1 Department of Biology, York University, Toronto, ON, M3J 1P3, Canada
2 Imperial Cancer Research Fund, South Hall Laboratories, South Mimms,
Hertfordshire, England EN6 3LD
3 Dept of Molecular Genetics, Albert Einstein College of Medicine, New York
10461 USA
* Author for correspondence (e-mail: moens{at}yorku.ca )
Accepted 11 December 2001
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Summary |
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Key words: Meiosis, Recombination proteins, Immunocytology, Synaptonemal complexes, BLM, RPA, MLH1, Mouse, Recombination nodules
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Introduction |
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Molecular models for the resolution of DNA-DNA interactions without
reciprocal recombination have been discussed by Gilbertson and Stahl
(Gilbertson and Stahl, 1996)
and involve helicase-topoisomerase activity to resolve joint molecules. The
acquisition of replication protein A, RPA and Bloom mutated protein, BLM, at
the RAD51/DMC1 sites might be the physical manifestation of the models
RPA may stimulate BLM-RecQ helicase activity
(Brosh et al., 2000
) in
concert with topoisomerase IIIa (Johnson
et al., 2000
) to resolve the early DNA-DNA interactions at the
RAD51/DMC1 sites.
The development of reciprocal genetic exchange events at meiosis in many
fungi, plants and animals can be monitored at several levels: (1) at the
chromosomal level by chiasmata, which are the sites of reciprocal
recombination (Jones, 1987);
(2) by the recombination nodules, RNs, which correlate with genetic and
cytological patterns of recombination
(Carpenter, 1975
;
Carpenter, 1979
;
Albini and Jones, 1988
); and
(3) by the MLH1p sites, which are associated with crossover sites
(Anderson et al., 1999
)
Nodules are SC-associated, electron-microscope-defined structures that have
been reported in the meiocytes of protists, fungi, plants and animals. In
early meiotic prophase, `nodules' refer to the several hundreds of small dense
bodies about 100 nm in diameter that are associated with chromosome cores and
SCs and contain the RAD51/DMC1 proteins in lily
(Anderson et al., 1997), mouse
and human (Haaf et al., 1995
;
Moens et al., 1997
;
Barlow et al., 1997
). Since the
correlation of these structures with reciprocal recombination is tenuous, they
are usually referred to as `early nodules', EN. The late RNs, as originally
defined by Carpenter (Carpenter,
1975
) in Drosophila melanogaster oocyte SCs, correspond
in number and location to reciprocal recombinant events in normal and mutant
D. melanogaster. In the rat, `late RNs' are well defined
electron-dense bodies of variable shape and size, 100 to 200 nm, located on
the mature SC at pachytene stage VII of the spermatogenic pathway but not in
earlier pachytene stages I to V (Clermont,
1972
; Moens,
1978
). Cross-sectioned SCs and whole-mount, shadow-cast EM
preparations show that RNs are located on the surface of the SC either along
the central element or obliquely across the SC
(Fig. 7). The reported number
of late RNs (19 to 22 per nucleus) in complete EM-reconstructed rat-pachytene
nuclei, their non-random distribution and their association with MLH1 (Mut L
homolog) protein in rat and mouse (this report), agree with their proposed
function in reciprocal recombination by Carpenter
(Carpenter, 1975
;
Carpenter, 1979
). A number of
publications have assigned various proteins to RNs but the assignments have
not previously been verified by EM demonstration of these proteins on the
RNs.
|
We report the events in individual mouse and rat spermatocyte nuclei from early to late meiotic prophase in terms of chromosome core behavior and associated protein complexes. The immunofluorescence observations are refined and detailed by immunoelectron microscopy of the recombination-related proteins and by EM visualization of RNs. These observations are interpreted in the context of the chromosome synapsis and reciprocal recombination and discussed in relation to reports by others on these events.
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Materials and Methods |
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Antibodies
Polyclonal rabbit antibodies against whole hamster SCs and polyclonal mouse
antibodies against the fusion proteins of the hamster 30 kDa chromosome core
protein, COR1p, and the 125 kDa synaptic protein SYN1p have been characterized
previously (Dobson et al.,
1994; Tarsounas et al.,
1997
; Tarsounas et al.,
1999a
) and have been used extensively by others
(Plug et al., 1997
;
Plug et al., 1998
;
Pittman et al., 1998
). The
equivalent rat 30/33 and 125 kDa SC proteins are named SCP3p and SCP1p
(Heyting et al., 1988
). From
J. Ingles, University of Toronto, we received the polyclonal rabbit anti-RPA
antibody (Henricksen et al.,
1994
; He et al.,
1995
), which has also been used for similar experiments on RPA at
meiosis (Plug et al., 1997
;
Plug et al., 1998
). From J.
Masson (ICRF, UK), we received the recombinant HsRPA with the 14, 32 and 70
kDa subunits identified with western blotting. Our mouse, 23LM, produced a
polyclonal anti-RPA serum against this recombinant HsRPA with good fluorescent
and EM immunocytology. Centromeres were labeled with a CREST serum as reported
earlier (Dobson at al., 1994
).
The rabbit anti-BLM antibody was raised against the purified fusion protein of
the last 380 amino acids of BLMp. The antiserum recognized the appropriate 180
kDa protein in western blots of HeLa whole-cell extracts, and no protein was
detected in the BS cell line that lacked the BLMp C-terminal portion
(Moens et al., 2000
). Mouse
full-length, (His)6-tagged DMC1 and RAD51 proteins were overexpressed in
Escherichia coli. The Ni-NTA-purified proteins were injected into
mice and rabbits that had negative pre-immune serum. Both types of sera
cross-react with RAD51p and DMC1p, but after immune depletion, they were
rendered specific for one or the other antigen. Because it was shown with
fluorescence and EM cytology of the purified antibodies that the two antigens
colocalize on mouse and rat SC-associated early nodules
(Tarsounas et al., 1999a
), we
used, for this study, the anti-DMC1 serum from our rabbit `Patch' or mouse
`17RB' to detect the mixed RAD51/DMC1 antigen in core/SC-associated early
nodules. These anti-RAD51/DMC1p antibodies differ from those used in a number
of earlier reports in that they do not contain anti-SC antibodies, which are
common contaminants of rabbit serum (Ashley
et al., 1995
; Kovalenko et
al., 1996
; Moens et al.,
1997
). The monoclonal anti-MLH1p antibody was obtained from BD
Pharmingen and has been characterized by Edelmann et al.
(Edelmann et al., 1996
) and
Anderson et al. (Anderson et al.,
1999
). The polyclonal anti-hMSH4 was generated in rabbit and has
been characterized by Santucci-Darmanin et al.
(Santucci-Darmanin et al.,
2000
). Antibodies against rat testis-specific histone H1t were
generated in a rabbit with E. coli-expressed H1t protein
(Kremer and Kistler, 1991
;
Moens, 1995
).
Imaging
Fluorescence from FITC- or rhodmamine-tagged secondary antibodies and
DAPI-stained chromatin was recorded by CCD camera or single or multiple
exposures of black and white or colour-positive film, ASA 400. Slides were
scanned at 1,000 dpi and recorded in TIFF format, and final images were
arranged with Adobe PhotoDeluxe software. Images were reproduced with an Epson
700 or 870 colour printer. To determine the colocation of RPA with BLM foci,
the images were recorded on black and white film and one positive and one
negative image were superimposed. Electron micrographs of cores/SCs and
immunogold were recorded at various magnifications, 1 k to 10 k, and
photographically enlarged. The contrast of SCs and nodules in EM preparations
was enhanced by staining for 30 minutes in 4% alcoholic PTA followed by a
rinse with 95% ethanol. Shadowcasting of SCs was achieved by gold-palladium
evaporation at a low angle of 7° on stationary or rotary moving grids
(Moens et al., 1987).
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Results |
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Time course of RAD51/DMC1p versus RPA foci on meiotic chromosome
cores/SCs
The RAD51/DMC1 foci of the meiotic prophase chromosomes are the sites of
single-strand DNA tails that are active in homology search and strand exchange
(Bishop, 1994;
Schwacha and Kleckner, 1997
).
The presence of RPA protein in relation to RAD51/DMC1 foci at successive
developmental stages of mouse spermatocyte nuclei at meiotic prophase is
illustrated in Fig. 1. The
number and intensity of immunofluorescent RPA foci increases from the time the
chromosome cores first form and synapse at the zygotene stage of meiosis until
late in the fully synapsed state at pachytene. The relative proportions of
RAD51/DMC1 and RPA foci in individual nuclei can be demonstrated with
simultaneous visualization of the two types of foci. In
Fig. 1A and B, the early
prophase nucleus to the left of the white line is in the leptotene/zygotene
stage, judging by the mostly unpaired centromeres (enhanced red). This nucleus
has an abundance of RAD51/DMC1 foci (about 200)
(Fig. 1A), but a maximum of
only 90 weak RPA foci (Fig.
1B). At a later stage, judging by the paired centromeres and the
aligned foci, there are fewer DMC1 foci (approximately 90) in the nucleus on
the right side of Fig. 1A,
while the number of RPA foci has increased to 180 in that same nucleus
(Fig. 1B).
|
|
RPA replaces RAD51/DMC1p
With the abundance of the two types of foci along the SCs, it is likely
that by chance alone the two types will be close together, and it is difficult
at the level of immunofluorescence resolution to decide how much actual
colocalization exists. At the high resolution of the electron micrographs in
Fig. 2, it is possible to
distinguish between pure RAD51/DMC1 foci (groups of 10 nm immunogold grains),
pure RPA foci (5 nm gold grains) and mixed foci (colocation of 5 and 10 nm
gold grains). The immunogold foci of 40 SCs were classified accordingly, and
the percentages of the three types at progressively later prophase stages are
shown for nine of the 40 SCs in the bar graph of
Fig. 1E. It is evident from the
brown middle segment of the first bar that most of the RPA protein initially
colocalizes with the RAD51/DMC1 foci. The remainder of the bars suggest that
the RPA gradually replaces the RAD51/DMC1 component of the foci to the point
where there is no RAD51/DMC1 protein (last bar in
Fig. 1E). While it is possible
that the mixed foci arise de novo, the gradient of the percentages of the two
types of proteins would favour a dynamic replacement process. The total
numbers of immunofluorescent foci per nucleus for successive developmental
stages is demonstrated in Fig.
1F. Many of these would be in mixed foci as shown in
Fig. 1E, Fig. 2C and
Fig. 4.
|
At high resolution, the abundance of RAD51/DMC1 foci and the paucity of RPA protein at the onset of chromosome synapsis is illustrated in the electron micrograph of a set of partially synapsed cores in Fig. 2A. There are six or seven groups of 10 nm RAD51/DMC1-associated immunogold grains but only one group and a few sporadic 5 nm, RPA-associated, immunogold grains. The reverse situation exists at a later stage where fully synapsed chromosome cores contain four RPA foci associated with the SC, but there is only one RAD51/DMC1 focus (Fig. 2B, RAD51/DMC1 10 nm immunogold grains, RPA 5 nm). At the onset of the pachytene stage, there are occasionally a few centromeric ends of chromosomes that have not completed synapsis. Such a `laggard' is shown in Fig. 2C, which demonstrates the combination of RAD51/DMC1 and RPA protein in single nodules, marked as `mix' in the figure. The arrow marks a small loop of aligned RPA gold grains, which is not uncommon, but the significance is unclear.
RPA at SC-associated transitional or terminal nodules
Where the chromosome cores are fully synapsed
(Fig. 2B), the 5 nm gold grains
tend to be concentrated on dense nodes of the central element of the SC,
suggesting that the RPA protein is present in some form of nodule
(Fig. 2B and insert;
Fig. 4D-G). These nodes do not
have RAD51/DMC1 protein and therefore do not conform to the concept of an
`early nodule'. Since they also do not have the MLH1 protein (see below), they
therefore do not qualify as `late nodules'. Few if any of these nodules will
persist and they therefore represent some sort of `terminal' or `transitional'
nodule, TN.
RPA at the X-Y pseudo autosomal region
The short Y chromosome becomes fully synapsed with the distal portion of
the long X chromosome during early meiotic prophase, but the two are
homologous only at their most distal regions, the pseudo-autosomal (PA) region
(Fig. 3). In the laboratory rat
and mouse, the PA region has one or more reciprocal recombinant events, which
are presumed to regulate the proper segregation of the X and the Y chromosome
at the first meiotic division (Soriano et
al., 1987). These crossovers are recognized as a chiasma at
diplotene and metaphase I. The RPA antigen, marked by green FITC
immunofluorescence in Fig. 3B,
is clearly present in the terminal segment of the PA region. The
immunofluorescence signal is frequently one of the brightest FITC foci of the
nucleus, and this may be caused by the presence of two or more proximal foci.
Similarly, the PA region has a pronounced BLM signal as reported previously
(Moens et al., 2000
).
|
When the X and the Y chromosomes have a long non-homologous pairing segment, there is RAD51/DMC1 antigen (10 nm gold grains) along the length of the paired segment (Fig. 3C) as well as along the core of the X chromosomes. However, RPA is concentrated in the terminal, PA, region (Fig. 3C) and along the X chromosome core (Fig. 3B). In the rat, which has better EM-defined, SC-associated nodules, the RPA can be detected at the nodule in the PA region (Fig. 3D; Fig. 7B).
RPA and BLMp colocalize
Because RPA (this report) and BLMp
(Walpita et al., 1999;
Moens et al., 2000
) are
frequently present on the small pseudoautosomal region of the X and Y
chromosomes, we tested the possibility that these two proteins regularly occur
together in the same protein complex. For this purpose, the BLM antigen was
detected by rabbit anti-BLM serum and the secondary antibody was tagged for
fluorescent microscopy or for EM observation. The RPA antigen was detected by
a mouse serum and differentially tagged secondary antibodies. To assess the
relative contribution of each antigen to the individual foci and to be able to
quantify the immunogold labeling of foci, we avoid the use of rabbit
antibodies against both BLM and RPA protein as in Walpita et al.
(Walpita et al., 1999
).
Superposition of the fluorescent images indicates that most RPA foci also have
BLM antigen (Fig. 4). Whereas
the RPA signals are generally of similar strength
(Fig. 4B), the BLM signals vary
widely in intensity (Fig. 4A).
For this reason, it is difficult to quantify the contribution of each antigen
to the foci by visual evaluation of subtle colour differences. Instead, we
represent the BLM signal intensity by black dots (negative image) and
superimpose these dots on the white RPA signals (positive image)
(Fig. 4C). This procedure
clearly demonstrates that at the level of resolution of immunofluorescence,
most BLM foci colocalize precisely with the RPA foci, and there is much
variation in the intensity of the BLM signals as indicated by the sizes of the
BLM signals.
The colocation of RPA and BLM was confirmed in immunogold-labeled foci, where the two types of gold grains, RPA 5 nm and BLM 10 nm, can be found together in the nodules (Fig. 4E-G). For the antibodies that we use, there are more RPA gold grains per focus than there are BLM gold grains (Fig. 4H).
MSH4 co-locates with RPA in transitional nodules
The meiosis-specific MSH4p homologue of E. coli MutSp is required
for synapsis and recombination. In mouse it is present along the paired
chromosome cores at meiotic prophase (Fig.
5) (Kneitz et al.,
2000) (S. Santucci-Darmanin, P.B.M., F. L. Lespinasse et al.,
unpublished). MSH4 fluorescent foci appear at the time that the early nodules
are losing the RAD51/DMC1 component. Biochemical evidence suggests that there
is an interaction between the two types of proteins (S. Santucci-Darmanian,
P.B.M., F. L. Lespinasse et al., unpublished), but it is relatively rare to
find co-location of the two types of proteins either with fluorescent
microscopy (Fig. 5C) or with
electron microscopy (Fig.
5D,E), when the two antigens are differentially labeled. However,
it is evident that MSH4 and RPA frequently co-locate to the same foci when
observed with fluorescent microscopy (Fig.
5A) or electron microscopy
(Fig. 5B). It is concluded that
MSH4 is mostly a component of the transition/terminal nodules. Because the TNs
vanish during meiotic prophase, it is possible that MSH4, in concert with RPA,
BLM and others, functions in the resolution of early DNA-DNA interactions.
|
Time course of RPA and BLM versus MLH1
Immunocytologically detectable MLH1 foci that are in association with the
SCs appear in the later part of meiotic prophase after RAD51/DMC1 foci are no
longer present, and their numbers, about 23 per nucleus, are small in
comparison with the numbers of other foci. In general, it appears that RPA
foci are already reduced to small numbers when MLH1 foci first appear
(Fig. 1F). The nucleus in
Fig. 6A has four RPA foci
marked by arrows and 22 MLH1 foci, nearly the full complement. There appears
to be no positional correspondence between RPA and MLH1 foci. Electron
microscopy, however, shows the occasional presence of RPA in RNs
(Fig. 3D;
Fig. 7B). The BLM foci persist
longer than the RPA foci (Fig.
1F), and they are still present when the MLH1 foci develop
(Fig. 6B). From
immunofluorescent observations, there is no clear indication that the two
types of foci colocate (Fig.
6B). The BLM foci decline in numbers, whereas the MLH1 foci remain
fairly constant (Fig. 6D).
Although the MLH1 foci normally remain until the onset of the diplotene stage
in male meiosis, they persist into the diplotene stage of female meiosis.
|
MLH1 protein is a component of EM-defined late recombination
nodules
EM of late pachytene mouse SCs treated with anti-MLH1 monoclonal antibody
and secondary goat anti-mouse antibody tagged with 5 nm gold grains show that
the MLH1 antigen is located at the EM-defined late RNs.
(Fig. 6C,E-G). The presence of
the MLH1 antigen is remarkably predictable. Where one SC of a given nucleus is
found to have an MLH1-labeled RN, all other SCs of that nucleus predictably
have one or two such RNs. This was confirmed in more than 20 nuclei. The exact
localization of an MLH1-labeled RN to the point of a presumptive chiasma is
demonstrated in Fig. 6F and G.
Normally RNs are no longer present at the diplotene stage in the male, but
when the diplotene configurations are induced precociously by the phosphatase
inhibitor okadaic acid in purified early prophase cells, the MLH1 foci are
still present at diplotene. The platinum/gold shadow casting enhances the
appearance of the RNs in the rat (Fig.
7), but the mouse nodules are somewhat smaller and are less well
defined with this methodology. Therefore the alternative, PTA staining, was
used to visualize mouse nodules and associated immunogold grains in Figs
2,3,4,5,6.
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Discussion |
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In the double-strand break model, the joint molecules can be resolved in a
number of ways by specific enzymatic complexes. Depending on how the two
Holliday junctions of a joint molecule are cleaved, there can be a 1:1 ratio
of reciprocal to non-reciprocal recombinants. Taking into account the
preferential resolution that causes deviations from the 1:1 ratio, there would
still be a large excess of reciprocal recombinations produced by the 250
RAD51/DMC1 sites in the mouse where an average of only about 23 reciprocal
events are observed. It has been argued, however, that the joint molecules can
also be resolved without reciprocal recombination along different pathways
(Gilbertson and Stahl, 1996).
These pathways require helicase and topoisomerase functions, and are therefore
of particular interest to us because of the observed association of Bloom RecQ
helicase with RAD51/DMC1 sites at the cores/SCs of meiotic prophase
chromosomes (Moens et al.,
2000
).
Bloom protein normally functions in chromosome stability by the removal of
DNA conformations that could lead to chromosome rearrangements or
recombination. BLM protein, as a member of the family of RecQ helicases,
belongs to a class of anti-recombinases
(Constantinou et al., 2000)
that could function to resolve the meiotic prophase chromosome interaction
without reciprocal recombination, analogous to the yeast RecQ helicase SGS1
that can resolve strand invasion events
(Frei and Gasser, 2000
).
Mutations of the BLM gene lead to chromosome rearrangements and
characteristically high levels of sister chromatid exchanges in somatic cells
(German, 1993
;
Watt et al., 1996
). BLM RecQ
helicase and topoisomerase IIIa interact physically and functionally to enable
passage of double-stranded DNA in somatic and meiotic human cells
(Harmon et al., 1999
;
Johnson et al., 2000
). Thus
the complex demonstrably suppresses recombination and is therefore an
appropriate candidate for the resolution of early DNA-DNA interactions at
meiotic prophase.
We observe that RAD51/DMC1 foci become associated with RPA and somewhat
later with BLMp. This observation can be related to the reports that RecQ
helicases interact with RPA. Brosh et al.
(Brosh et al., 2000) have
shown that in vitro the presence of RPA stimulates the helicase activity of
BLMp, and Constantino et al. (Constantino et al., 2000) show that the RecQ
helicases WRN and BLM localize to RPA foci, where they promote translocation
of the Holliday junctions and dissociate recombination intermediates.
Similarly, Sakamoto et al. (Sakamoto et
al., 2001
) report that WRNp colocalizes with RPA foci at sites of
induced DNA damage. Taken together, these observations provide support for the
hypothesis that early meiotic prophase DNA-DNA interactions are resolved,
without reciprocal recombination, by a protein complex that includes RPA and
BLMp, which probably acts in conjunction with topoisomerase IIIa.
Although it is well established that MSH4p is a partner of the RPA/BLM complex and the TNs (Fig. 5C,D,E), its function in the complex is uncertain (S. Santucci-Darmanin, P.B.M., F. L. Lespinasse et al., unpublished). At later stages when few MSH4 foci remain, it has been reported that they colocalize with MLH1p but here, too, the functions are still speculative (Santucci-Darmanin, 2000).
Origin of recombination nodules
Because MLH1 protein is associated with recombinant events and because the
MLH1 foci are correlated in number and distribution with reciprocal
recombinant events, it has been hypothesized that the MLH1 foci represent the
RNs in mice (Anderson et al.,
1999). We provide evidence in support of that hypothesis by
showing that the MLH1 antigen is present in EM-defined RNs
(Fig. 6C,E) and that the RN and
the MLH1 protein are present at the site of a chiasma
(Fig. 6F,G). In
MLH1-/- mice, chromosome synapsis is normal. The repulsion of
homologues at the onset of meiosis appears normal but subsequently there is a
complete or near complete lack of chiasmata, and the result is an accumulation
of univalents in the nucleus (Baker, 1996;
Edelmann et al., 1996
).
Apparently, reciprocal recombinant events are mostly absent.
It is evident that most early DNA-DNA interactions that are recognized as RAD51/DMC1 foci are resolved by mid prophase. At a later stage, the foci that correspond to RNs are observed. The question arises whether (1) a subset of the early interactions persist and become late RNs or (2) whether the late RNs arise de novo. There is support for both propositions.
These observations on the origins of RNs are clearly related to but do not yet resolve the issue of genetic and crossover interference. They suggest that in the appropriate organisms or experimental conditions, the immunocytology of recombination-related proteins could elucidate the mechanisms of interference that limit and position the crossovers.
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Acknowledgments |
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