spoll, rad50, xrs2 and m r e l l , have
also been shown to abolish meiotic DSBs (CAO et al.
1990; IVANOV
et al. 1992;JOHZUKA and OGAWA
1995).
MER2 is not required for mitotic recombination or
DNA damage repair: The observation that the MER2
gene is transcribed in vegetative cells raisesthe possibility that the Mer2 protein plays a role during vegetative
growth. The low level of spliced MER2 RNA produced
in the absence of Mer1 might generate sufficient Mer2
TABLE 8
Mapping of the MER2 gene
Tetrad Types
Interval
PD
'IT
NPD
Total
tetrads
distance
MErn-IMEl
IMElSUP4
SUP4-MER2
89
58
95
53
84
50
1
2
0
143
144
145
20.6 cM
33.3 cM
17.2 cM
Tetrad data were obtained by dissection of strain
The order of markers is CEhX"IMEl-MER2-SUP4.
Map
S2703.
protein to perform its mitotic function. Alternatively,
the truncated protein (131 amino acids) resulting from
translation of the unspliced MER2 transcript might perform a function in vegetative cells, whereas the fulllength protein (291 amino acids) resulting from translation of the spliced mRNA functions in meiotic cells.
Several genes required for meiotic recombination are
also essential for DNA damage repair duringvegetative
growth (FRIEDBERG
et al. 1991).We therefore examined
the effect of MER2 mutations on spontaneous recombination, recombination induced by W and MMS, and
survival after exposure to W or MMS. Neither deletion
of the MER2 gene noroverproduction of the Mer2 protein had any effect on spontaneous recombination or
response to DNA damage (Table 7). Thus, the reason
for MER2 gene expression in vegetative cellsremains a
mystery.
mer2 confers defects in SC formation and homolog
pairing: Electron microscopic analysis of silver-stained
meiotic chromosomes demonstrates that the mer2 mutant is defective in SC formation (Figures 2 and 3). A
mer2 diploid assembles short to full-lengthaxialelements, but these elements do not synapse. In wild-type
yeast, axial element development and synapsis are concurrent events (PADMORE
et al. 1991); mer:! is one of
several mutants in whichthese processes are uncoupled.
In terms of its effect on SC formation, m 2 is similar to
several other mutations that abolish meiotic recombination. The mei4 (MENEESet al. 1992), red02 (BHARGAVA
et al. 1991), spoll (LOIDLet al. 1994) and rad50 (ALANI
et al. 1990) null mutants all assemble unsynapsed axial
elements. In the case of spoll, limited formation of SC
has been observed ( LOIDLet al. 1994).
FISH analysis indicates that the mer2 mutant displays
a substantial defect in homolog pairing (Table 6). Recent studies have demonstrated that homologous chromosomes are paired in a substantial fraction of cells
before introduction into sporulation medium (LOIDL
et al. 1994;WEINER
and KLECKNER 1994). Chromosomes
Yeast Meiotic Recombination Gene
become unpaired during premeiotic DNA replication
andthen reassociate (WEINERand KLECKNER 1994).
These observations led to the proposal that vegetative
and meiotic cells employ the same mechanism of homolog alignment (WEINER
and KLECKNER 1994). Because
pairing in vegetative cells is unlikely to involve breaks
in DNA, WEINER
and KLECKNER (1994) proposed that
homologs initiallyassociate
by unstable paranemic
joints between intact DNA helices. During meiosis,
chromatin condensation might disrupt these unstable
associations (KLECKNER et al. 1991; KLECKNER and
WEINER1993); on the other hand, SC formation and
perhaps also recombination establishes more stable
connections between homologs. In the mer2 mutant,
chromatin condensation proceeds, but there is no SC
formation or recombination. Thus, one interpretation
of the mer2 phenotype is that the mutant successfully
carries out thehomology search, but is unable to stabilize the associations between homologs. If the connections between homologs are unstable, then thepossibility should be considered that chromosome pairing
can
be disrupted during spreading. Homolog pairing measured in spread preparations might therefore provide
a minimum estimate of the level of pairing in vivo.
An alternative interpretation of the mer2 defect in
homolog pairing is that the Mer2 protein plays a role
in homology searching and recognition. If so, there
must be an alternative pathway that operates in its a b
sence and is responsible for the pairingobserved in the
mer2 null mutant. The level of homolog pairingin mer2
strains is significantly higher than in the spoll mutant,
consistent with two previous studies in which the spoll
mutant had the lowest level of pairing of several mutants characterized (LOIDLet al. 1994;WEINER and
KLECKNER 1994).
It is very unlikely that most or all of the homolog
pairing observed in the mer2 mutant reflects residual
premeiotic pairing in unsporulated cells for two reasons. First, pairing was scored among nuclei in which
individual chromosomes were evident as condensed
“sausages” as determined by a DNA-specific dye.Such
chromosome morphology is not observed in premeiotic
cells. Second, the level of pairing observed in the mer2
mutant is higher than in the spoll mutant, yet these
mutants enter meiosiswith the same efficiency (i.e.,
similar to wild type; Figure 2) (KLAPHoLz et al. 1985).
Even if all of the pairing observed in the spoll mutant
is due to residual premeiotic pairing, then the mer2
mutant stilldisplays a significant level of meiotically
induced homolog pairing, as determined by the difference (16%)between the mer2 and $1011pairing values.
Possible functions for theMer2protein: The predicted amino acid sequence of the Mer2 protein provides little insight into Mer2 function (ENGEBRECHT
et
al. 1991). The Mer2 protein is 291amino acids in length
and is predicted to haveseveral a-helical segments.
There is a potential nuclear localization signal (MOLL
57
et al. 1991; ROBBINS
et al. 1991) near the carboxy terminus of the protein (amino acids 250-265).
The mer2 null mutation is pleiotropic, affecting meiotic recombination, homolog pairing and SC formation. It is possible that Mer2 acts indirectly, by regulating the activity of several gene products. For example,
Mer2 might be a transcription factor that activates the
expression of genes involved in recombination and others required for synapsis. An alternative to the view
that Mer2 is a transcriptional (or posttranscriptional)
regulator is that Mer2 directly affects a specific aspect
of meiotic DNA metabolism and the other aspects of
the mutant phenotype are secondary consequences of
this primary defect. If so, then what might be the primary function of the Mer2 protein?
Two observations argue against a primary role for
Mer2 in homology searching or recognition. First, the
mer2 mutant undergoes a significant amount of meiotically induced homolog pairing. Thus, if the defects in
recombination and SC formation result from the defect
in pairing, DSB formation and synapsis should be reduced, but not completely eliminated. Furthermore,
three studies have shownthat meiotic DSBs are formed
during meiosis in haploid yeast, indicating that DSB
formation does not depend on prior interactions between homologous chromosomes (DE MASSYet al. 1994;
GILBERTSON
and STAHL1994; FANet a2. 1995).
It also seems unlikely that Mer2 is a structural component of the SC. Mutations in genes that encode SC
components effect quite modest reductions in recombination, demonstrating that the SC is not absolutely requiredfor meiotic recombination (HOLLINGSWORTH
and BYERS1989; HOLLINCSWORTH
et al. 1990; ROCKMILL
and ROEDER1990; HOLLINGSWORTH
and JOHNSON
1993; FRIEDMAN
et al. 1994; SYM et al. 1993; SYM and
ROEDER1994). Furthermore, a role in SC formation
cannot account for the requirement for theMer2 protein in meiotic intrachromosomal recombination in the
rDNA array, where there is no SC formation (DRESSER
and GIROUX1988).
We favor the view that the primary defect in the mer2
mutant is DSBformation. If synapsis initiates at the sites
of some or all DSBs as suggested (KLECKNER et al. 1991;
PADMORE et al. 1991), then a defect in DSB formation
can account, not only for the failure of recombination,
but also for the defect in SC formation. A number of
observations suggest that an early step in the recombination pathway is required for synaptic initiation. First,
DSB formation precedes the initiation of synapsis and
DSBs disappear concurrent with the formationof tripartite sc (PADMORE et al. 1991). Second,of the numerous
yeast mutants characterized, none assembles SCs in the
absence of DSBs. Finally, studies of chromosome rearrangements in maize havesuggested a causal relationship between the establishment of a crossover site and
the initiation of synapsis (MAGUIFW 1977; MACUIRE and
RIESS 1994).
58
B. Rockmill et al.
Chromatin structure plays a major role in determining the sites of meiotic DSBs. The breaks occur preferentially in intergenic regions that contain transcription
promoters and are hypersensitive to nuclease digestion
in chromatin isolated from both vegetative and meiotic
cells (WU and LIGHTEN1994). Cleavage at these sites
must be catalyzed either by a meiotically induced endonuclease or by a constitutive nuclease that is somehow
activated or recruited by meiosis-specific gene products.
The Mer2 protein could itself be the endonuclease or
it could interact with the nuclease to modify its activity.
As noted above, mutations in several different genes
confer meiotic phenotypes that are thus far indistinguishable from that of w 2 . Some or all of the encoded
proteins might act togetheras a complex. Indeed, there
is evidence that Mrell interacts physically with Rad50
and Xrs2 ( J ~ H Z U K Aand OGAWA
1995); these three gene
products participate in DSB repair during both vegetative growth and meiosis (IVANOVet al. 1992; AJIMURA
et
al. 1993;JoHzUKA and OGAWA
1995). Mer2 may modify
the activity of this complex to effect its meiosis-specific
function in DSB formation.
We thank JOHN GAMEfor providing yeast strains, KEVIN BENTLEY
for helpwith chromosome gels and AURORA
STORLAZZI
for providing
pSG315. KAREN VOELKEL-MEIMAN
provided excellent technical assistance. Thisinvestigation was supported by Public Health Service grant
GM-28904from the National Institute of General Medical Sciences
and by a grant from the Jane
Coffin Childs MemorialFund for Medical Research. J.E. was a Fellow of the Jane Coffin Childs Memorial
Fund for Medical Research.
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