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Molecular and Cellular Biology, October 2001, p. 6984-6998, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.6984-6998.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Novel Meiosis-Specific Isoform of Mammalian
SMC1
E.
Revenkova,1
M.
Eijpe,2
C.
Heyting,2
B.
Gross,1 and
R.
Jessberger1,*
Institute for Gene Therapy and Molecular
Medicine, Mount Sinai School of Medicine, New York, NY
10029,1 and Laboratory of Genetics,
Wageningen University, NL-6703HA Wageningen, The
Netherlands2
Received 30 May 2001/Returned for modification 9 July 2001/Accepted 12 July 2001
 |
ABSTRACT |
Structural maintenance of chromosomes (SMC) proteins fulfill
pivotal roles in chromosome dynamics. In yeast, the SMC1-SMC3 heterodimer is required for meiotic sister chromatid cohesion and DNA
recombination. Little is known, however, about mammalian SMC proteins
in meiotic cells. We have identified a novel SMC protein (SMC1
),
which
except for a unique, basic, DNA binding C-terminal motif
is
highly homologous to SMC1 (which may now be called SMC1
) and is not
present in the yeast genome. SMC1
is specifically expressed in
testes and coimmunoprecipitates with SMC3 from testis nuclear extracts,
but not from a variety of somatic cells. This establishes for mammalian
cells the concept of cell-type- and tissue-specific SMC protein
isoforms. Analysis of testis sections and chromosome spreads of various
stages of meiosis revealed localization of SMC1
along the axial
elements of synaptonemal complexes in prophase I. Most SMC1
dissociates from the chromosome arms in late-pachytene-diplotene cells.
However, SMC1
, but not SMC1
, remains chromatin associated at the
centromeres up to metaphase II. Thus, SMC1
and not SMC1
is likely
involved in maintaining cohesion between sister centromeres until
anaphase II.
 |
INTRODUCTION |
The eukaryotic,
evolutionarily highly conserved SMC (Structural Maintenance of
Chromosomes) proteins are involved in several key DNA and chromatin
dynamic processes (for recent reviews, see references 11, 21, 26,
27, 31, 48, 60, and 62). The best-documented processes are
chromosome condensation and sister chromatid cohesion. Evidence is also
accumulating for a function in DNA recombination and repair. A fourth
role of SMC proteins is in gene dosage compensation in
Caenorhabditis elegans. The phylogenetic tree comprises five
subfamilies (32): SMC1 to SMC4 and an ancestral family
that includes the recently defined SMC5 and SMC6 groups with the Rad18
and Spr18 proteins of Schizosaccharomyces pombe
(16), which act in recombinational repair.
SMC proteins share a characteristic design. Coiled-coil domains
are flanked by globular N- and C-terminal domains and are divided in
the central region by a flexible hinge domain of about 150 aa. The N-
and C-terminal domains of about 100 to 150 aa are highly conserved and
carry important motifs. The N-terminal domain includes an NTP binding
motif (Walker A box [68]), which has been shown to bind the ATP
analogue azido-ATP (1). The C-terminal domain contains a
DA box (68). The C-terminal and second coiled-coil domains, but not the N terminus, bind DNA (1, 2). It has been proposed that the antiparallel, heterodimeric SMC1-SMC3 protein with an N and C terminus at each end may connect two DNA molecules, such as sister chromatids, and may directly contribute to their alignment in cohesion and to recombination between sister chromatids (2, 26, 62).
In eukaryotes the SMC1-SMC3 or SMC2-SMC4 heterodimers form large
multiprotein complexes. One of these complexes is condensin, which,
besides the SMC2-SMC4 heterodimer, contains several non-SMC subunits.
Condensin is necessary for mitotic chromosome condensation in
Saccharomyces cerevisiae (61), S. pombe (64), and Xenopus laevis egg
extracts (25) and has also been described in human cells
(57). The MIX-1 and DPY-27 proteins of C. elegans, proteins homologous to SMC2 and SMC4, are present in a
different multiprotein complex, which regulates gene dosage
compensation on the X chromosomes of the hermaphrodite nematode
(8, 39).
The other pair of SMC proteins, SMC1 and SMC3, is present in at
least two protein complexes with distinct, albeit partially connected,
functions. Genetic studies of S. cerevisiae revealed a
requirement for Smc1p and Smc3p in mitotic sister chromatid cohesion
(19, 45). The respective protein complex is called cohesin, contains two other polypeptides besides Smc1p and Smc3p, and
interacts with several other factors required for sister chromatid cohesion and its release (reviewed in references 11, 48,
and 62). One of the non-SMC cohesin subunits is the
S. cerevisiae Scc1p (Mcd1p) protein, homologous
to Rad21 in S. pombe (4, 19, 45). The
rad21-45 mutation (in S. pombe) also
causes X-ray sensitivity and a mitotic hyperrecombination phenotype
(4, 18). A similar cohesin complex was identified from
X. laevis cell extracts, extensively characterized, and
found to be required for sister chromatid cohesion in this system
(41, 42). We have identified the SMC1 and SMC3 proteins as
constituents of the mammalian recombination complex RC-1, which is
present in a variety of somatic cells (29, 30, 63). This
complex catalyzes SMC protein-dependent cell-free transfer of duplex
DNA molecules, which mimics recombinational repair of gaps and
deletions (29, 30). The presence of the SMC1 and SMC3
proteins in these multiprotein complexes furthered speculations
about an SMC-mediated functional link between sister chromatid cohesion
and recombinational repair (26, 31, 62). Recent evidence
from studies of yeast supports this concept. Klein et al.
(36) reported that S. cerevisae Smc3p is
required not only for meiotic sister chromatid cohesion, but also for
meiotic DNA recombination.
Sister chromatid cohesion and DNA recombination are both essential for
meiosis (for reviews, see references 35, 54, and 66). In mitotic cells, DNA recombination is primarily a
means to repair DNA damage, and the role of cohesins may include the direction of recombinational repair towards the sister chromatid rather
than the homologous chromosome (if there is one) (33, 58).
In meiosis, recombination and sister chromatid cohesion are essential,
but the relationship between the two processes has been modified.
Whereas meiotic recombination has to be directed towards the homologue
rather than the sister chromatid, cohesion between sister chromatids
has to be maintained to ensure the proper orientation and disjunction
of homologues at meiosis I (66). During the meiotic
prophase, a characteristic, zipperlike protein structure, the
synaptonemal complex (SC), is formed between homologues and likely
plays an important but not entirely clarified role in adapting
recombination and cohesion for meiosis (20, 23). SCs
consist of two axial elements (AEs), which are connected by numerous
transverse filaments along their lengths. Each AE structurally supports
the two sister chromatids of one homologue.
In budding yeast, Smc3p colocalizes with an AE component during the
meiotic prophase (36). In meiotic yeast cells, the cohesin protein Scc1p (Mcd1p) is largely replaced by its meiosis-specific homologue, Rec8p, or its homologues in other organisms (36, 47,
69). In S. cerevisiae, Rec8p localizes along the AEs
of SCs (36).
These observations also suggest for mammalian meiotic cells an
association of cohesion proteins with the SC. In mammals, two AE
components have been identified, SCP2 (49) and SCP3
(37), which are specifically expressed in the meiotic
prophase. Recently, we have shown that mammalian SMC1 is present in
meiotic nuclei throughout prophase I. Upon permeabilization of
spermatocytes in the presence of Triton X-100, SMC1 is specifically
retained in a dotlike pattern along the AEs of SCs. We also showed that mammalian SMC1 and SMC3 proteins associate with AE components, e.g.,
SCP2 and SCP3 (13). Thus, it is intriguing to hypothesize about an essential role of mammalian SMC proteins in meiotic sister chromatid cohesion
its establishment, maintenance, and resolution
and in meiotic DNA recombination. Differences in mitotic sister chromatid cohesion, however, extend beyond meiotic prophase I. Sister chromatids separate only in the chromosome arms, but not at the centromeres, during meiosis I, leaving centromeric cohesion intact until anaphase II, when all cohesion is finally removed. An important question is
whether there is specific adaptation of SMC proteins and their complexes to their specific meiotic functions. The differences mentioned above in the interplay of sister chromatid cohesion and DNA
recombination between meiosis and mitosis render such adaptation
likely. One way to achieve this adaptation is through expression of a
meiosis-specific homologue or isoform of a somatic protein.
Here, we demonstrate the existence of a meiosis-specific isoform of
mammalian SMC1, named SMC1
, which we describe in this report.
 |
MATERIALS AND METHODS |
Cloning of SMC1
cDNA.
The 150-kDa protein was isolated
from testis nuclear extracts by immunoprecipitation with anti-SMC3
antibody (63) and separation by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis. Sequences from seven
peptides, generated by tryptic digest of the protein, were determined.
Based on this sequence information, oligonucleotides were designed for
the screening of a mouse testis 5'-STRETCH cDNA library
(Clontech Inc.). Several overlapping clones were isolated, which
covered a 1,735-bp fragment at the 5' end of the cDNA. To recover the
missing 3' end, rapid amplification of cDNA ends (RACE)
(15) was performed using a SMART RACE cDNA amplification
kit (Clontech Inc.). Several independent clones have been obtained and
sequenced. The sequence of the C terminus was further verified by
performing nested PCR on 3' RACE PCR products from testis RNA. The PCR
used a gene-specific primer located at the positions corresponding to
aa 1171 to 1179 of SMC1
, and the Nested Universal Primer
(Clontech Inc.), located downstream of poly(A) within the region
defined by the RACE universal primer. Twenty cloned PCR products were
analyzed by restriction analysis, and five clones were sequenced. All
belonged to the same gene and encoded identical C termini. The 4,056-bp
full-length cDNA was assembled using standard cloning protocols.
Northern blot analysis.
Eight-microgram aliquots of total
RNA from various mouse tissues (Ambion) were separated
electrophoretically and transferred to a nylon membrane (Hybond N;
Amersham) using standard protocols (55). Hybridization was
performed as described previously (9). The probe
corresponded to the first 616 bp of the SMC1
cDNA.
Protein purification and antibody generation.
The C-terminal
domain of SMC1
(SMC1
-C) was subcloned into the Escherichia
coli expression vector pQE32 (Qiagen Inc.). The His6-tagged protein, with a molecular mass of 33 kDa, was overexpressed and purified on Ni-nitrilotriacetic acid agarose
resin (Qiagen) and eluted in steps with increasing imidazole.
The yield was 200 to 600 µg per liter of E. coli culture.
About 80% of the total SMC1
-C protein was found to be insoluble
under native conditions and had to be dissolved in 8 M urea. The
remaining 20%, native soluble protein, was mixed with an equal amount
of denatured protein and injected into mice. Monoclonal antibodies were
generated by standard hybridoma techniques. Hybridoma tissue culture
supernatants were screened by enzyme-linked immunosorbent assay using
96-well plates that were coated with the C-terminal protein.
Subsequently, the positive supernatants were tested for their
specificities by Western blotting on testis versus somatic cell
extracts and by immunofluorescence on testis versus liver sections. At
least six independent hybridoma clones produced antibodies specific in
both procedures for SMC1
.
The rabbit anti-SMC1 serum SMC1-C1 and the anti-SMC3 serum (13,
63), the hamster anti-SCP3 serum H1 (14), the
rabbit anti-SCP3 serum 175 (37), and the rabbit anti-SCP2
serum (49) have been described. For labeling of
kinetochores, we used a human autoimmune serum from a patient with
CREST (calcinosis, Raynaud syndrome, esophageal dismobility,
sclerodactyly, and telangiectasia) syndrome; this serum reacts with
kinetochore proteins and has been described by Moens et al.
(46).
Immunoprecipitation.
Nuclear extracts from tissue or cells
were prepared as described previously (29), with the
concentration of dithiothreitol (DTT) lowered to 0.1 mM. Five
micrograms of affinity-purified anti-SMC3 antibody was incubated for 4 to 16 h at 4°C with nuclear extract (50 µg of protein unless
otherwise indicated), which was diluted 1:5 in buffer IP
(phosphate-buffered saline plus 0.75% Brij 58, and 500 mM NaCl). A
20-µl slurry of protein A beads was added, and the mixture was
further incubated for 1 h. The beads were washed six times with
buffer IP or variations of it as described in the text and were boiled
with SDS gel sample buffer before being loaded on reducing 5%
polyacrylamide SDS gels. Detection of proteins was done by silver
staining according to a published protocol (29).
Preparation of spreads and agar filtrates.
Spreads of mouse
spermatocytes were prepared by the dry-down technique of Speed
(59), as modified by Peters et al.
(51). Agar filtrates of lysed rat spermatocytes
were prepared as described by Heyting and Dietrich (22).
Immunofluorescence labeling.
Immunofluorescence labeling of
dry-down preparations and agar filtrates was performed as described
previously (22). The slides were mounted in Vecta Shield
(Vector Laboratories Inc., Burlingame, Calif.). The monoclonal
anti-SMC1
antibodies in tissue culture supernatant (from two
independent hybridomas) were diluted 1:1, serum 175 (rabbit anti-SCP3)
was diluted 1:500, CREST serum (anti-kinetochore) was diluted 1:1,000,
and serum H1 (hamster anti-SCP3) was diluted 1:50. Goat anti-rabbit
immunoglobulin G (IgG) conjugated with aminomethylcoumarin acetate
(AMCA) (Vector) or with Texas red (Jackson ImmunoResearch Laboratories,
West Grove, Pa.), goat anti-mouse IgG conjugated with
fluorescein isothiocyanate (FITC) (Jackson), goat anti-hamster IgG
conjugated with AMCA, and goat anti-human IgG conjugated with Texas red
were used as secondary antibodies and were diluted according to the
instructions of the suppliers.
Microscopy.
Spread preparations were examined with a Zeiss
Axioplan research microscope equipped with epifluorescence illumination
and Plan-Neofluar optics. Selected images were directly photographed on
an ISO 400 color negative film using single-band-pass emission filters
(for DAPI [4',6'-diamidino-2-phenylindole]-AMCA, FITC, and Texas red
fluorescence) with separated excitation filters. Negatives were scanned
at high resolution, and their computer images were processed and
combined using the CorelDraw and Corel Photopaint software
packages.

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FIG. 1.
Immunoprecipitation of SMC3 and associated SMC1 proteins
with anti-SMC3 antibodies. (A) Immunoprecipitation from various bovine
tissue nuclear extracts. Thym., thymus. (B) Immunoprecipitation
from mouse and bovine testis nuclear extracts. (C) Control
immunoprecipitation from bovine testis nuclear extract, with (+Ab) and
without ( Ab) anti-SMC3 antibody included. (D) Immunoprecipitation
from nuclear extracts prepared from actively proliferating
(lipopolysaccharide-induced; Activ.) and resting (Restg.) mouse spleen
cell cultures and from an actively growing mouse pre-B-cell line. (E)
Immunoprecipitation from bovine testis nuclear extract gel filtration
(BioGel A15m resin) fractions (20 µg of protein each) representing
1-, 3-, and 6-MDa molecular-mass positions. (F) Immunoprecipitation
from mouse kidney, rat testis, and purified rat spermatocyte (Purif.
Sp.) nuclear extracts (25 µg of protein each). Controls without
antibody (no Ab) are included. All precipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and silver staining. M,
molecular mass marker.
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Isolation of meiotic cells.
Spermatocytes were isolated from
rat testes by cell elutriation and density centrifugation according to
the method of Heyting and Dietrich (22), and the
composition of the isolated cell fraction was analyzed by differential
counts of Giemsa-stained preparations, as described previously
(38). The cell fraction that was used in this study had
the following composition: Sertoli cells, 0.3%; spermatogonia, 0.7%;
and spermatocytes, 99%, of which 1.2% was in leptotene-zygotene, 29%
was in early-mid pachytene, 54% was in late pachytene or prediffuse
diplotene, and 16% was in diffuse or postdiffuse diplotene.
DNA interaction assays.
DNA concentrations are expressed as
nucleotide equivalents. The assay for retention of double-stranded DNA
on nitrocellulose filters through binding to protein (filter binding
assay) was performed essentially as described elsewhere
(3).
Linear double-stranded DNA fragments were generated by digestion of
pBluescript SK(+) (Stratagene Inc.) with
AluI and were
labeled at their 5' ends with
32P. Reactions were
performed in 20-µl mixtures containing 25 mM
Tris-HCl (pH 7.5), 10 mM
KCl, 0.2 mM EDTA, 1 mM DTT, 10 ng of
linear DNA, and various amounts of
peptide. After 20 min at room
temperature, the reaction mixtures were
diluted by adding 1 ml
of reaction buffer containing 10 mM sodium
pyrophosphate. The
reaction mixtures were filtered through prewashed
0.1-µm-pore-size
nitrocellulose filters (Whatman Inc.). The filters
were washed
thrice with 1 ml of the reaction buffer containing sodium
pyrophosphate,
and the radioactivity retained on the filters was
measured in
a liquid scintillation
counter.
The gel shift (gel retardation) assay was performed as described
previously (
1,
2). For a DNA substrate, we used a 200-bp
DNA fragment of the 5S rRNA gene or a 230-bp DNA fragment derived
from
the double-stranded form of M13mp8, which we know are good
binding
substrates for SMC protein domains (
1,
2). The reaction
buffer contained 0.8 pmol of
32P 3'-end-labeled
DNA (5,000 to 10,000 cpm) in 20 mM HEPES (pH
7.5), 1 mM DTT, and 0.1 mg
of bovine serum albumin (ultrapure;
Amersham-Pharmacia Inc.)/ml. The
SMC1

peptide consisted of the
C-terminal 28 aa
(TEDQEGSRSHRKPRVPRVSMSPKSPQSR; theoretical pI,
11.4). The
positive control peptide was derived from mouse Rad54L
(
34), aa 152 to 181 (KVCRPHQREGVKFLWECVTSRRIPGSHGLI; theoretical
pI, 10.09). The
negative control peptide was derived from human
Rad21
(
44), aa 612 to 631 (TQEEPYSDIIATPGPRFHII;
theoretical
pI, 4.65).
The assay for network formation was done as described for RecA
(
7) or mammalian DNA binding proteins (
17),
with some
modifications. The assay measures coaggregation of DNA into
DNA-protein
complexes that sediment rapidly. The DNA substrate was
AluI-digested
(blunt-ended),
5'-
32P-labeled plasmid DNA as used for the filter
binding assay. Various
amounts of peptide were incubated with the DNA
(1.5 pmol) in a
volume of 50 µl for 20 min at room temperature in the
DNA binding
buffer also used for gel shift experiments. The reaction
mixture
was then centrifuged for 3 min at 14,000 ×
g.
The supernatant
was transferred to a scintillation vial, and the pellet
was solubilized
in 100 µl of 0.1% SDS and also transferred to a
scintillation
vial. The supernatant (nonaggregated) and pellet
(aggregated)
DNAs were measured. All experiments were done in
triplicate.
Nucleotide sequence accession numbers.
The GenBank accession
number of mSMC1
is AF303827.
 |
RESULTS |
A novel SMC protein complex from testis.
Earlier, we used
affinity-purified polyclonal anti-SMC3 antibodies, raised against
the C-terminal domain of bovine SMC3, in immunoprecipitation
experiments with nuclear extracts of mitotically dividing cells of
human, mouse, hamster, and bovine origin (reference 63 and
unpublished observations). Under stringent precipitation conditions,
the SMC1 and SMC3 proteins were observed as the only strong bands in
silver-stained SDS polyacrylamide gels, used to analyze the
precipitates. A weaker 120-kDa band was often visible and probably
represented the Rad21 protein. Immunoprecipitation experiments with
nuclear extracts prepared from a variety of mouse and bovine tissues,
however, revealed a marked difference between extracts from testes and
all other extracts. From testes, we coimmunoprecipitated a hitherto
undescribed protein that migrates at an approximately 145- to 155-kDa
position between SMC1 and SMC3, depending on the particular gel
electrophoresis conditions (Fig. 1). The 150-kDa protein was also not
observed in immunoprecipitates from various human, mouse, and hamster
cell lines, nor was it present in cytoplasmic extract fractions
(63; data not shown). However, a faint 150-kDa band
was visible in immunoprecipitates from ovary extracts (Fig. 1A).
Comparable results were obtained with mouse tissues (Fig. 1B and
F). The coprecipitated protein from mouse testis extract migrates at
155 kDa, a position slightly higher than that of the bovine protein.
The 150-kDa protein is present neither in resting nor in activated,
proliferating somatic mouse cells (Fig. 1D).
Incubation of the precipitates with bacterial alkaline phosphatase or
the inclusion of ATP (1 mM) or of the phosphatase inihibitor
o-vanadate (0.5 mM) in the extracts and all buffers did not
alter
the result (not shown). The characteristic band pattern also did
not change upon variation of the precipitation and wash conditions,
e.g., the use of different detergents, differently pretreated
protein A
or protein G beads, or protein G-precleared extracts
(not
shown). Association of the 150-kDa protein with SMC3 was
found to be as
resistant to stringent precipitation reaction conditions
as that of
SMC1 with SMC3. The 150-kDa protein was also immunoprecipitated
from
testis nuclear extract fractions that had been obtained by
gel
filtration of the extract through a large BioGel A15m chromatography
column. Similar chromatography experiments were done before for
either
purification of RC-1 from thymus or other analysis of testis
extract
fractions (
13,
30) (Fig.
1E). The protein was found
together with SMC3 in fractions that represent molecular masses
of
globular proteins of around 1 MDa and

albeit in smaller
amounts

at
3 MDa. At 3 MDa, more of SMC1-SMC3 was visible, possibly
indicating
differences in masses between complexes based on SMC1-SMC3
or
the 150-kDa protein and SMC3. Thus, the 150-kDa protein
coprecipitates
and copurifies with SMC3 and is likely a component of a
large
multiprotein complex, similar but not identical to that
containing
the SMC1-SMC3 heterodimer (
13).
In immunoblotting, neither a monoclonal nor an affinity-purified
polyclonal antibody, both raised against the C-terminal domain
of
SMC1, recognizes any protein migrating between the SMC1 and
SMC3
proteins (
13,
63). Likewise, anti-SMC3 antibodies do
not
react with the protein in immunoblotting. Thus, the 150-kDa
protein is
not a degradation product of SMC1 or very homologous
to
SMC3.
From immunoblotting and immunoprecipitation experiments, we estimate an
approximate relative abundance of SMC1

, 150-kDa protein,
and SMC3 of
1:2:3 in total testis nuclear extracts. In extracts
from purified
spermatocyte preparations, which consist of >99%
meiotic cells (70%
late pachytene-diplotene [
22,
38]), only
a small amount
of SMC1

was seen, while the 150-kDa protein and
SMC3 precipitated in
an approximately equimolar ratio (Fig.
1F).
Large-scale immunoprecipitation followed by SDS-polyacrylamide gel
electrophoresis and amino acid sequencing of the 150-kDa
protein
allowed us to deduce seven peptide sequences 6 to 12 aa
in length. This
information was used to generate oligonucleotides
with which a mouse
testis library was screened. By a combination
of further screening and
reverse transcription-PCR from mouse
testis RNA, the entire cDNA
was cloned and then sequenced. This
cDNA encodes a protein that has not
been reported previously and
that shows a high level of homology to
mammalian SMC1 (Fig.
2A).
The homology is highest in the conserved functional domains of
SMC
proteins, the N-terminal, C-terminal, and hinge domains. Lower
degrees
of homology were found with SMC4 and SMC3. Dendrogram
analyses of the
N- and C-terminal domains confirmed the close
relationship to SMC1
(Fig.
2B). Therefore, we call the protein
SMC1

, indicating an SMC1
variant or isoform. The "classical"
SMC1 may be termed SMC1

.
SMC1

bears a unique C-terminal sequence
of 28 aa that has been found
neither in any other SMC protein
nor in the databases. Among the 28 aa
are 4 proline residues and
7 arginine and lysine residues. This
C-terminal peptide is very
basic, with a theoretical pI of 11.4. In
contrast, the entire
C-terminal domain of SMC1

, with 186 aa, has a
theoretical pI
of 6.9. This C-terminal motif was present in all
independently
isolated clones and was confirmed by RT-PCR of mouse
testis and
subsequent sequencing of PCR products. SMC1

shows all
motifs
characteristic of SMC proteins, including the N-terminal Walker
A box, the C-terminal Walker B box, and the signature motif typical
of
ABC-ATPases (
28,
68), as well as the extended coiled-coil
domains with heptad repeats and the hinge region.


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FIG. 2.
Amino acid sequence comparison and dendrogram of
SMC1 . (A) N-terminal domains of SMC1 and mammalian SMC proteins
representing the four most closely related SMC subfamilies. (B)
C-terminal domains of SMC1 and mammalian SMC proteins representing
the most closely related SMC subfamilies. The program Megalign
(DNAStar) was used. The accession numbers for the related SMC
proteins are as follows: mSMC1 (mouse SMCB; AF047600),
bSMC1 (bovine SMC1; AF072712), hSMC1 (human SB1.8; S78271), XSMC1
(X. laevis; AF051784), mSMC3 (mouse SMCD; AF047601),
bSMC3 (bovine; AF072713), hSMC4 (human CAP-C; AB019987), and hSMC2
(human CAP-E; AF092563). mSMC1 , mouse SMC1 ; hSMC1 , predicted
human protein (CAB41703; partial sequence). Identical residues are
shaded. Dashes indicate gaps in alignment.
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Specific expression of SMC1
in meiotic cells.
Northern
blotting of RNA from a variety of mouse tissues was performed using a
616-bp 5' DNA fragment of SMC1
as a probe (Fig.
3). This experiment confirmed
testis-specific expression of the gene. The specific signal of about
4.5 kb was not seen in RNA from any of the other tissues. We also used
the same probe to analyze RNA prepared from purified spermatocytes. The
same 4.5-kb signal was observed, and no other band was detected (not shown).

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FIG. 3.
Tissue specificity of SMC1 RNA expression. (Top)
Northern blot of total RNA extracted from different mouse tissues and
hybridized to an SMC1 -specific probe. (Bottom) Corresponding agarose
gel stained with Radiant Red fluorescent RNA stain prior to transfer
for loading control.
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Next, we generated monoclonal antibodies for the analysis of protein
expression. The antibodies were raised in mice against
a C-terminal
33-kDa fragment of mouse SMC1

, expressed in
E. coli,
and
purified by affinity chromatography on Ni-Sepharose (Fig.
4A). The C-terminal protein was partially
soluble under native
conditions, and the insoluble precipitates were
solubilized in
8 M urea. A mixture of native and denatured protein was
used for
immunization. We obtained several hybridoma lines that produce
antibodies that specifically recognize SMC1

but not SMC1

(Fig.
4B). No cross-reaction with even large amounts of the purified
C-terminal domain of SMC1

, or with SMC proteins present in somatic
cell nuclear extracts, was observed (Fig.
4B and C). The tissue
specificity of SMC1

was also confirmed for a large variety of
bovine
tissues (Fig.
4D). We obtained several antibodies that
recognize
SMC1

of mouse, rat, and bovine origin. Immunoprecipitation
using the anti-SMC1

antibody with testis extracts confirmed the
association of SMC1

with SMC3 (Fig.
4E). We never observed SMC1
to coimmunoprecipitate with anti-SMC1

antibodies.

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FIG. 4.
Generation and specificity of anti-SMC1 monoclonal
antibodies. (A) Coomassie blue-stained SDS-polyacrylamide gel loaded
with the C-terminal domains of SMC1 (approximately 67 kDa) and
SMC1 (approximately 33 kDa), with their positions indicated by and . (B) Immunoblot of a gel identical to that in panel A probed
with a monoclonal anti-SMC1 antibody. (C) Immunoblot of nuclear
extracts from mouse testis and kidney probed with a monoclonal
anti-SMC1 antibody. (D) Immunoblot of nuclear extracts from a
variety of bovine tissues probed with a monoclonal anti-SMC1
antibody. M, marker. (E) Anti-SMC1 immunoprecipitates from testis
nuclear extracts probed in Western blotting with anti-SMC3 or
anti-SMC1 antibodies. AB, no anti-SMC1 ; +AB, with
anti-SMC1 .
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We then used these monoclonal antibodies to investigate expression and
localization of SMC1

in testis. Mouse testis sections
and, for
control, liver sections were prepared and immunoprobed
with
FITC-labeled anti-SMC1

and stained with propidium iodide
(after RNase treatment) to visualize nuclear DNA (Fig.
5). No
specific anti-SMC1

signal was
obtained with liver sections. In
testis, however, strong staining of
prophase I nuclei was observed.
The antibodies stained the compact
chromosomal axes within the
meiotic nuclei, indicative of the presence
of SMC1

along the
SCs. In these sections, only weak staining was
observed in cells
of later stages. These results were confirmed with
three other
anti-SMC1

antibodies (not shown).

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FIG. 5.
Testis and liver section stained with anti-SMC1 .
Sections of mouse testis or liver were incubated with either propidium
iodide (PI)-RNase or anti-SMC1 , FITC-labeled, to visualize DNA or
SMC1 . The merged images are at the bottom, and two magnifications
are shown as indicated. Bar = 10 µm.
|
|
By immunoblotting, SMC1

was also found in preparations of SCs from
rat spermatocytes (
13) (not shown). As these preparations
contain only a very limited number of proteins (<20) and are prepared
under stringent conditions, this indicates a close association
of
SMC1

with
SCs.
Chromosomal localization of SMC1
throughout meiosis.
High-resolution analysis of rat spermatocyte nuclear spreads confirmed
and significantly extended our initial observations (Fig.
6 and
7). Several different
anti-SMC1
antibodies were used in immunofluorescence, all yielding
identical results. Spreads of cells in consecutive stages of meiosis up
to anaphase II were analyzed for SMC1
, the AE-specific
protein SCP3, and kinetochores (Fig. 6). SMC1
tightly colocalizes
with SCP3 from early prophase I (leptotene and zygotene) on along the
entire AEs of the chromosomes in presynapsed (leptotene-zygotene [Fig.
6A]), synapsed (pachytene), unsynapsed (XY bivalent [Fig. 6B]), and
desynapsed (diplotene [Fig. 6C]) regions. The distribution of SMC1
appears rather uniform along the AEs, with occasional more intense
dots. Until diplotene, there is no concentration of SMC1
around the
centromeres (Fig. 6A and B). SCP3 and SMC1
remain tightly associated
with the AEs throughout pachytene. Upon desynapsis of the homologous
chromosomes in diplotene, SCP3 and SMC1
start to accumulate around
the centromeres and to dissociate from the chromosome arms (Fig. 7C to
E). Staining for both proteins is also visible at sites of bridges
between the homologues, which possibly represent sites of crossover
(Fig. 6C).

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FIG. 6.
Immunolocalization of SMC1 and SMC1 in
successive stages of meiosis. (A to J) Images of immunofluorescence
triple labeling of SCP3 (blue), SMC1 (green), and kinetochores (red)
in dry-down preparations of rat spermatocytes. (A to F and H) Shown on
the left are the merged images of SCP3 (blue) and kinetochores (red),
and shown on the right are the merged images of the same cell of
SMC1 (green) and kinetochores (red). (A) zygotene (the long arrows
indicate asynapsed segments of AEs, the short arrows point to regions
of presynaptic alignment, and the arrowheads designate paired segments
of AEs); (B) pachytene (the XY bivalent is indicated by arrows); (C)
diplotene (the arrows point to connections containing SCP3 and SMC1
between AEs of homologous chromosomes); (D) diakinesis (the
arrowheads point to partial splitting of AEs); (E) metaphase I (the
arrowheads indicate a weak signal for SCP3 in the chromosome arms,
whereas SMC1 is hardly detectable); (F) metaphase II; (H) anaphase
II. (G) Enlargement of the area indicated in panel F, with the merged
images of SCP3 (blue) and kinetochores (red) (left); SMC1 (green)
and kinetochores (red) (middle); and SCP3 (blue), SMC1 (green), and
kinetochores (red) (right). (I and J) Enlargements of areas indicated
in panel H, with the merged images of SCP3 (blue) and kinetochores
(red) (left); SMC1 (green) and kinetochores (red) (middle); and SCP3
(blue), SMC1 (green), and kinetochores (red) (right). (K to M)
Images of immunofluorescence triple labeling of SCP3 (blue), SMC1
(green), and SMC1 (red). Shown are a pachytene SC (K), a diplotene
SC (L), and a metaphase I bivalent (M) in a dry-down preparation of rat
spermatocytes. The tops of panels K to M show the merged images of SCP3
(blue), SMC1 (red), and SMC1 (green); the middles show SCP3
(blue), SMC1 (green), and SMC1 (red); and the bottoms show SCP3
(blue), SMC1 (green), and SMC1 (red). Bars = 10 µm (A to F
and H) and 1 µm (G, I, J, and K to M).
|
|

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FIG. 7.
Immunolocalization of SMC3 and SMC1 in
successive stages of meiosis. (A to C) Images of immunofluorescence
triple labeling of SMC1 (red), SMC3 (green), and SCP3 (blue) in a
frozen section of rat testis. (A) Merged images of SMC1 (red) and
SMC3 (green). (B) Merged images of SCP3 (blue) and SMC1 (red). (C)
Merged images of SMC1 (red), SMC3 (green), and SCP3 (blue). The
pictures show parts of two testicular tubules. lp, late-pachytene
spermatocytes; pl, preleptotene spermatocytes; i, interstitial zone
between the two tubules; lz, late-zygotene spermatocytes. (D) Images of
immunofluorescence triple labeling of SMC1 (red), SMC3 (green), and
SCP3 (blue) in a rat pachytene nucleus, spread by agar filtration; on
the left, the merged images of SCP3 (blue) and SMC1 (red) are shown,
and on the right, the merged images of SMC3 (green) and SMC1 (red)
are shown. (E to K) Images of immunofluorescence triple labeling of
SCP3 (blue), SMC3 (green), and kinetochores (red) in dry-down
preparations of rat spermatocytes. (E to I) On the left are the merged
images of SCP3 (blue) and kinetochores (red), and on the right are SMC3
(green) and kinetochores (red). (J) Enlarged images of the area
indicated in H. (K) Enlarged images of the area indicated in I. On the
left are the merged images of SCP3 (blue) and kinetochores (red), in
the middle are SMC3 (green) and kinetochores (red), and on the right
are SCP3 (blue), SMC3 (green), and kinetochores (red). Bars = 10 µm (A to I) and 1 µm (J and K).
|
|
The further meiosis I continues, the less SCP3 and SMC1

we find in
the chromosome arms, but intense staining remains present
at the
centromeres. This is obvious in diakinesis (Fig.
6D), metaphase
I (Fig.
6E), and up to metaphase II (Fig.
6F). In anaphase II,
SMC1

is not
visibly associated with the chromosome anymore. Aggregates
containing
SCP3 can still be seen in anaphase II, but most of
these have detached
from the kinetochores (Fig.
6H to J). The
colocalization of SCP3 and
SMC1

is also not perfect in other
respects. The SMC1

signal on
the AEs has a more granular appearance
(though far less dotty than that
of SMC1

[
13]), and there is
a limited SMC1

but no
SCP3 signal present on peripheral chromatin
loops from leptotene until
metaphase II. Moreover, some SCP3,
but very little if any SMC1

,
persists in the chromosome arms
in metaphase I (Fig.
6E).
To compare the chromosomal localization of SMC1

with that of
SMC1

, we performed additional immunofluorescence experiments
on agar
filtrates of rat spermatocytes (
13), using anti-SMC1

,
anti-SMC1

, and anti-SCP3 (Fig.
6K to M). As described by us earlier
(
13), SMC1

is present throughout spermatocyte nuclei in
frozen
sections (Fig.
6A). However, if spermatocytes are lysed in
buffers
containing Triton X-100 (as is done for agar filtration), or if
they are permeabilized in such buffers (as we have done previously
with
frozen sections [
13]), SMC1

is preferentially
retained
in intensely labeled dots along the AEs. Thus, the
localization
of SMC1

differs from that of SMC1

, which is almost
uniformly
distributed along the AEs. Also, the SMC1

dots appear not
to
be as close to the AEs as the SMC1

staining (Fig.
6K and L).
Thus, SMC1

seems to localize most closely to the SC, while SMC1
also appears SC associated, albeit not centered as much towards
the
AEs. Strikingly, and unlike SMC1

, SMC1

did not accumulate
around
the centromeres in diakinesis and metaphase I (Fig.
6 M).
We also analyzed the localization of SMC3 in spermatocytes by triple
labeling of SMC3, SCP3, and SMC1

, using frozen sections
that had not
been exposed to Triton X-100 (Fig.
7A to C) or agar
filtrates (Fig.
7D), and by triple labeling of SMC3, SCP3, and
kinetochores, using
dry-down preparations (Fig.
7E to K). Along
the AEs, SMC3, like
SMC1

, occurred mainly homogeneously distributed
on the AEs. From
late diplotene up to metaphase II, SMC3 was concentrated
at the
kinetochores, very similar to the localization seen for
SMC1

(Fig.
7F to H and J), whereas SMC3

like SMC1


was not detectable
anymore
at the kinetochores in anaphase II (Fig.
7I and K). In
SMC1

-SMC3-SCP3 triple labelings of frozen sections, SMC3 does
not
colocalize with SMC1

, indicating yet another difference between
SMC1

and SMC1

. Unexpectedly, we could not demonstrate the tight
colocalization of SMC3 with AEs if we labeled the AEs with anti-SCP2
rather than anti-SCP3 (not shown and reference
13).
Apparently,
the anti-SCP2 antibodies interfere with the immunolabeling
of
SMC3 in AEs, perhaps implying a close association of SMC3 with
SCP2.
Initial analysis of the C-terminal motif.
As noted above,
SMC1
carries an unusual, basic C-terminal amino acid sequence of 28 aa that has not been found in any other SMC protein. Analyzing this
sequence, we found a nuclear localization signal (NLS) sequence (RKPR
[24]), and therefore, the C-terminal motif may
contribute to nuclear import of the protein. In addition, the basic pI
of the peptide and two consecutive SP motifs (10) renders
interaction with DNA likely. We tested both hypotheses.
To test for an NLS function, we cloned the 28-aa motif in frame with
the enhanced green fluorescent protein (EGFP) gene into
a mammalian
expression vector. The construct was transfected into
293 cells, and
the intracellular distribution of EGFP was monitored
by fluorescence
microscopy. For a positive control, we used the
EGFP-Nuc protein, a
variant of EGFP fused to three copies of the
NLS of the simian virus 40 large T antigen (Clontech Inc.), and
the unaltered EGFP was used for a
negative, cytoplasmic control.
Screening several thousand cells, we did
not detect EGFP expression
in the nucleus (not shown). Thus, the 28-aa
motif does not confer
NLS activity on EGFP and therefore is not likely
to decisively
contribute to the nuclear import of SMC1

.
Preliminary evidence for an interaction of the 28-aa peptide with DNA
was obtained in filter binding and gel shift (gel retardation)
assays.
For a positive control in these assays, we used a peptide
derived from
the Rad54L protein (
34); for a negative control,
we used a
Rad21-derived peptide from a non-DNA binding region
(
44).
In filter binding assays (
43,
53), we used
AluI-digested (blunt-ended) plasmid DNA, radioactively
labeled at the 5' ends.
The DNA was efficiently retained by the peptide
and the Rad54
control peptide but not by the Rad21 control peptide. The
amount
of DNA retained on the nitrocellulose filters was directly
proportional
to the amount of peptide used in the assay (not
shown).
In gel shift experiments, end-labeled DNA fragments, either from
M13mp18 or from the 5S rRNA gene, were used (
1,
2,
40) and
incubated with increasing amounts of the peptide. The
results (Fig.
8A) show efficient binding of the
SMC1

-C peptide
to the DNA substrate. The migration distance from the
start position
linearly decreased with increasing amounts of peptide
added, with
no indication of cooperative binding. Surprisingly, almost
all
DNA was shifted to a higher position even with submolar amounts
of
peptide (the ratio of peptide to DNA was 1:12 in nucleotide
equivalents
or 1:2 in DNA molecules). This may indicate binding
of one peptide
molecule to more than one DNA molecule, i.e., network
formation.

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FIG. 8.
DNA interaction of the 28-aa SMC1 C-terminal motif.
(A) In a gel shift assay, increasing amounts of the 28-aa peptide were
incubated with 0.8 pmol of a 5'-32P-labeled 200-bp
ribosomal DNA fragment as indicated. R54 and R21, Rad54 and Rad21
control peptides. (B) Assay for protein-DNA network formation. Bound
(pelleted) and unbound (supernatant) DNA was measured after incubation
of 1.5-pmol of DNA substrate with increasing amounts of the 28-aa
peptide.
|
|
To further test this hypothesis, we used an assay for nucleoprotein
network formation that was used primarily in studies of
DNA
recombination proteins, such as
E. coli RecA or mammalian
proteins (
7,
17). Nucleoprotein network formation activity
was found to be required for homologous DNA pairing (
7).
Radioactively
labeled DNA is incubated with protein, and aggregates are
pelleted
by centrifugation. The pellet (bound DNA) and supernatant
(unbound)
are measured in a scintillation counter. The results (Fig.
8B)
show network formation activity of the SMC1

peptide. The Rad21
peptide did not yield a signal above background, and the Rad54L
peptide
was active but eightfold less efficient than the SMC1

-C
terminal
peptide (not shown). Network formation as seen in this
assay starts at
a minimal molar ratio of peptide to DNA of 1:1
(in nucleotides).
However, to be efficiently pelleted, DNA-protein
aggregates of
considerable mass have to be created. Therefore,
the true minimum ratio
required for one peptide molecule to bind
several DNA molecules may be
significantly lower. Future molecular
studies will have to determine
the details of the reaction requirements,
specificities, and mechanism,
as well as the function of the motif
in the context of the entire
protein or protein
complex.
Together, these initial results demonstrate DNA binding activities of
the unique SMC1

C-terminal
motif.
 |
DISCUSSION |
In this report, we demonstrate for the first time the existence of
a tissue- and cell-type-specific isoform of a mammalian SMC protein,
SMC1
. Specific expression of SMC1
was found in meiotic cells and
tissue from several mammalian species, including mouse, rat, and cow.
This, and the high degree of evolutionary conservation generally found
among SMC proteins, renders SMC1
likely to exist in humans as well.
Indeed, the working draft sequence of human chromosome 22 contains a
gene highly homologous to the mouse SMC1
gene (GenBank accession
number NT011522). This gene was mapped to the region 22q13.31.
Currently, only one locus in this region is known to be associated with
human disease, i.e., methemoglobin reductase deficiency, which
apparently has no direct connection to chromatin dynamics. We could
not, however, identify a corresponding gene in the S. cerevisiae genome. While there exist a few open reading frames
with low homology, no certain assignment to SMC1
could be made.
Likewise, no C. elegans or S. pombe orthologues
were detected in the database. Thus, the SMC1 gene may have diversified
in higher eukaryotes throughout evolution into a universally and a
meiotically expressed isoform. Swapping of SMC proteins in somatic
cells has been reported for the gene dosage compensation complex in
C. elegans (39), but no SMC protein isoform,
and no meiosis-specific SMC protein, has been described.
While up to 70% homologous to SMC1
in the globular domains, the new
SMC1
displays a characteristic feature that distinguishes it from
SMC1
: the highly basic C-terminal domain of 28 aa. Distinct C-terminal protein sequences, albeit not of such unusual composition, that are specific for meiotic isoforms of somatic proteins have been
described, for example, for mammalian DNA ligase III (6). We predicted that this unique C-terminal sequence would contribute important functions to SMC1
. It may, for example, affect
interactions of the protein with DNA, or it may act as an NLS. The
latter proved to be unlikely, since the peptide on its own did not
confer nuclear localization on EGFP. An NLS function may also not be
necessary: in SMC1
, there are seven more predicted NLSs distributed
all over the protein. The C-terminal peptide, however, interacts with DNA. The basic pI and the presence of two SP motifs, known to constitute DNA binding motifs (e.g., in histone H1
[10]), rendered this likely. Indeed, initial evidence
from three independent assays, filter binding, gel shift, and
protein-DNA network formation, suggests that the peptide efficiently
binds DNA. The gel shift experiments indicated binding of one peptide
molecule to more than one DNA molecule, i.e., possible network
formation. Preliminary experiments with network formation confirmed the
capacity of the peptide molecules to link several DNA molecules, i.e.,
to form DNA-peptide aggregates. The ability of short peptides to
promote networking-related reactions, such as homologous DNA pairing, has been reported, for example, for a 20-aa peptide derived from the
E. coli RecA protein (67). A detailed
molecular analysis of these DNA interactions, and the demonstration of
their significance in vivo, are important subjects for future studies.
Together, the unique C-terminal sequence of SMC1
is likely to
codetermine the DNA binding properties of the protein.
The new evidence for specialization of mammalian SMC proteins is
reminiscent of what has been reported for a limited number of other
proteins that are also involved in DNA dynamics. Examples in yeast are
the Scc1-type proteins
also collectively called gordin proteins
(48)
and Rec8p proteins, as well as the Rad51p and Dmc1p
proteins (5, 47, 49, 65, 69). Recently, a meiosis-specific homologue of the Scc3 protein, STAG 3, has been described
(52). Whereas Rad51p and MCD1p-Scc1p-RAD21 exist in both
somatic and meiotic cells, Dmc1p and Rec8p of yeast and of higher
eukaryotes are restricted to meiotic cells (although human Rec8p
transcripts were also found in spermatids and the thymus
[50]) and postmeiotic cells (47). In
meiosis, the relationship between cohesion and recombination is
modified, and apparently this is accompanied by replacement of
components of the protein complexes involved, like the gordins. Our
results suggest that in mammals one of the SMC components of cohesin,
SMC1
, is replaced by a meiosis-specific isoform, SMC1
. As had
been found for the gordins and Rad51 in yeast, SMC1
is only
partially replaced in mammals: SMC1
is present in both somatic and
meiotic cells, whereas SMC1
exists only in meiotic cells.
Earlier, we demonstrated the association of SMC1
with meiotic
chromatin in rat spermatocytes, and we have shown its presence in
preparations of SCs (13). Although SMC1
and SMC1
are
coimmunoprecipitated from total testis extracts by anti-SMC3
antibodies, a fraction of SMC1
in spermatocytes does not colocalize
with SMC3. Thus, two different SMC3-containing complexes exist in
testis. The slightly different behavior of SMC1
-SMC3 and
SMC1
-SMC3 in gel filtration, with the latter eluting predominantly
at a lower-molecular-mass position around 1 MDa, further indicates two
different higher-order complexes of different masses or stabilities
that share SMC3 but contain either one of the two SMC1 isoforms.
Furthermore, very little SMC1
, but an equimolar amount of SMC1
,
is coimmunoprecipitated with SMC3 from late-prophase I spermatocytes.
More evidence for this hypothesis originated from chromosomal
localization studies.
As for SMC1
, expression of SMC1
is regulated during meiotic cell
development, i.e., most protein is observed in prophase I of meiosis.
There are, however, important differences in chromosomal localization
between SMC1
and SMC1
. While SMC1
is distributed throughout
the meiotic prophase nucleus and
upon permeabilization or lysis of
cells in the presence of Triton X-100
is preferentially retained in a
dotlike pattern along AEs of SCs (reference 13 and this
paper), SMC1
is more tightly associated and more uniformly distributed along the AEs. In late prophase I (pachytene-diplotene), SMC1
also associates with bridges between the AEs of homologues, which possibly represent the sites of crossover. This has not been
observed for SMC1
. In addition, the association of SMC1
with the
AEs of SCs seems to be even closer than that of SMC1
with the SC.
Thus, there may be a structural organization in layers, with SMC1
constituting the inner and SMC1
an outer SMC-containing layer at the
SC. Finally, in diplotene and diakinesis, SMC1
and SMC3 accumulate
around the centromeres, where the proteins persist until anaphase II,
whereas SMC1
does not concentrate at the centromeres in any stage of
meiosis. This strongly suggests that SMC1
, and not SMC1
, is
involved with SMC3 in the maintenance of centromere cohesion during the
first meiotic division in mammals. In S. cerevisiae, a
similar conclusion was reached for another meiosis-specific component
of cohesin, Rec8 (36). SCP3 (reference 12 and
this paper) and SCP2 (56) also accumulate at the
centromeres from late diplotene until anaphase II. It remains to be
investigated whether these SC proteins contribute directly to
maintenance of centromere cohesion. Most likely, the dissociation of
SMC1
and SMC1
from the chromosome arms in late prophase
contributes to the release of sister chromatid arm cohesion, while
centromeric cohesion is further supported by SMC1
.
In addition, we observed that preparations of nuclear extracts and
total cell lysates, especially those from purified pachytene-diplotene spermatocytes (22), contained a characteristic 85-kDa
degradation product of SMC1
but no SMC1
or SMC3 degradation
products (not shown). Northern blotting showed the same 4.5-kb
transcript in RNA from purified spermatocytes as in testis RNA,
rendering alternative splicing unlikely. Thus, SMC1
appears to be
more sensitive to proteolysis than the other SMC proteins. One may
speculate that such proteolysis may be required for the release of arm
cohesion, similar to degradation of Rec8. The SMC1
degradation
product does not coprecipitate with SMC3, indicating that the 85-kDa
fragment of SMC1
is not present in a complex with SMC3.
Alternatively, it may also be cleaved quickly after synthesis and thus
prevented from associating with SMC3. The nature of the protease that
cleaves SMC1
, and whether such cleavage is necessary for meiotic
progression, are among the questions now to be addressed.
In summary, we propose the existence of two multiprotein complexes in
meiotic cells that are based on two different SMC1-SMC3 cores:
SMC1
-SMC3 and SMC1
-SMC3. Both complexes associate with meiotic
chromatin and should contribute to meiotic sister chromatid cohesion.
The "
-complex," however, appears more loosely chromosome associated, in a punctate pattern, and also in the chromatin loops. This complex dissociates and is released from the chromatin in late
prophase I. The "
-complex" closely localizes to the SC and remains chromosome associated at the centromeres beyond prophase I
until metaphase-anaphase II. Therefore, the
-complex, and not the
-complex, is likely responsible for centromeric cohesion until
anaphase II. The interplay among meiotic sister chromatid cohesion, DNA
recombination, and the new meiosis-specific SMC1
is now amenable to
future studies.
 |
ACKNOWLEDGMENTS |
We thank Mirjam van Aalderen for expert technical assistance and
David Avila for protein sequencing. E.R. was supported by a grant from
the Human Frontier of Science Foundation and by NIH grant GM62517. M.E.
was financially supported by grant no. 901-01-097 of The Netherlands
Society for Scientific Research (NWO).
An initial part of this work was done at the Basel Institute for
Immunology, Basel, Switzerland.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, Box 1496, New York, NY 10029. Phone: (212) 859-8259. Fax: (212) 803-6740. E-mail: rolf.jessberger{at}mssm.edu.
 |
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Molecular and Cellular Biology, October 2001, p. 6984-6998, Vol. 21, No. 20
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.20.6984-6998.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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