1 Department of Physiology, University of Maryland School of Medicine, Baltimore
21201, USA
2 Genetics of Development and Disease Branch, 10/9N105, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892, USA
3 Laboratory of Biosystems and Cancer, 37/5016, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, MD 20892,
USA
4 Department of Biology, York University, Toronto, Ontario, M3J 1P3,
Canada
5 Department of Oncology, Lombardi Cancer Center, Georgetown University Medical
Center, Washington, DC 20007, USA
* Author for correspondence (e-mail: xiaolingx{at}intra.niddk.nih.gov)
Accepted 24 January 2003
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SUMMARY |
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Key words: BRCA1, MLH1, H2AX, Crossing-over, Apoptosis
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INTRODUCTION |
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Germline mutations of BRCA1 are found in approximately half of
familial breast cancer cases and the majority of kindreds with combined
familial breast and ovary cancer (Alberg
and Helzlsouer, 1997; Brody and
Biesecker, 1998
; Paterson,
1998
). BRCA1 contains 24 exons that encode proteins of
1863 and 1812 amino acids in human and mouse, respectively
(Lane et al., 1995
;
Miki et al., 1994
). It has
been shown that BRCA1 contains multiple functional domains that interact with
numerous molecules including products of tumor suppressor genes, oncogenes,
DNA damage-repair proteins, cell-cycle regulators, ubiquitin hydrolases, and
transcriptional activators and repressors (reviewed by
Deng and Brodie, 2000
). These
observations indicate that BRCA1 plays important roles in multiple biological
processes and pathways. Mutations of mouse Brca1 using various
gene-targeting constructs that introduce null, hypomorphic and tissue-specific
mutations result in embryonic lethality, cellular growth defects, increased
apoptosis, premature aging and/or tumorigenesis
(Bachelier et al., 2003
;
Cao et al., 2003
;
Gowen et al., 1996
;
Hakem et al., 1996
;
Hohenstein et al., 2001
;
Liu et al., 1996
;
Ludwig et al., 1997
;
Ludwig et al., 2001
;
Shen et al., 1998
;
Xu et al., 1999a
;
Xu et al., 1999b
;
Xu et al., 2001
).
Although pleiotropic effects are associated with Brca1 mutations,
it is generally believed that a major function of BRCA1 is to maintain genome
integrity. For example, tumorigenesis following loss of BRCA1 function is a
consequence of genome instability (Deng,
2001). Brca1-mutant embryos and embryonic fibroblast
cells display hypersensitivity to
-irradiation, demonstrating
chromosome structural and numeric alterations, loss of G2/M cell-cycle
checkpoint and centrosome amplification
(Aprelikova et al., 2001
;
Shen et al., 1998
;
Xu et al., 1999a
). Studies of
somatic cell lines carrying mutations in either human BRCA1 or mouse
Brca1 reveal essential functions of this gene in multiple DNA
damage-repair pathways. This includes nuclear excision repair
(Hartman and Ford, 2002
),
transcription-coupled repair of oxidative DNA damage
(Abbott et al., 1999
;
Gowen et al., 1998
),
homologous recombinational repair
(Moynahan et al., 1999
;
Moynahan et al., 2001
;
Snouwaert et al., 1999
) and
nonhomologous end-joining (Baldeyron et
al., 2002
; Zhong et al.,
2002a
; Zhong et al.,
2002b
). In conclusion, genetic instability associated with BRCA1
deficiency is caused, at least in part, by impaired DNA-damage repair.
Significantly, mice homozygous for a hypomorphic mutation of Brca1
survive to adulthood in a p53 (Trp53 Mouse Genome
Informatics) null background at low frequency
(Cressman et al., 1999).
However, the two Brca1 and p53 double homozygous mutant
(Brca1-/-p53-/-) male mice examined for this
report were infertile because of azoospermia. Moreover, the number of
apoptotic cells in the testes was increased, indicating that the germ cell
death observed in Brca1-/-p53-/- mice was
p53-independent. However, the reason why Brca1 deficiency causes failure of
spermatogenesis was not examined because of the difficulty in producing
Brca1-/-p53-/- mice.
The Brca1 gene is expressed in meiotic germ cells. Expression
levels increase in the late pachytene and diplotene stages of meiosis I
(Zabludoff et al., 1996). We
hypothesized that the defect in spermatogenesis observed in Brca1
mutant mice was secondary to inadequate DNA repair during meiosis. A mouse
model that carried an in-frame deletion of Brca1 exon 11
(Brca1
11/
11) plus functional deletions of
either one or both p53 alleles was utilized to test this hypothesis.
Although Brca1
11/
11 mice carrying two wild
type p53 alleles die in late gestation, they can survive to adulthood
if one or both p53 alleles
(Brca1
11/
11p53+/- or
Brca1
11/
11p53-/-) are mutated
(Xu et al., 2001
). In this
study, these mice were used to demonstrate that deletion of Brca1
exon 11 disrupts spermatogenesis but not oogenesis. Disruption of
spermatogenesis was accompanied by both p53-independent and p53-dependent
apoptosis, associated with altered RNA expression levels of a number of genes
that are involved in DNA-damage repair and mislocalization of Rad51 and Mlh1.
These defects are accompanied by prolonged localization of
H2AX, a DNA
damage sensor, in nuclear foci, suggesting that the meiotic chromosomes from
Brca1 mutant mice accumulate unrepaired DNA damage.
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MATERIALS AND METHODS |
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Preparation of chromosome spreads for immunofluorescence
Chromosome spreads were prepared from testes of P10-28 males and ovaries of
embryonic day (E) 16.5-17.5 female embryos as described
(Romanienko and Camerini-Otero,
2000). The slides bearing chromosome spreads were either used
fresh or within two months of freezing at -20°C (slides thawed at room
temperature for
5 minutes). After washing with phosphate-buffered saline
(PBS) three times for 5 minutes each, slides were processed for
immunofluorescent staining using standard procedures. The first antibodies
used were: RAD51 (SC-8349, Santa Cruz Biotechnology, Inc. Santa Cruz, CA at
1:100 dilution), DMC1 (SC-8973, Santa Cruz Biotechnology at 1:100 dilution),
MLH1 (a mouse monoclonal antibody from Pharmingen, San Diego, CA at 1:100
dilution), rabbit anti-phospho-histone H2AX (a mouse monoclonal antibody from
Upstate Biotechnology, Lake Placid, NY at 1:500 dilution) and rabbit anti-SCP3
antibody (Dobson et al.,
1994
). Alex-red 560 and green 488-conjugated secondary antibodies
(Molecular Probes, Eugene, OR) were used at 1:500 dilution.
Western blotting
Western blots were carried out using standard procedures using the
following antibodies: goat anti-DMC1 at 1:1000 dilution (Santa Cruz
Biotechnology); rabbit anti-RAD51 at 1:1000 dilution (Santa Cruz
Biotechnology); mouse anti-MLH1 at 1:1000 dilution; and mouse anti-Pms2 at
1:1000 dilution (Pharmingen, San Diego, CA).
Semiquantitative RT-PCR
Total RNA was extracted from testes at different developmental stages,
including P14, P16, P21 and P22. Reverse-transcription reactions were carried
out using a 1st strand cDNA-synthesis kit (Roche, Indianapolis, IN). The cDNA
samples were stored at -20°C. One µg of RNA from each sample was used
as template for each reaction and 1 µl of cDNA from each sample was used
for PCR reaction. The optimal number of cycles for amplification was
determined according to the cycle number that yielded the strongest band in
the linear range. The ranges of cycles varied from 25-28, depending on the
specific RNA target and primer set. The samples were heated to 94°C for 2
minutes and then run through 25-28 cycles of 94°C for 30 s, 60°C for
30 s and 72°C for 1 minute, followed by 72°C for 10 minutes and then
4°C. Primers used in this study and the DNA lengths amplified are shown
below.
TUNEL assay and BrdU-incorporation assay
Testis sections were analyzed for apoptosis using the ApoTag kit as
recommended by the manufacturer (Intergen Company, Purchase, NY). To evaluate
cellular proliferation rates, BrdU incorporation was measured using a cellular
proliferation kit following the manufacturer's directions (Amersham
Bioscience, England).
-Irradiation
Mice at 1 month of age were irradiated at 80 Gy (1Gy minutes-1,
Gammacell 40) and were euthanized 10 minutes, 30 minutes, 2 hours and 4 hours
after irradiation. Testes were removed and processed for chromosome spreads
and immunofluorescence staining as described above.
Statistical analyses
Student's T test was used to compare differences of pachytene and
diplotene spermatocytes, rates of apoptosis, numbers of foci for Mlh1 and
Rad51, and numbers of sex bodies for H2AX between Brca1 mutant
and control mice at ages specified in the text. P
0.05 was
considered statistically significant.
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RESULTS |
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Testes of Brca1 mutant mice exhibit increased rates of
apoptosis
The presence of prominent dark Hematoxylin-stained spermatocyte nuclei in
the testes of Brca1 mutant mice
(Fig. 1K) suggested that the
Brca1 mutation caused increased rates of spermatocyte apoptosis.
Tissue sections from testes of
Brca111/
11p53+/- and
p53+/- control mice at P10, P16 and P21 were analyzed by
TUNEL assay to quantify these differences. At P10, <2% of seminiferous
tubules in control and mutant testes contained apoptotic cells. This did not
change in testes from control mice at P16 and P21. However, in Brca1
mutant mice the number of seminiferous tubules that contained apoptotic
spermatocytes increased significantly at P16 and P21 (22% and 30%,
respectively) (Fig. 3A). To
directly compare rates of cellular apoptosis, the percentage of apoptotic
cells were determined in TUNEL-positive seminiferous tubules that were
selected randomly from testes of ten control and ten Brca1 mutant
mice. At P10, in testes from both control and Brca1 mutant mice fewer
than 4% of cells were apoptotic (Fig.
3B). At P16 and P21 the percentage of apoptotic cells did not
change in testes from control mice, but increased significantly to 40% at P16
and 50% at P21 (both P
0.0001 compared to control mice) in testes
from Brca1 mutant mice (Fig.
3B). Thus, the premature termination of spermatogenesis at the
pachytene stage of meiosis I in Brca1 mutant mice was accompanied by
significantly increased rates of apoptosis.
|
The majority of homologous chromosomes from the testes of
Brca1 mutant mice are able to synapse but do not form
crossing-overs
To determine whether or not the homologous synapsis process was impaired by
Brca1 mutation, testicular chromosome spreads were stained with an
antibody to synaptonemal complex protein 3 (SCP3). SCP3 is one of the
components of the axial and lateral element of the synaptonemal complex. It
appears in leptotene stage spermatocytes and disappears in late meiotic cells
(Dobson et al., 1994).
Analysis of >2000 spermatocytes from
Brca1
11/
11p53+/- and control mice
at P10-P21 demonstrated that the chromosomes from spermatocytes of
Brca1 mutant mice behaved normally until the pachytene stage
(Fig. 4A-D). Only 3% of the
cells showed delayed pairing of one or two chromosomes in Brca1
mutant mice (data not shown). Chromosomal spreads prepared from P16
Brca1 mutant and control mice demonstrated that approximately one
third of spermatocytes in both strains completed synapses of homologous
chromosomes (Fig. 4G). These
observations were consistent with our earlier histological findings
demonstrating that mutant spermatocytes developed relatively normally until
the pachytene stage.
|
Absence of the Brca1 full-length isoform diminishes Mlh1 foci on
chromosomes
Many DNA damage-repair proteins, including MLH1, RAD51 and DMC1, are
involved in the crossing-over process. To investigate the molecular mechanisms
underlying the failure of crossing-over formation in spermatocytes from
Brca1 mutant mice, the expression patterns and sites of cellular
localization of Mlh1, Rad51 and Dmc1 were determined.
In control mice, Mlh1 first appeared as distinct spots along the
chromosomes of late zygotene spermatocytes and was maintained until the
mid-pachytene stage in spermatocytes (Fig.
5A, n=267). By contrast, no Mlh1 foci were observed on
the majority (156 out of 162) of chromosome spreads prepared from late
zygotene to mid-pachytene stage spermatocytes of Brca1 mutant mice.
The remaining six spermatocytes showed very weak staining
(Fig. 5B). Western blotting was
performed to determine whether the abnormal distribution of Mlh1 on the
chromosomes from Brca1 mutant mice was due to reduced protein
concentrations of either Mlh1 or its binding partner Pms2. There were
no obvious differences in the concentrations of these proteins between
Brca1 mutant and control mice at any of the stages examined
(Fig. 5D). In conclusion,
although the distribution of Mlh1 on chromosomes was abnormal in
Brca1 mutant mice, overall expression levels were normal. Because
Brca1 mutant females were fertile, localization of Mlh1 was also
checked in female mice. Nearly all of the chromosomes isolated from 200 late
zygotene to mid-pachytene stage oocytes taken from E16.5-17.5
Brca111/
11p53+/- female embryos
had Mlh1 foci. There were no obvious differences in either the localization
pattern or intensity of the foci between Brca1 mutant
(Fig. 5C) and control (not
shown) oocytes.
|
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|
Steady-state RNA levels of RuvB-like DNA helicase, XPB, p62 and TFIID
are reduced in Brca1 mutant spermatocytes
Unwinding of parental DNA strands is a prerequisite for DNA replication,
repair and recombination. The unwinding of duplex DNA is catalyzed by DNA
helicases, which destabilize the hydrogen bounds between complementary double
strands of DNA (Kanemaki et al.,
1999). The absence of crossing-over in
Brca1
11/
11 p53+/- mutant
spermatocytes prompted us to measure the expression of genes that encode
proteins with DNA helicase, branch migration and basic transcription activity.
RNA samples from P14, P16, P21 and P22 were prepared from
p53+/- control and Brca1
11/
11
p53+/- mutant mice. An appropriately sized RT-PCR product for
p47, which encodes a RuvB-like DNA helicase, was detected in testes from both
control and Brca1 mutant mice. In RNA isolated from control
testicular tissue, the RNA encoding p47 began to increase at P16 and was
maintained at high levels through P22 (Fig.
8A). By contrast, the concentration of p47 RNA did not increase in
testicular tissue from Brca1 mutant animals
(Fig. 8A). The absence of an
increase in p47 RNA during spermatogenesis in the absence of full-length Brca1
is consistent with the possibility that Brca1 could be a positive regulator of
p47 expression during spermatogenesis.
|
Because defective spermatogenesis began at P16 in Brca1 mutant animals, one possible reason for the lack of increased expression of these genes in Brca1 mutant mice could be the lack of spermatocytes at later ages (such as diplonema). If this were the case, lower expression levels would not necessarily be associated with absence of full-length Brca1. To test if lower expression levels directly correlated with absence of full-length Brca1, gene expression was examined in Brca1 mutant embryos isolated from E9.5, E10.5 and E16.5 (Fig. 8C). These analyses demonstrated a direct correlation between the absence of full-length Brca1 and lower expression levels of p47, p62, XPB and TFIID, which is consistent with the notion that Brca1 is a positive regulator of these genes.
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DISCUSSION |
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The majority of homologous chromosomes synapsed normally at the pachytene
stage in spermatocytes isolated from Brca1 mutant mice, indicating
that Brca1 is not required for this process. However, spermatocytes
from Brca1 mutant mice were arrested at the late pachytene stage and
failed to enter the diplotene stage. This defect correlated with the absence
of Mlh1 foci on the homologous chromosomes. Because Mlh1 foci localize to
crossing-over sites on meiotic chromosomes and serve as a molecular marker for
this event (Baker et al., 1996;
Moens et al., 2002
), it
appears that full-length Brca1 is required for crossing-over. Crossing-over is
a multi-step process that is initiated by a DSB in DNA, followed by
exonucleolytic digestion, strand invasion, Holliday junction formation, branch
migration and junction resolution, and, finally, heteroduplex DNA formation
(reviewed by Cohen and Pollard,
2001
; Roeder,
1997
). It is possible that Brca1 is not required for the early
steps of crossing-over because the phenotype of Brca1-deficient spermatocytes
appeared at a later stage than those reported for SPO11-deficient
(Romanienko and Camerini-Otero,
2000
), ATM-deficient (Barlow et
al., 1998
), DMC1-deficient
(Yoshida et al., 1998
) and
MSH4-deficient (Kneitz et al.,
2000
) spermatocytes.
Our data indicate a link between BRCA1 function and the proper localization
of MLH1. Previous investigations revealed that MLH1, together with either
MSH4/MSH5/PMS2 or MSH4/MSH5/MLH3, are involved in both mismatch repair and
crossing-over (Cohen and Pollard,
2001). BRCA1 also displays an affinity for branched DNA structures
and forms protein-DNA complexes cooperatively between multiple DNA strands
without DNA sequence specificity (Paull et
al., 2001
). The results of the experiments presented here are
consistent with the possibility that BRCA1 acts to recruit MLH1 to sites of
recombination.
There are both similarities and differences between the phenotypes of
Brca1 and Mlh1 deficient spermatocytes
(Baker et al., 1996;
Eaker et al., 2002
). In both
strains, the majority of homologous chromosomes undergo synapsis. A 10-100
fold reduction in chiasma formation is found in Mlh1 mutant mice
whereas <1% of spermatocytes in Brca1 mutant mice show any sign of
chiasma formation and spermatocytes deficient in either protein have high
rates of apoptosis. However, although Brca1 mutant spermatocytes died
at the end of the pachytene stage, the majority of Mlh1 mutant
spermatocytes die during metaphase (Eaker
et al., 2002
). It is interesting to note that the effect of
Brca1 mutation on formation of Mlh1 foci is male specific because
Mlh1 foci are formed normally in oocytes.
In an attempt to further define the interaction between BRCA1 and MLH1, we
performed co-immunoprecipitation experiments between BRCA1 and MLH1 in human
spermatocytes but could not detect any direct binding between these proteins
(data not shown). Thus, the interaction between BRCA1 and MLH1 may be mediated
indirectly through other proteins. Consistent with this, a mass-spectrometry
study showed that BRCA1 and other proteins, including MSH2, MSH6, MLH1, ATM,
BLM, RAD50, MRE11, NBS and RFC, form a complex called BRCA1-assocated
genome-surveillance complex (BASC) (Wang
et al., 2000) that is postulated to be involved in the recognition
and repair of aberrant DNA structures. Wang et al.
(2000
) were able to
demonstrate binding of BRCA1 to MSH2 and MSH6, both factors that bind directly
to MHL1.
A candidate approach was used to study the localization and expression of
several genes and gene products that are involved in various aspects of
DNA-damage repair. We showed that distribution of Rad51 on chromosomes was
disrupted in testes from Brca1 mutant mice. This finding is
significant because Rad51 is known to interact with Brca1 either directly or
indirectly after treatment with DNA-damaging reagents
(Huber et al., 2001;
Scully et al., 1997
).
Therefore, the abnormal distribution of Rad51 in mutant chromosomes provides
strong supporting evidence for the hypothesis that full-length BRCA1 is
required for the repair of DNA during meiosis in spermatocytes. Notably, the
localization of Dmc1, a closely related partner of Rad51
(Masson and West, 2001
;
Tarsounas et al., 1999
), was
not altered, which indicates that loss of full-length Brca1 disrupts the
function of some but not all DNA damage-repair proteins.
Recent investigations have highlighted the interaction between H2AX
and BRCA1 and the importance of
H2AX in DNA-damage repair. After using
a laser beam to generate a DSB,
H2AX becomes phosphorylated and moves
to the site of the lesion within minutes
(Paull et al., 2000
). BRCA1
moves to the same site approximately 45 minutes later. This suggests that
H2AX serves as a DNA damage sensor and that it is responsible for
recruiting BRCA1 to sites of DNA damage. In fact, BRCA1 foci do not form on
chromosomes from mice that lack H2AX, which results in increased genetic
instability (Celeste et al.,
2002
).
At the late zygotene and early pachytene stages of normal spermatogenesis,
H2AX relocalizes from sites of DSBs on chromosomes to the XY body. This
did not occur in the absence of full-length Brca1. Instead,
H2AX
remained as multiple, distinct foci on chromosomes. Because
H2AX binds
to DNA prior to binding Brca1 (Paull et
al., 2000
), it is likely that the binding of
H2AX to DSBs
is independent of Brca1 and that the absence of full-length Brca1 did not
alter this process in spermatocytes. However, loss of full-length Brca1 did
block relocalization of
H2AX to the XY body. There are two possible
explanations for this observation. First, recruitment of Brca1 by
H2AX
may be essential for repairing DNA damage in spermatocytes. Therefore, in the
absence of full-length Brca1, unrepaired DNA damage accumulates in the
spermatocytes, which traps
H2AX and prevents it from moving to the XY
bodies. Experimental support for this possibility comes from the observation
that the phenotypes of control spermatocytes that sustained extensive DNA
damage from
-irradiation and that of Brca1 mutant spermatocytes were
similar. Alternatively, Brca1 could play a role that actively recruits
H2AX to the XY body. The absence of Brca1 consequently results in the
failure of
H2AX localization to the XY body. Recently, it as been
proposed that Brca1 is involved in X-chromosome inactivation
(Ganesan et al., 2002
). Future
studies are needed to determine whether loss of localization of
H2AX to
the XY body in Brca1 mutant spermatocytes affects X-inactivation.
Although BRCA1 appears to regulate the expression of many genes that are
involved in multiple biological processes
(Chen et al., 1999;
Deng and Brodie, 2000
), only
three of these genes are known to play a role in DNA-damage repair. These are
Gadd45 (a DNA damage-repair and response gene)
(Harkin et al., 1999
),
Ddb2 (which is defective in Xeroderma Pigmentosum group E cells and
encodes the p48-damaged DNA-binding protein)
(Takimoto et al., 2002
) and
Xpc (a gene encoding Xeroderma Pigmentosum group C complementing
protein) (Hartman and Ford,
2002
). All three genes are regulated by p53 and are involved in
the nuclear excision-repair pathway
(Hartman and Ford, 2002
;
MacLachlan et al., 2002
;
Takimoto et al., 2002
). In
this study, we found that loss of full-length Brca1 correlated with decreased
expression of several genes that encode proteins involved in the repair of
DSBs, including RuvB-like DNA helicase, TFIIH (p62) and TFIID. Additional
investigations will determine if these genes are transcriptional targets of
BRCA1 and if this regulation is direct or indirect.
The p53-dependent apoptotic pathway plays a dominant role in some but not
all organs (reviewed by Burns and El-Deiry,
1999). We showed that spermatocyte apoptosis triggered by loss of
full-length Brca1 was mediated by p53-dependent and p53-independent pathways.
The most likely proximal trigger for both p53-dependent and p53-independent
apoptosis in Brca1-deficient spermatocytes is the development of genetic
instability. Thus, mutant spermatocytes that escaped the first wave of
p53-dependent apoptosis survived slightly longer but still failed to progress
into the diplotene stage. This suggests that activation of more than one
apoptotic pathway ensures that spermatogenesis cannot be completed in the
presence of significant genetic instability.
In summary, this study revealed that Brca1 plays an essential role in DNA-damage repair during spermatogenesis (Fig. 9). Brca1 functions through at least two different mechanisms, the recruitment of DNA damage-repair proteins to sites of DNA damage, and the regulation of the expression of DNA damage-repair genes. Examples of the first mechanism include the diminished numbers of Rad51 and Mlh1 foci on chromosomes in the absence of full-length Brca1. Examples of the second mechanism are the altered expression levels of RuvB-like DNA helicase, XPB, p62 and TFIID in the absence of full-length Brca1. Thus, Brca1-deficient spermatocytes unavoidably accumulate unrepaired DNA damage that triggers both p53-dependent and p53-independent apoptosis, and failure of spermatogenesis.
|
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbott, D. W., Thompson, M. E., Robinson-Benion, C., Tomlinson,
G., Jensen, R. A. and Holt, J. T. (1999). BRCA1 expression
restores radiation resistance in BRCA1-defective cancer cells through
enhancement of transcription-coupled DNA repair. J. Biol.
Chem. 274,18808
-18812.
Alberg, A. J. and Helzlsouer, K. J. (1997). Epidemiology, prevention, and early detection of breast cancer. Curr. Opin. Oncol. 9,505 -511.[Medline]
Aprelikova, O., Pace, A. J., Fang, B., Koller, B. H. and Liu, E.
T. (2001). BRCA1 is a selective co-activator of 14-3-3 sigma
gene transcription in mouse embryonic stem cells. J. Biol.
Chem. 276,25647
-25650.
Bachelier, R., Xu, X., Wang, X., Li, W., Naramura, M., Gu, H. and Deng, C. X. (2003). Normal lymphocyte development and thymic lymphoma formation in Brca1 exon 11-deficient mice. Oncogene (in press).
Baker, S. M., Plug, A. W., Prolla, T. A., Bronner, C. E., Harris, A. C., Yao, X., Christie, D. M., Monell, C., Arnheim, N., Bradley, A. et al. (1996). Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nat. Genet. 13,336 -342.[Medline]
Baldeyron, C., Jacquemin, E., Smith, J., Jacquemont, C., De Oliveira, I., Gad, S., Feunteun, J., Stoppa-Lyonnet, D. and Papadopoulo, D. (2002). A single mutated BRCA1 allele leads to impaired fidelity of double strand break end-joining. Oncogene 21,1401 -1410.[CrossRef][Medline]
Barlow, C., Liyanage, M., Moens, P. B., Tarsounas, M.,
Nagashima, K., Brown, K., Rottinghaus, S., Jackson, S. P., Tagle, D., Ried, T.
et al. (1998). Atm deficiency results in severe meiotic
disruption as early as leptonema of prophase I.
Development 125,4007
-4017.
Brody, L. C. and Biesecker, B. B. (1998). Breast cancer susceptibility genes. BRCA1 and BRCA2. Medicine (Baltimore) 77,208 -226.[CrossRef][Medline]
Burns, T. F. and El-Deiry, W. S. (1999). The p53 pathway and apoptosis. J. Cell. Physiol. 181,231 -239.[CrossRef][Medline]
Cao, L., Li, W., Kim, S., Brodie, B. G. and Deng, C. X.
(2003). Senescence, ageing and malignant transformation mediated
by p53 in mice lacking Brca1 exon 11 isoform. Genes
Dev. 17,201
-213.
Celeste, A., Petersen, S., Romanienko, P. J.,
Fernandez-Capetillo, O., Chen, H. T., Sedelnikova, O. A., Reina-San-Martin,
B., Coppola, V., Meffre, E., Difilippantonio, M. J. et al.
(2002). Genomic instability in mice lacking histone H2AX.
Science 296,922
-927.
Chen, Y., Lee, W. H. and Chew, H. K. (1999). Emerging roles of BRCA1 in transcriptional regulation and DNA repair. J. Cell. Physiol. 181,385 -392.[CrossRef][Medline]
Cohen, P. E. and Pollard, J. W. (2001). Regulation of meiotic recombination and prophase I progression in mammals. BioEssays 23,996 -1009.[CrossRef][Medline]
Cressman, V. L., Backlund, D. C., Avrutskaya, A. V., Leadon, S.
A., Godfrey, V. and Koller, B. H. (1999). Growth retardation,
DNA repair defects, and lack of spermatogenesis in BRCA1-deficient mice.
Mol. Cell. Biol. 19,7061
-7075.
Dasika, G. K., Lin, S. C., Zhao, S., Sung, P., Tomkinson, A. and Lee, E. Y. (1999). DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene 18,7883 -7899.[CrossRef][Medline]
Deng, C. X. (2001). Tumorigenesis as a consequence of genetic instability in Brca1 mutant mice. Mutat. Res. 477,183 -189.[Medline]
Deng, C. X. and Brodie, S. G. (2000). Roles of BRCA1 and its interacting proteins. BioEssays 22,728 -737.[CrossRef][Medline]
Dobson, M. J., Pearlman, R. E., Karaiskakis, A., Spyropoulos, B.
and Moens, P. B. (1994). Synaptonemal complex proteins:
occurrence, epitope mapping and chromosome disjunction. J. Cell
Sci. 107,2749
-2760.
Eaker, S., Cobb, J., Pyle, A. and Handel, M. A. (2002). Meiotic prophase abnormalities and metaphase cell death in MLH1-deficient mouse spermatocytes: insights into regulation of spermatogenic progress. Dev. Biol. 249, 85-95.[CrossRef][Medline]
Enders, G. C. and May, J. J., 2nd (1994). Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female mice. Dev. Biol. 163,331 -340.[CrossRef][Medline]
Ganesan, S., Silver, D. P., Greenberg, R. A., Avni, D., Drapkin, R., Miron, A., Mok, S. C., Randrianarison, V., Brodie, S., Salstrom, J. et al. (2002). BRCA1 Supports XIST RNA Concentration on the Inactive X Chromosome. Cell 111,393 -405.[Medline]
Gowen, L. C., Avrutskaya, A. V., Latour, A. M., Koller, B. H.
and Leadon, S. A. (1998). BRCA1 required for
transcription-coupled repair of oxidative DNA damage.
Science 281,1009
-1012.
Gowen, L. C., Johnson, B. L., Latour, A. M., Sulik, K. K. and Koller, B. H. (1996). Brca1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities. Nat. Genet. 12,191 -194.[Medline]
Hakem, R., de la Pompa, J. L., Sirard, C., Mo, R., Woo, M., Hakem, A., Wakeham, A., Potter, J., Reitmair, A., Billia, F. et al. (1996). The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. Cell 85,1009 -1023.[Medline]
Harkin, D. P., Bean, J. M., Miklos, D., Song, Y. H., Truong, V. B., Englert, C., Christians, F. C., Ellisen, L. W., Maheswaran, S., Oliner, J. D. et al. (1999). Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell 97,575 -586.[Medline]
Hartman, A. R. and Ford, J. M. (2002). BRCA1 induces DNA damage recognition factors and enhances nucleotide excision repair. Nat. Genet. 32,180 -184.[CrossRef][Medline]
Hohenstein, P., Kielman, M. F., Breukel, C., Bennett, L. M., Wiseman, R., Krimpenfort, P., Cornelisse, C., van Ommen, G. J., Devilee, P. and Fodde, R. (2001). A targeted mouse Brca1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage. Oncogene 20,2544 -2550.[CrossRef][Medline]
Huber, L. J., Yang, T. W., Sarkisian, C. J., Master, S. R.,
Deng, C. X. and Chodosh, L. A. (2001). Impaired DNA damage
response in cells expressing an exon 11-deleted murine Brca1 variant that
localizes to nuclear foci. Mol. Cell. Biol.
21,4005
-4015.
Kanemaki, M., Kurokawa, Y., Matsu-ura, T., Makino, Y., Masani,
A., Okazaki, K., Morishita, T. and Tamura, T. A. (1999).
TIP49b, a new RuvB-like DNA helicase, is included in a complex together with
another RuvB-like DNA helicase, TIP49a. J. Biol. Chem.
274,22437
-22444.
Kneitz, B., Cohen, P. E., Avdievich, E., Zhu, L., Kane, M. F.,
Hou, H., Jr, Kolodner, R. D., Kucherlapati, R., Pollard, J. W. and Edelmann,
W. (2000). MutS homolog 4 localization to meiotic chromosomes
is required for chromosome pairing during meiosis in male and female mice.
Genes Dev. 14,1085
-1097.
Lane, T. F., Deng, C., Elson, A., Lyu, M. S., Kozak, C. A. and Leder, P. (1995). Expression of Brca1 is associated with terminal differentiation of ectodermally and mesodermally derived tissues in mice. Genes Dev. 9,2712 -2722.[Abstract]
Liu, C. Y., Flesken-Nikitin, A., Li, S., Zeng, Y. and Lee, W. H. (1996). Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development. Genes Dev. 10,1835 -1843.[Abstract]
Ludwig, T., Chapman, D. L., Papaioannou, V. E. and Efstratiadis, A. (1997). Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos. Genes Dev. 11,1226 -1241.[Abstract]
Ludwig, T., Fisher, P., Ganesan, S. and Efstratiadis, A.
(2001). Tumorigenesis in mice carrying a truncating Brca1
mutation. Genes Dev. 15,1188
-1193.
MacLachlan, T. K., Takimoto, R. and El-Deiry, W. S.
(2002). BRCA1 directs a selective p53-dependent transcriptional
response towards growth arrest and DNA repair targets. Mol. Cell.
Biol. 22,4280
-4292.
Mahadevaiah, S. K., Turner, J. M., Baudat, F., Rogakou, E. P., de Boer, P., Blanco-Rodriguez, J., Jasin, M., Keeney, S., Bonner, W. M. and Burgoyne, P. S. (2001). Recombinational DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27,271 -276.[CrossRef][Medline]
Masson, J. Y. and West, S. C. (2001). The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem. Sci. 26,131 -136.[CrossRef][Medline]
Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., Harshman, K., Tavtigian, S., Liu, Q., Cochran, C., Bennett, L. M., Ding, W. et al. (1994). A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266, 66-71.[Medline]
Moens, P. B., Kolas, N. K., Tarsounas, M., Marcon, E., Cohen, P.
E. and Spyropoulos, B. (2002). The time course and
chromosomal localization of recombination-related proteins at meiosis in the
mouse are compatible with models that can resolve the early DNA-DNA
interactions without reciprocal recombination. J. Cell
Sci. 115,1611
-1622.
Moynahan, M. E., Chiu, J. W., Koller, B. H. and Jasin, M. (1999). Brca1 controls homology-directed DNA repair. Mol. Cell 4,511 -518.[Medline]
Moynahan, M. E., Cui, T. Y. and Jasin, M.
(2001). Homology-directed DNA repair, mitomycin-c resistance, and
chromosome stability is restored with correction of a Brca1 mutation.
Cancer Res. 61,4842
-4850.
Paterson, J. W. (1998). BRCA1: a review of structure and putative functions. Dis. Markers 13,261 -274.[Medline]
Paull, T. T., Cortez, D., Bowers, B., Elledge, S. J. and
Gellert, M. (2001). From the cover: direct DNA binding by
Brca1. Proc. Natl. Acad. Sci. USA
98,6086
-6091.
Paull, T. T., Rogakou, E. P., Yamazaki, V., Kirchgessner, C. U., Gellert, M. and Bonner, W. M. (2000). A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10,886 -895.[CrossRef][Medline]
Redon, C., Pilch, D., Rogakou, E., Sedelnikova, O., Newrock, K. and Bonner, W. (2002). Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12,162 -169.[CrossRef][Medline]
Roeder, G. S. (1997). Meiotic chromosomes: it
takes two to tango. Genes Dev.
11,2600
-2621.
Romanienko, P. J. and Camerini-Otero, R. D. (2000). The mouse Spo11 gene is required for meiotic chromosome synapsis. Mol. Cell 6,975 -987.[Medline]
Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and Livingston, D. M. (1997). Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 88,265 -275.[Medline]
Shen, S. X., Weaver, Z., Xu, X., Li, C., Weinstein, M., Chen, L., Guan, X. Y., Ried, T. and Deng, C. X. (1998). A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene 17,3115 -3124.[CrossRef][Medline]
Snouwaert, J. N., Gowen, L. C., Latour, A. M., Mohn, A. R., Xiao, A., DiBiase, L. and Koller, B. H. (1999). BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a brca1 transgene. Oncogene 18,7900 -7907.[CrossRef][Medline]
Takimoto, R., MacLachlan, T. K., Dicker, D. T., Niitsu, Y., Mori, T. and el-Deiry, W. S. (2002). BRCA1 transcriptionally regulates damaged DNA binding protein (DDB2) in the DNA repair response following UV-irradiation. Cancer Biol. Ther. 1, 177-186.[Medline]
Tarsounas, M., Morita, T., Pearlman, R. E. and Moens, P. B.
(1999). RAD51 and DMC1 form mixed complexes associated with mouse
meiotic chromosome cores and synaptonemal complexes. J. Cell
Biol. 147,207
-220.
Tarsounas, M. and Moens, P. B. (2001). Checkpoint and DNA-repair proteins are associated with the cores of mammalian meiotic chromosomes. Curr. Top. Dev. Biol. 51,109 -134.[Medline]
Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S. J. and
Qin, J. (2000). BASC, a super complex of BRCA1-associated
proteins involved in the recognition and repair of aberrant DNA structures.
Genes Dev. 14,927
-939.
Xu, X., Qiao, W., Linke, S. P., Cao, L., Li, W. M., Furth, P. A., Harris, C. C. and Deng, C. X. (2001). Genetic interactions between tumor suppressors Brca1 and p53 in apoptosis, cell cycle and tumorigenesis. Nat. Genet. 28,266 -271.[CrossRef][Medline]
Xu, X., Weaver, Z., Linke, S. P., Li, C., Gotay, J., Wang, X. W., Harris, C. C., Ried, T. and Deng, C. X. (1999a). Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol. Cell 3,389 -395.[Medline]
Xu, X., Wagner, K. U., Larson, D., Weaver, Z., Li, C., Ried, T., Hennighausen, L., Wynshaw-Boris, A. and Deng, C. X. (1999b). Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation. Nat. Genet. 22, 37-43.[CrossRef][Medline]
Yoshida, K., Kondoh, G., Matsuda, Y., Habu, T., Nishimune, Y. and Morita, T. (1998). The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis. Mol. Cell 1,707 -718.[Medline]
Zabludoff, S. D., Wright, W. W., Harshman, K. and Wold, B. J. (1996). BRCA1 mRNA is expressed highly during meiosis and spermiogenesis but not during mitosis of male germ cells. Oncogene 13,649 -653.[Medline]
Zhong, Q., Boyer, T. G., Chen, P. L. and Lee, W. H.
(2002a). Deficient nonhomologous end-joining activity in
cell-free extracts from Brca1-null fibroblasts. Cancer
Res. 62,3966
-3970.
Zhong, Q., Chen, C. F., Chen, P. L. and Lee, W. H.
(2002b). BRCA1 facilitates microhomology-mediated end joining of
DNA double strand breaks. J. Biol. Chem.
277,28641
-28647.
Zickler, D. and Kleckner, N. (1999). Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33,603 -754.[CrossRef][Medline]