Waksman Institute and Department of Genetics, Rutgers, the State University of New Jersey, 190 Frelinghuysen RD, Piscataway, New Jersey 08854-8020, USA
Author for correspondence (e-mail:
mckim{at}rci.rutgers.edu)
Accepted 14 April 2003
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Summary |
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Key words: Double-strand break, Meiotic recombination, Drosophila, DNA repair, Synaptonemal complex, Oogenesis
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Introduction |
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Another hallmark of meiotic prophase is the synaptonemal complex (SC), a
specialized protein-chromosome structure that physically connects aligned
homologous chromosomes. Completion of synapsis between homologous chromosomes
is marked by the presence of SC along their entire lengths. SC can form in the
absence of DSBs in Drosophila
(Liu et al., 2002;
McKim et al., 1998
) and
Caenorhabditis elegans (Dernburg
et al., 1998
), but not in budding yeast
(Roeder, 1997
) or mouse
(Baudat et al., 2000
;
Romanienko and Camerini-Otero,
2000
). The relative timing of DSBs and SC formation in the mouse
was determined using an antibody that recognizes the phosphorylated form of a
histone H2A variant, H2AX (
-H2AX). On induction of DSBs in mammalian
mitotic and meiotic cells, H2AX is rapidly phosphorylated
(Rogakou et al., 1999
).
-H2AX staining was detected before the appearance of SC proteins,
suggesting that DSBs appear before SC formation during meiotic prophase in the
mouse (Mahadevaiah et al.,
2001
), consistent with time-course studies of DSB formation in
S. cerevisiae (Padmore et al.,
1991
).
Drosophila has a single H2A variant (HIS2AV) that, like H2AX, is
phosphorylated at a conserved SQ motif within an extended C-terminal tail
following chromosome breakage in mitotic cells
(Madigan et al., 2002).
Phosphorylation of Drosophila HIS2AV (
-HIS2AV) in mitotic
cells is rapid, reaching its maximum within five minutes of exposure to agents
that induce DSBs but not single-strand nicks
(Madigan et al., 2002
). Here
we provide evidence that HIS2AV is phosphorylated in response to meiotic DSB
formation. Using
-HIS2AV staining as a marker for DSB formation, we
found evidence that DSB formation occurs after synapsis and is partially
dependent on the SC protein C(3)G. In addition,
-HIS2AV staining
suggests that DSB repair is delayed in okr (Rad54 homolog) and
spnB (Rad51/Dmc1 homolog) mutants, but it does occur eventually.
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Materials and Methods |
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Cytology
For immunolocalization experiments, females were aged for 16-40 hours at
room temperature, dissected and fixed using either the `Buffer A' protocol
(Belmont et al., 1989) or the
PBS-based protocol described by Page and Hawley
(Page and Hawley, 2001
). To
detect DSBs, the Anti-phospho-H2AX (Ser139) rabbit polyclonal antibody
(Upstate Biotechnology) was used at 1:100 and a Cy3-labeled goat anti-rabbit
secondary antibody (Amersham) was used at 1:250. The guinea pig C(3)G antibody
(Page and Hawley, 2001
) was
used at 1:500 and a FITC-labeled goat anti-guinea pig secondary antibody
(Vector) was used at 1:250. A combination of two Orb antibodies (4H8 and 6H4)
(Lantz et al., 1994
) was used
at 1:150 and a FITC-labeled goat anti-mouse secondary antibody (Vector) was
used at 1:250 or a Cy5-labeled goat anti-mouse (Amersham) was used at 1:100.
Chromosomes were stained with 4',6'-diamidino-2-phenylindole hydrochloride
(DAPI) (0.2 µM) for 10 minutes or Hoechst (0.1 µl/ml of a 10 mg/ml
solution) for 5 minutes. Images were collected using a Leica TCS SP2 confocal
microscope or a Zeiss Axioplan II imaging microscope equipped with a Cooke
Corp. Sensicam CCD camera using a 63x or 100x objective. Sections
were collected at 0.2 µm or 0.3 µm intervals.
The counts of -HIS2AV foci were made from a subset of germaria where
the foci were clear. Foci were manually counted by examining the full series
of optical sections containing an oocyte nucleus. This method might
underestimate foci because two foci that were in the same X-Y position but in
consecutive Z-sections may have been counted as a single focus. In addition,
the foci were not always the same intensity, which may reflect their
asynchronous development. It was not difficult to distinguish the nuclear foci
from background because the latter was cytoplasmic. For example, the
cytoplasmic background was variable between germaria but uniform throughout
the germarium. By contrast, nuclear foci in wildtype were only present in
region 2a and early 2b. Nuclei at other stages were generally free of
staining.
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Results |
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Consistent with the hypothesis that the foci disappear because of DSB
repair, a dramatic change in the -HIS2AV staining pattern was observed
in spnB (a meiosis-specific Rad51 homolog) and okr (a Rad54
homolog) mutants, which are proposed to be defective in DSB repair
(Ghabrial et al., 1998
).
Although the wild-type
-HIS2AV foci were limited to early pachytene
(region 2a and occasionally 2b, Fig.
1B,C) and were always absent from late pachytene oocytes (region
3, Fig. 1D,
Table 2), the
okrWS and spnBBU mutant germaria
always exhibited foci in late pachytene (region 3 and early vitellarium)
(Fig. 3A-D,
Fig. 4). In addition, the
number of
-HIS2AV foci in region 3 oocytes of the DSB repair-defective
mutants was consistent and usually higher than in wildtype, often in excess of
20
-HIS2AV foci (average 21.1, Table
1). The foci persisted in the okr and spnB
mutants until stage 4 of the vitellarium, at which point they disappeared,
just before the dissolution of the SC (Fig.
5). The persistent and numerous foci in these mutants may result
from the failure to efficiently repair DSBs. Indeed, the
-HIS2AV
staining in okrWS and spnBBU mutants
gradually became brighter until region 3, suggesting that the
-HIS2AV
was accumulating (Fig.
3C,D).
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To determine whether the foci detected by the -H2AX antibody were
dependent on phosphorylation of HIS2AV, we stained germaria from females
carrying a mutant form of the protein that lacks the phosphorylation site.
Homozygotes for the null allele His2Av810 are lethal
(van Daal and Elgin, 1992
).
However, when this homozygote is coupled with a transgene expressing
His2Av
CT, which lacks the last 14 amino acids
inclusive of the phosphorylation site, the flies are viable and fertile
(Clarkson et al., 1999
). When
the His2Av
CT; His2Av810 females were
stained with the
-H2AX antibody, the nuclear foci were absent (data not
shown). Because the spnBBU mutation causes an increase of
-HIS2AV foci in late pachytene oocytes, we used it as a tool to confirm
the absence of foci in the His2Av
CT mutant. Indeed,
His2Av
CT; spnBBU
His2Av810 females exhibited no nuclear foci of
-HIS2AV
staining (Fig. 3E). The
dependence of staining on the C-terminal tail of the His2Av gene is
consistent with the conclusion that the
-H2AX antibody detects the
phosphorylated form of HIS2AV.
To determine whether the foci of -HIS2AV were dependent on DSB
formation, we examined staining patterns in mei-W68 mutant oocytes,
where meiotic recombination does not occur
(McKim et al., 1998
). We did
not observe nuclear
-HIS2AV staining in either a mei-W68
single mutant (compare with wildtype where foci were normally observed early
in pachytene), or okrWS mei-W681 and
mei-W684572; spnBBU double mutants (compare
with okrWS or spnBBU single mutants
where a large number of
-HIS2AV foci are normally observed in late
pachytene oocytes) (Fig. 3D,F).
The dependence of staining on mei-W68 shows that the
-H2AX
antibody is detecting a response to DSBs in wild-type pachytene. In
conjunction with western blot analysis showing that HIS2AV is phosphorylated
when DSBs are induced (Madigan et al.,
2002
), our results suggest that Drosophila HIS2AV is
phosphorylated in response to meiotic DSBs.
Relationship of the SC to -HIS2AV formation
To correlate DSB repair with the prophase events of synapsis and SC
formation, we used an antibody to C(3)G, which is proposed to be a structural
component of the transverse elements (Page
and Hawley, 2001). Formation of the SC occurs early in region 2a
(Carpenter, 1979
), and C(3)G
staining is visible in pro-oocytes of region 2a cysts at the same time or
earlier than ORB staining (Page and
Hawley, 2001
).
-HIS2AV foci were only observed in
pro-oocytes with extensive C(3)G staining, indicative of pachytene. In some
germaria, earlier stage (more anterior) pro-oocytes had patches of C(3)G
staining, indicative of zygotene where the chromosomes have not fully
synapsed. These pro-oocytes lacked
-HIS2AV foci
(Fig. 2,
Table 2). The appearance of
-HIS2AV foci only after the appearance of C(3)G staining suggests that
DSBs are not phosphorylated until after SC formation. As in the experiments
using ORB staining, the
-HIS2AV foci were consistently absent from
region 3 oocytes (Fig. 4A,
n=13, and Table 2),
suggesting the completion of an important step in DSB repair by this
stage.
Carpenter previously described the distribution of early recombination
nodules (RN) in Drosophila, which are predicted to be protein
complexes situated at the sites of DSB repair
(Anderson et al., 1997;
Bishop, 1994
;
Carpenter, 1979
;
Plug et al., 1996
;
Tarsounas et al., 1999
;
Terasawa et al., 1995
). The
-HIS2AV foci and early RN patterns are similar, both appearing at the
same stages of cyst development. Both the
-HIS2AV foci and early RNs
usually appear in three to six consecutive cysts, first appearing in small
numbers, then increasing in the following cyst or two, and then decreasing in
number again before disappearing altogether
(Table 2). For example, in
germarium B-2, the cyst with the most foci in the pro-oocytes (the fourth) had
two cysts before it and two cysts after it with fewer foci. A similar pattern
was also observed for early RNs
(Carpenter, 1979
). Considering
that the cysts are usually arranged in temporal order, this pattern probably
reflects that, as was suggested for early RNs, the appearance and
disappearance of
-HIS2AV foci is asynchronous. Asynchrony in appearance
of
-HIS2AV foci means that the average number observed per cell in
wildtype is probably less than the total number of DSB sites. Exceptions to
the temporal ordering of cysts do occur, as noted by Carpenter
(Carpenter, 1979
), such as the
fourth cyst in germarium C-1, which is probably out of order.
Complete SC formation (pachytene) was not necessary for -HIS2AV foci
to be observed. Although the most numerous foci were observed in the two
pro-oocytes with thread-like C(3)G staining
(Fig. 2B), foci were also
observed in nurse cells, which have only short segments of C(3)G staining
(Fig. 2C). For example, cyst 3
in the germarium shown in Fig.
1A had one cell with 16 foci (some shown in the single section of
Fig. 1B) and at least five
nuclei with four or less foci. Cyst 4 had nuclei with 17 and 19 foci but also
five nuclei with five or less foci. Thus, the
-HIS2AV foci are not
uniformly distributed among the nuclei in each cell of a 16-cell cyst, but
instead the largest numbers were found in the pro-oocytes. That the largest
number of
-HIS2AV foci are in the pro-oocytes parallels Carpenter's
(Carpenter, 1979
) observation
that there is a gradient of SC formation; the pro-oocytes form complete SC and
the adjacent nurse cells form progressively less SC. Similarly, in
spnB and okr mutants, it was clear that several cells in a
16-cell cyst stained with
-HIS2AV (e.g.
Fig. 4B,D). These data indicate
that DSBs are induced in the nurse cells that do not complete SC formation, in
agreement with previous studies showing early RNs
(Carpenter, 1975b
) and MEI-P22
localization, a protein required for DSBs
(Liu et al., 2002
), in nurse
cell nuclei.
DSB formation occurs in the absence of C(3)G
To test directly the dependence of DSB formation on the SC, we examined
-HIS2AV staining in c(3)G68 mutants, which lack SC.
Surprisingly, a small number of
-HIS2AV foci were consistently observed
in early prophase (region 2a) of c(3)G68 mutant germaria
(data not shown). No more than three foci were counted in a nucleus (average
2.2, Table 1) but, due to the
absence SC staining, we could not determine whether these cells were
pro-oocytes. To enhance the detection of
-HIS2AV foci, we constructed
okrWS; c(3)G68 double mutant females with the
expectation that any DSBs would persist and be visible by antibody staining in
late prophase (region 3) oocytes. This experiment confirmed the presence of
-HIS2AV foci in c(3)G68 mutant oocytes; however, in
comparison to the okr single mutant (average 21.1, see above), there
were clearly fewer foci in region 2b or 3 oocytes (average 5.4,
Table 1; compare
Fig. 3D and 3G). On the basis
of these observations, DSBs appear to be induced in a c(3)G mutant,
but possibly less frequently than in wildtype (see Discussion).
Phosphorylation of HIS2AV is delayed in DSB repair mutants
okr and spnB
The onset of SC formation consistently appears at the same time (early in
region 2a of the germarium) in both wildtype and the DSB-repair defective
mutants (data not shown). Furthermore, the SC disappears at the same
developmental time point as wildtype, vitellarium stage 5-6, suggesting there
are no delays in the cell cycle of DSB repair-defective mutants. However,
there was a slight delay in the onset of -HIS2AV staining in the DSB
repair mutants relative to wildtype. In wildtype,
-HIS2AV foci were
observed in the first or second cyst with C(3)G staining
(Table 2). In all 13
okrWS and eight spnBBU mutant germaria
examined,
-HIS2AV foci were not observed until the third or fourth
C(3)G staining cysts (Figs 3,
4).
Comparing the defects of SC-deficient and DSB repair-defective
mutants
The few -HIS2AV foci in a c(3)G mutant were observed in the
same developmental stages as in wildtype, present in regions 2a and 2b but
absent in region 3. The c(3)G pattern of fewer foci appearing and
disappearing in an otherwise normal time course is a different phenomenon than
that observed with DSB-repair defective mutants. In okr and
spnB mutants, we suggest that all DSB sites are phosphorylated but in
a time course different from wildtype. The absence of SC does not appear to
delay the phosphorylation of HIS2AV as in the okr and spnB
mutants, and the smaller number of foci in c(3)G mutants probably
reflects a reduction in DSB formation. This conclusion also makes it likely
that, in wildtype, the appearance of foci after SC formation reflects when
DSBs are induced.
The failure to repair DSBs does not induce sterility in a weak allele
of okr
Mutations in DSB repair genes like okr and spnB confer
sterility as a consequence of a checkpoint-like mechanism that is triggered by
the presence of unrepaired DSBs (Ghabrial
and Schupbach, 1999). Surprisingly, c(3)G mutants
suppress okr mutants (Ghabrial
and Schupbach, 1999
), despite our evidence for DSBs in the
okrWS; c(3)G68 double mutant. One explanation
for this result is that the low number of unrepaired DSBs (implied from the
low number of observed foci) is not sufficient to activate the checkpoint
mechanism. We tested this hypothesis by examining
-HIS2AV staining in a
hypomorphic allele of okr, okrZ0682, which causes a
reduction in meiotic crossing over (to 30% of wildtype) but not the sterility
associated with amorphic okr alleles (D.S. and K.M., unpublished). In
all okrZ0682 late pachytene (e.g. region 3) and
vitellarium oocytes that were examined (n=15 ovarioles),
-HIS2AV foci were present (Fig.
3H). The presence of staining indicated that there were unrepaired
DSBs in late pachytene oocytes but their presence was not sufficient to cause
sterility. There were, however, obviously fewer
-HIS2AV foci in region
3 okrZ0682 oocytes than in okrWS or
spnBBU mutants (compare
Fig. 3D and 3H). Because
okrZ0682 is a hypomorph, the reduced number of foci
relative to a null allele may indicate that a significant number of breaks are
repaired before region 3. We suggest that okrZ0682 females
are fertile and c(3)G mutants suppress strong okr mutants
for the same reason. In both cases there is a low number of unrepaired DSBs
and these are not sufficient to trigger the cell-cycle response that leads to
sterility.
Deleting the HIS2AV phosphorylation site has negligible effects on
meiotic recombination
We performed genetic tests on the His2AvCT;
His2Av810 females to investigate the consequences of failing
to phosphorylate HIS2AV following meiotic DSB formation. The frequency of
X-chromosome nondisjunction was not significantly elevated relative to
wildtype (0.2%, n=954). Elevated levels of X-chromosome
nondisjunction correlate with a decrease in meiotic crossing over
(Hawley, 1988
); therefore,
these results suggest that there are no defects in repairing meiotic DSBs as
crossovers. This was confirmed by direct analysis of crossing over: the
frequency of 2nd chromosome crossing over in His2Av
CT;
His2Av810 females within the al dp
(12.4%) and dp b (24.3%, n=420) intervals
was similar to controls (al dp=13.5%; dp
b=25.5%). The results of these two experiments indicate that the lack of
-HIS2AV is not a serious detriment to meiotic DSB repair.
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Discussion |
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The rapid onset of the phosphorylation makes -HIS2AV a useful
cytological marker for DSB formation
(Madigan et al., 2002
).
However, there are two important caveats to interpreting the cytological data.
First, the rate at which HIS2AV is phosphorylated during Drosophila
meiosis is not known. We also expect mutations that directly or indirectly
affect the activity of relevant kinases such as the Drosophila ATM
homolog (Burma et al., 2001
)
would prevent
-HIS2AV from appearing, even though DSBs are present.
Second, in meiotic cells we do not know that the removal of phosphorylation
always corresponds to repair of the break. However, we have no reason to
suspect that the relationship between phosphorylation and the presence of a
DSB is different than mitotic cells. Furthermore, phosphorylation and
dephosphorylation of H2AX appears to be rapid in mouse meiotic cells
(Mahadevaiah et al., 2001
).
Thus, we favor the simplest interpretation of the cytological data; the
appearance of
-HIS2AV closely reflects the presence or absence of
DSBs.
In wild-type Drosophila females, the number of foci observed in
the pro-oocytes is probably less than the total number of DSBs because of
asynchrony in either DSB formation or phosphorylation. In the DSB
repair-defective mutants, however, the total number of foci could be close to
the number of DSBs if most or all of the breaks are not repaired. In fact, the
average number of foci in a okr null mutant (21.1) is close to the
number of initiation events predicted from genetic data. Chovnick and
colleagues estimated that only 20% of gene conversion events at ry
are associated with a crossover (Hilliker
et al., 1988). Taking into account that the rosy locus is
in a relatively crossover-depressed region of the genome
(McKim et al., 2002
), we
predict from the genetic data that there are three or four initiation events
per chromosome arm or 15-20 per nucleus.
Does DSB formation occur after SC formation?
Genetic studies in Drosophila showed that meiotic recombination is
not required for SC formation (McKim et
al., 1998), but did not rule out the possibility that the two
events are independent.
-HIS2AV foci are absent at zygotene (short
segments of C(3)G in the oocyte) but are present at early pachytene (once
C(3)G accumulates into filaments), supporting the model that, in wildtype, DSB
formation does not occur until after SC formation. This is the same
relationship between the SC and early RNs in Drosophila
(Carpenter, 1979
). We have
not, however, ruled out the possibility that DSBs are initiated earlier but
phosphorylation occurs after SC formation. This is an unlikely scenario for
two reasons. First, SC formation, or C(3)G accumulation in particular, appears
to stimulate DSB formation (see below). Second, while the absence of SC
reduces the number of foci, it does not prevent or delay HIS2AV
phosphorylation. A direct comparison to our approach of using a phosphorylated
histone to monitor DSB formation is a similar study of male mouse meiosis
(Mahadevaiah et al., 2001
). In
contrast to our results, but like S. cerevisiae, mouse meiotic DSB
formation occurs before and is required for SC formation
(Baudat et al., 2000
;
Roeder, 1997
;
Romanienko and Camerini-Otero,
2000
).
The observation of -HIS2AV foci in c(3)G mutant females is
the first evidence for SC-independent DSB formation in Drosophila.
This result was unexpected because gene conversion
(Carlson, 1972
) and crossing
over (Hall, 1972
) have been
reported to be virtually eliminated in c(3)G mutants. However, the
data from the gene conversion study were also consistent with a reduction in
DSB frequency, which is consistent with our observation that the number of
-HIS2AV foci is reduced in c(3)G mutant females (10-25% of
wildtype). Therefore, the presence of SC probably has a significant
stimulatory role in DSB formation. The absence of crossing over, despite our
evidence that DSBs form in c(3)G mutants, can also be explained;
genetic evidence suggests that c(3)G is required for repairing DSBs
as crossovers (Roberts, 1969
)
(R.B. and K.M., unpublished). Thus, even if DSBs are present in c(3)G
mutants (either endogenous or from X-irradiation), they cannot be repaired as
crossovers. Surprisingly, the dependence of meiotic DSB formation and crossing
over on a central element component is not a conserved feature in all
organisms. In mutants of the S. cerevisiae c(3)G homolog
zip1, DSB formation occurs at a normal frequency and the production
of crossovers is only mildly reduced (Sym
et al., 1993
; Xu et al.,
1997
). The effects of c(3)G mutants on DSB formation are
more reminiscent of mutations in the S. cerevisiae axial element
components hop1 and red1, where the frequency of DSB
formation is reduced to 10-12% and 25-47%, respectively, of wild-type levels
(Schwacha and Kleckner, 1997
;
Woltering et al., 2000
;
Xu et al., 1997
).
DSB repair is delayed, but not eliminated, in the absence of
spnB and okr
The simplest hypothesis to explain the disappearance of -HIS2AV foci
in wild-type oocytes before mid-pachytene is that a crucial step in DSB repair
has been completed. The presence of
-HIS2AV foci at a later stage of
prophase in okr and spnB mutants, in which DSB repair is
defective, supports the conclusion that the disappearance of the foci
corresponds to a step in DSB repair. Similar to the conclusions drawn from the
physical studies of DSB sites in S. cerevisiae rad51 and
dmc1 mutants (Bishop et al.,
1992
; Shinohara et al.,
1992
), DSBs probably persist longer in okr and
spnB mutants. The
-HIS2AV foci in spnB and
okr mutants disappear at a consistent developmental time point,
vitellarium stage 4, just before dissolution of the SC. One explanation for
the late disappearance of foci is that an alternative mechanism to repair the
DSBs in spnB and okr mutants becomes available late in
meiotic prophase. One reason we favor this explanation is that the high
fertility of okrZ0682 mutants, which also have
-HIS2AV foci that persist until stage 4 oocytes, can only be explained
if all of the DSBs are repaired. Furthermore, a similar observation was made
in S. cerevisiae rad51 mutants, where the repair of DSBs was delayed
but did eventually occur (Shinohara et
al., 1992
). Nonetheless, we have not proven that the disappearance
of foci in spnB and okr mutants corresponds precisely to the
repair of DSBs. Thus, another, more complex, explanation for these
observations is that the HIS2AV phosphorylation is removed even though the DSB
has not been repaired.
Phosphorylation of H2AX in the mouse is one of the earliest responses to
DSB formation in the mouse, and has been observed before accumulation of the
DSB repair proteins Rad50, Rad51 and Dmc1
(Mahadevaiah et al., 2001;
Paull et al., 2000
). However,
the relationship between the phosphorylation of H2A variants and the
recruitment of DSB repair proteins may be complex. H2AX is required for Brca1,
Nbs1 and 53BP1 (Bassing et al.,
2002
; Celeste et al.,
2002
; Fernandez-Capetillo et
al., 2002
) but not Rad51 foci formation
(Celeste et al., 2002
). Our
observation that the onset of
-HIS2AV foci is delayed in okr
and spnB mutants suggests that phosphorylation of Drosophila
HIS2AV is influenced by some DNA repair proteins. We have not, however, ruled
out the possibility that okr and spnB mutants have a delay
in the DSB formation, although such an activity for these proteins has not
previously been described.
The abnormal development of the oocyte in spnB and okr
mutants is proposed to be caused by a checkpoint response to unrepaired DSBs;
there is a failure to translate gurken with subsequent defects in
dorsal-ventral (D-V) polarity of the oocyte
(Ghabrial and Schupbach,
1999). Our
-HIS2AV experiments revealed this DSB repair
defect in spnB and okr mutants. However, the
-HIS2AV
foci disappear in spnB and okr mutants before the D-V
phenotype is visible. If indeed the disappearance of foci corresponds to DSB
repair, cell cycle events in the germarium or early in the vitellarium, such
as a checkpoint response, may not have developmental effects until later in
oogenesis. Interestingly, Abdu et al. (Abdu
et al., 2002
) suggested that the cell cycle and developmental
effects reflect a divergence in the checkpoint pathway. It is possible that
one pathway regulates the DNA repair functions, allowing repair by a
relatively early stage (vitellarium stage 4), whereas the second pathway has
effects on oocyte patterning later in oocyte development.
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Acknowledgments |
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Footnotes |
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References |
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