1 Institute of Cell and Molecular Biology, University of Edinburgh, King's
Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
2 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The
Chancellor's Building, University of Edinburgh, 49 Little France Crescent,
Edinburgh, EH16 4SB, UK
3 Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine
Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU,
UK
* Present address: Faculty of Dentistry, National Yang-Ming University, Taiwan,
ROC
Author for correspondence (e-mail:
David.Melton{at}ed.ac.uk)
Accepted 23 October 2002
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SUMMARY |
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Key words: Meiosis, Nucleotide excision repair, Oxidative DNA damage, Recombination, Spermatozoa, Xeroderma pigmentosum
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INTRODUCTION |
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Unlike other NER proteins Ercc1 and Xpf are also involved in homologous
recombination, double-strand break repair and the repair of interstrand
cross-links. Their role in recombination was deduced originally from studies
on the Saccharomyces cerevisiae homologues, RAD10 (Ercc1) and RAD1
(Xpf) (Schiestl and Prakash,
1990). In mammalian cells Ercc1 is required for the correct
processing of heteroduplex intermediates formed during homologous
recombination (Adair et al.,
2000
; Sargent et al.,
2000
) and also acts to remove protruding single-stranded ends in
the single-stranded annealing pathway for homologous recombination and
double-strand break repair (Davies et al.,
1995
; Fishman-Lobell and
Haber, 1992
). In addition, Ercc1- or
Xpf-deficient mammalian cells are characteristically hypersensitive
to interstrand cross-linking agents. In a reaction distinct from NER, the
Ercc1/Xpf complex has the ability to cut adjacent to such cross-links
(Kuraoka et al., 2000a
). The
key role of the Ercc1/Xpf complex in all these repair pathways is the ability
to cleave single-stranded 3' tails projecting from DNA duplexes.
Ercc1 was the first NER gene to be inactivated in the mouse
(McWhir et al., 1993).
Subsequently Xpa (de Vries et
al., 1995
; Nakane et al.,
1995
) and Xpc (Sands
et al., 1995
) knockouts have proved to be good models for XP and
this has led to the suggestion that it is the recombination, rather than the
NER deficit that is the key to the Ercc1 knockout phenotype
(Weeda et al., 1997
). A number
of different repair genes are highly expressed in testis [e.g. Polb
(Alcivar et al., 1992
);
Lig3 (Chen et al.,
1995
); Xrcc1 (Walter
et al., 1996
)] and as part of our investigation of the importance
of the recombination repair functions of Ercc1 we decided to study
spermatogenesis and oogenesis in Ercc1-deficient mice. Functional
homologous recombination pathways are essential for the successful completion
of meiosis. In yeast, unresolved double-strand breaks are thought to trigger a
checkpoint leading to pachytene arrest and, in the mouse, the apoptotic
elimination of spermatocytes with synaptic errors occurs via a p53-independent
pathway (Odorisio et al.,
1998
). Knockouts for a number of mismatch repair genes have been
shown to lead to a specific failure of spermatogenesis at the pachytene stage,
consistent with the requirement for mismatch repair to process heteroduplex
recombination intermediates [Pms2 (Baker et al., 1950; Mlh1
(Edelmann et al., 1996
);
Msh5 (de Vries et al.,
1999
); Msh4 (Kneitz
et al., 2000
)]. If Ercc1 were essential at the same
stage, a similar phenotype would be anticipated. Support for the notion that
Ercc1 may be required for meiosis comes from the Drosophila
melanogaster homologue of Xpf, mei-9, where mutation causes
reduced meiotic recombination and increased non-disjunction as well as
defective NER (Sekelsky et al.,
1995
).
Within the testis several germ cell stages, including pachytene
spermatocytes, have been shown to produce high levels of reactive oxygen
species (Fisher and Aitken,
1997), which induce a variety of DNA lesions, with one of the most
abundant being 7,8-dihydro-8-oxoguanine (8-oxoG) (for a review, see
Lindahl, 1993
). This lesion is
strongly mutagenic and also acts as a block to transcription by RNA polymerase
II (Le Page et al., 2000a
). A
complex anti-oxidant defence system has been described in the rat testis
(Bauche et al., 1994
).
Traditionally base excision repair (BER) was considered to have the key role
in removing 8-oxoG and 8-oxoG DNA glycosylase (Ogg1) is highly expressed in
the testis (Rosenquist et al.,
1997
). However, the discovery of transcription-coupled repair of
8-oxoG and the observation that this process continues to operate in
Ogg1-null cells (Le Page et al.,
2000b
) has, belatedly, led to the recognition that NER may also
have an important role to play in the repair of 8-oxoG.
Ercc1 knockout mice die before the first wave of spermatogenesis
has been completed in control littermates, so in this study we have also used
animals where the Ercc1-deficient liver phenotype has been corrected
by an Ercc1 transgene under the control of a liver-specific promoter
(Selfridge et al., 2001). The
increased lifespan of these animals means that the consequences of
Ercc1 deficiency can now be studied in other tissues. Here we show
that Ercc1 is essential for normal spermatogenesis and oogenesis, but
that the premeiotic lesions and DNA damage observed are consistent with a
general role for the repair functions of Ercc1 throughout
gametogenesis rather than a specific requirement at meiotic crossing over.
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MATERIALS AND METHODS |
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Collection of tissues for histology
Testes and ovaries were immersion fixed, either in Bouins for 6 hours
(ovaries) or 10 hours (testes), or in 4% neutral buffered formaldehyde (NBF)
overnight at 4°C, or in Collidine overnight (testes only). Tissues fixed
in Bouins or NBF were processed into paraffin wax using a standard 16 hour
cycle. Testes fixed in Collidine were processed manually into Araldyte and
stained with 1% Toluidine Blue containing 1% borax (BDH, Poole, Dorset, UK) at
60°C until a suitable staining intensity was obtained
(Kerr et al., 1993). Sections
(5 µm) of Bouins-fixed testes were stained with Haematoxylin and Eosin, or
using the Apotag method (Sharpe et al.,
1998
). Epididymides from adult mice were minced in Biggers,
Whitten and Whittingham medium (BWW)
(Biggers et al., 1971
) and
allowed to stand for 5 minutes before recovering the supernatant, avoiding
tissue debris. Cells were resuspended at approx. 3x106
cells/ml in BWW, aliquoted and frozen at -20°C.
Antibodies
A rabbit polyclonal antibody to mitochondrial core protein II of bovine
complex III was kindly supplied by Dr Hermann Schaegger (University of
Frankfurt). An affinity-purified rabbit polyclonal antibody raised against a
peptide specific for the RNA binding protein Dazl
(Ruggiu et al., 1997;
Ruggiu et al., 2000
) was a
gift from Dr Nicola Reynolds (MRC Human Genetics Unit, Edinburgh). The
antibody to mouse Ercc1 was raised in a rabbit against a His-tagged
recombinant protein containing a central fragment (amino acids 36-175) of
mouse Ercc1 (K.-T. Hsia and D. Melton, unpublished). The antibody was
affinity-purified from crude serum using antigen immobilised on nitrocellulose
membrane (Robinson et al.,
1988
). Pilot experiments with the Ercc1 antibody found that no
immunopositive signal was obtained when Bouins-fixed wild-type testes were
used so all further experiments were carried out using testes fixed in
NBF.
Immunohistochemistry
Briefly, sections (5 µm) were mounted on slides coated with
3-aminopropyl triethoxy-silane (Sigma Chemical Co., Poole, Dorset, UK), dried
overnight (50°C), dewaxed and rehydrated. Thereafter, sections were
incubated with 3% hydrogen peroxide in methanol for 30 minutes to block
endogenous peroxidase, washed once each (5 minutes) in distilled water and TBS
(0.05 M Tris-HCl pH 7.4, 0.85% NaCl) and blocked for 30 minutes using normal
rabbit serum diluted in TBS (1:5, NSS-TBS). Sections were incubated with
primary antibody diluted in TBS (anti-Ercc1, 1 in 100; anti-Dazl, 1 in 100)
overnight at 4°C. Sections were washed twice in TBS (5 minutes each),
incubated for 30 minutes with biotinylated swine anti-rabbit immunoglobulin,
diluted 1:500 in NSS-TBS for 30 minutes, then washed again in TBS (2 times 5
minutes). Bound antibodies were detected according to standard methods
(Saunders et al., 2001).
Images were captured using an Olympus Provis microscope (Olympus Optical Co.,
London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak, London, UK)
and assembled on a Macintosh PowerPC computer using Photoshop 6 (Adobe).
Comet assay on sperm DNA
The alkaline single cell gel electrophoresis (Comet) assay was performed
using the CometAssayTM Kit from R&D Systems (Abingdon, Oxon., UK),
adapted as follows. Cells were defrosted at room temperature and 5 µl of
cells at 3x106/ml were mixed with 25 µl low melt
agarose (37°C). This cell suspension was then dropped into one of the
wells of a CometSlide (R&D Systems), immediately covered and allowed to
set at 4°C. The coverslips were removed and the cells lysed in 0.75% (w/v)
SDS (Sigma, Poole, Dorset, UK) and 1% (v/v) DMSO (Sigma) in lysis solution
supplied with the kit for 30 minutes at 37°C. The lysis solution was
replaced with alkali solution [0.3 M NaOH, 1 mM EDTA (Sigma) and 20 µg/ml
Proteinase K (Amresco-Anachem Ltd., Luton, Bedfordshire, UK)] for 30 minutes
at 4°C to denature the DNA. Slides were placed in a horizontal
electrophoresis tank filled with alkaline electrophoresis buffer (300 mM NaOH,
1 mM EDTA, pH 12.3), left for 20 minutes, electrophoresed for 10 minutes at 25
V (300 mA) and dehydrated in ice-cold methanol (100% - 5 minutes), then
ethanol (100% - 5 minutes) and allowed to air-dry overnight at room
temperature. The slides were then stained using 20 µl per well of ethidium
bromide (15 µg/ml; Sigma) and covered. Slides were viewed using a Leitz
DMRB microscope fitted with a N2.1 filter block, providing an excitation
filter of 515-560 nm from a 50 W mercury lamp and a barrier filter of 580 nm.
Cells were analysed using Komet 4 software (Kinetic Imaging Ltd., Liverpool,
UK).
HPLC analysis of testis DNA
HPLC of DNA from fresh and frozen testis samples was performed as described
(Selfridge et al., 2001).
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RESULTS |
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In mice, the first wave of spermatogenesis occurs synchronously during
testis maturation with spermatocytes at the leptotene stage of prophase I
detectable on about day 10 (Bellve et al.,
1977). To see if the high levels of Ercc1 expression were
associated with meiotic crossing over, RNA and protein samples from testes of
wild-type mice (p8-42) were subjected to northern
(Fig. 1B) and western analysis
(Fig. 1C). The levels of
Ercc1 mRNA increased with age, but when the filter was reprobed for
actin mRNA, the Ercc1/actin ratio determined by phosphorimagery was
essentially constant from p8-35 and only increased slightly at p42. The levels
of Ercc1 protein were more variable, perhaps because they were determined by
densitometry rather than phosphorimagery. The Ercc1/mitochondrial core protein
II ratios shown in Fig. 1C are
the means of two separate determinations on two independent samples. Ercc1
protein was also present from the earliest stages examined with an increase in
the p23 and p35 samples. This increase could reflect expression in round
spermatids detected by immunohistochemistry (see later section on
Ercc1 expression in male germ cells), as they would be present at
these times. However, there was no suggestion at the mRNA or protein level
that Ercc1 expression was linked with the first wave of meiotic
crossing over.
Ercc1 was absent from the testes and all other tissues of
Ercc1-deficient mice examined (data not shown). The Ercc1
transgene used to correct the liver phenotype is under the control of the
transthyretin (Ttr) gene promoter
(Selfridge et al., 2001). The
endogenous Ttr gene is expressed strongly in the liver, but also in
the choroid plexus of the brain. Northern analysis of transgene-containing
Ercc1 nulls with a Ttr probe revealed the liver-specific
pattern of transgene expression, with no transgene transcripts detectable in
other tissues (Fig. 1D).
However, low levels of Ercc1 protein were detected in the testes and brain of
some transgene-containing Ercc1 nulls analysed, including the one
shown in Fig. 1D. The level of
Ercc1 protein in the testes of these animals was variable, but was always
<10% of the wild-type level.
Infertility in Ercc1-deficient mice
Ercc1 nulls die by 3 weeks of age, but it was possible to assess
the fertility of transgene-positive Ercc1 nulls, which live for up to
12 weeks (Selfridge et al.,
2001). Although copulation plugs were observed, no pregnancies
were ever detected from matings between male or female transgene-positive
Ercc1 nulls and wild-type partners and we conclude that
transgene-positive Ercc1 nulls of both sexes are infertile.
Individual testis weights, expressed as a percentage of body weight, from 3-
and 6-week transgene-positive Ercc1 nulls were compared with
wild-type littermates (see Fig.
2 for 6-week data). This correction was made because body weights
of transgene-positive Ercc1 nulls are, on average, only 60% of wild
type (Selfridge et al., 2001
).
Even correcting for body weight, mean testis weights from transgene-positive
Ercc1 nulls were only 50% of controls (3-week: wild type
0.27±0.07(s.d.)%, transgene-positive Ercc1 null
0.16±0.03%, P=2.8x10-5 by Student's
t-test. 6-week: wild type 0.44±0.08%, transgene-positive
Ercc1 null 0.19±0.05%,
P=4.9x10-9).
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Morphological appearance of testes from Ercc1-null mice
The germ cell complement of testes from wild-type and Ercc1 nulls
were examined using immunohistochemistry for the germ cell-specific protein
Dazl (Ruggiu et al., 1997). On
days 3, 7 (not shown), 10 (not shown), 12 and 22, mutant animals contained
less germ cells within the seminiferous epithelium than their wild-type
littermates (Fig. 3, compare A, C, and E,
with B, D, and F, respectively). The number of germ cells in
individual tubules in the nulls was highly variable, even within the same
testis, when compared to the uniform appearance of tubules from control
littermates. Tubules devoid of germ cells (Sertoli cell only, SCO;
Fig. 3 asterisk) and germ cells
with abnormal morphology (arrows) were seen at all ages in the
Ercc1-null testes. In 22-day old wild-type mice the seminiferous
epithelium was well developed (Fig.
3E), a lumen had formed and, in agreement with published data
(Bellve et al., 1977
), the most
advanced germ cell stage was an early round spermatid. In age-matched
Ercc1-null littermates (Fig.
3F) germ cells were limited to spermatogonia and a very few
pachytene spermatocytes while the majority of tubules appeared to be SCO.
Formation of tubule lumens was either incomplete, or absent and the Sertoli
cell cytoplasm occupied the centre of the tubules.
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Morphological appearance of testes from transgene-positive
Ercc1-null mice
The introduction of the Ercc1 transgene onto the null background
resulted in partial restoration of the early wave of spermatogenesis and germ
cells up to and including pachytene spermatocytes were observed in 3-week-old
animals (Fig. 4B, labelled P).
Although some tubules appeared to be SCO (asterisks), tubule lumens had begun
to form. At 6-7 weeks of age the testes of wild-type mice contained a full
complement of germ cells with spermatozoa ready for release into the tubule
lumens (Fig. 4C, labelled s).
In age-matched transgene-positive Ercc1 nulls the testicular
phenotype was variable, both between animals and within the testes of
individual males. Although, in every case the number of germ cells within the
seminiferous tubules was substantially reduced
(Fig. 4D) and SCO tubules were
observed (asterisk), all stages of germ cell development from spermatocytes
(arrowheads) to mature elongated spermatids () were detected. In tubules
containing a substantial population of germ cells `gaps' within the epithelium
(arrows) were often observed. Critically, the loss of germ cells within any
individual tubule did not appear to be confined to a single stage of
development and the net result was a more disordered arrangement of germ cells
than seen in normal spermatogenesis
(Oakberg, 1956
).
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The reduction in the diameter of the seminiferous tubules (around 50%) and in the height of the seminiferous epithelium in transgene-positive Ercc1 nulls compared with controls was most evident when tissue embedded in Araldyte was examined (Fig. 4E compared with F, double-headed arrows). Toluidine Blue staining revealed occasional lipid droplets close to the basement membrane (Fig. 4E, arrows) of 10-week controls. In the 10-week transgene-positive Ercc1 nulls (Fig. 4F) there was an accumulation of numerous lipid droplets in the cytoplasm of Sertoli cells (arrows) and interstitial Leydig cells (L). The lipid in the Sertoli cells is consistent with accumulation of waste products following phagocytosis of germ cell remnants.
Apoptosis within testes of Ercc1-deficient mice
Germ cells with damaged DNA or aberrant meioses are eliminated by apoptosis
(for a review, see Baarends et al.,
2001). Testicular sections were stained using the Apotag method
that has been used previously on fixed testis sections
(Sharpe et al., 1998
). In
7-week wild-type mice immunopositive germ cells were detected at stages XI and
XII (Fig. 5A, arrows). These
cells were infrequent and located close to the basement membrane, consistent
with previous reports that most cells undergoing apoptosis in the normal
testis are mitotically active A type spermatogonia
(Krishnamurthy et al., 1998
).
In age-matched transgene-positive Ercc1 null littermates, although
considerably fewer germ cells were present than in controls, the
immunopositive staining was sometimes associated with groups of germ cells
(Fig. 5B). We did not detect
elevated levels of apoptotic cells in Ercc1 nulls between days 7 and
22, consistent with rapid elimination of cells containing abnormal DNA (not
shown).
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Ercc1 expression in male germ cells
Northern analysis on extracts from whole testes had not indicated a clear
association between Ercc1 expression and a particular stage in
spermatogenesis. To investigate Ercc1 expression in individual germ
cells, immunohistochemistry was undertaken on testis sections from 9-week-old
animals using an affinity-purified Ercc1-specific antiserum. In wild-type
males Ercc1 immunopositive staining was barely detectable in Sertoli and
Leydig cells and, while some immunopositive reaction was present in all germ
cells, the most intense immunoreaction was in germ cells from late pachytene
spermatocytes (stage IX) to round spermatids (stage VIII)
(Fig. 5C). These data,
summarised on a diagram of the stages of the spermatogenic cycle in
Fig. 5E, are consistent with
the increased levels of Ercc1 protein detected in p23 and p35 testes by
western blotting (Fig. 1C).
Some non-specific staining of sperm tails was seen in all samples and, in
age-matched transgene-positive Ercc1-null littermates very faint
immunopositive staining was observed in some germ cells
(Fig. 5D, arrows), consistent
with the low levels of transgene-encoded Ercc1 expression detected on
western blots (Fig. 1D). No
immunopositive staining was seen in 3-week-old Ercc1 nulls (data not
shown).
Analysis of spermatozoa from Ercc1-deficient mice
Consistent with the deficit in spermatogenesis observed, the number of
sperm recovered from the epididymides of 7-week-old transgene-positive
Ercc1 nulls was very limited. While sperm from age-matched controls
(Fig. 6A) were morphologically
normal, those from transgene-positive Ercc1-null littermates
(Fig. 6C) showed a range of
head () and tail (*) malformations.
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Alkaline comet analysis was undertaken to assess the level of DNA damage (single-and double-strand breaks and alkali-labile sites) in sperm from the different Ercc1 genotypes. Transgene-positive Ercc1 nulls (Fig. 6D) consistently showed much larger comet tails than wild-type littermates (Fig. 6B). Statistical analysis of the comet data (summarised in Fig. 6E) confirmed that the increase in the median percentage of DNA in comet tails in transgene-positive Ercc1 nulls compared to wild type was statistically significant (P<0.05 by Mann-Whitney U test). Sperm comet tails in Ercc1 heterozygous males were intermediate between the other two genotypes, presumably reflecting a 50% reduction in Ercc1 levels. Thus, the limited number of sperm produced in transgene-positive Ercc1 null animals contained DNA with high levels of damage.
Oxidative DNA damage in Ercc1-deficient testis
The level of oxidative DNA damage in testis DNA was assessed by measuring
the commonest oxidised base, 8-oxoG, by HPLC with electrochemical detection.
Levels were calculated as moles of 8-oxoG/105 moles of
deoxyguanosine. The mean value obtained for 6- to 10-week-old control testis
was 0.16±0.05 (n=7). The value for transgene-positive
Ercc1-null littermates was 0.54±0.30 (n=4). The
3-fold increase in oxidative DNA damage levels in Ercc1-deficient
testis was statistically significant (P=0.008 by Student's
t-test).
Morphological appearance of ovaries from Ercc1 null
females
Ovaries were analysed from both Ercc1 nulls (days 8, 10, 14) and
transgene-positive Ercc1 nulls (day 16 and adult). All
transgene-positive Ercc1 null adult females were infertile and no
fully mature antral follicles were detected in any of the ovaries examined
(e.g. Fig. 7F). Female germ
cells enter meiotic prophase during fetal life
(Hartung and Stahl, 1977) and,
after birth, the oocyte remains arrested in diplotene (the so-called dictyate
stage) during an extended growth phase within the follicle whilst it matures
in size. At all ages examined, in both types of Ercc1-deficient
animal, the number of oocytes was reduced although some variation in numbers
was observed between animals and between ovaries from the same animal.
Although some large oocytes, enclosed in follicles with multiple layers of
granulosa cells, were seen even in null females, oocytes often appeared to be
in the process of degeneration (asterisks in
Fig. 7B,D,F). Thus, contrary to
the situation in spermatogenesis, oogenesis was similarly affected in
Ercc1 null and transgene-positive Ercc1 nulls. The most
striking difference between ovaries from immature Ercc1-null and
transgene-positive Ercc1-null females and their wild-type littermates
was the absence of primary follicles from the periphery of the ovary.
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DISCUSSION |
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We have shown that Ercc1, like its partner Xpf
(Shannon et al., 1999), is
expressed at the highest level in the testis. This alone does not imply a key
role for the recombination repair functions of Ercc1/Xpf, because many other
DNA repair proteins are also highly expressed in testis. In particular, the
expression of four genes involved in NER only is also elevated in the testis
[Xpb (Weeda et al.,
1991
); Xpc (Li et
al., 1996
); Mhr23a and b (Rad23a and
b) (van der Spek et al.,
1996
)]. Meiotic crossing over during the first synchronous wave of
spermatogenesis coincides with a sharp increase in the expression of some
mismatch repair genes, such as Mlh1, which are essential for the
repair of heteroduplex intermediates formed during homologous recombination
(Edelmann et al., 1996
). In
contrast similar levels of Ercc1 expression were detected in testes
of all ages examined. This and a previous study
(Shannon et al., 1999
) are in
agreement that Ercc1/Xpf do not show a mismatch repair protein-like
surge in expression coincident with the first wave of meiotic crossing
over.
The highest levels of immunostaining for Ercc1 were seen in meiotic cells
from late pachytene in prophase I, through the second meiotic division, to
round spermatids. These results are consistent with the western blot data
where an increase in the total amount of Ercc1 protein was noted in samples
from day 23 onwards at a time when pachytene spermatocytes and round
spermatids are the most abundant cell types within the testis. Earlier germ
cell stages contained less intense immunostaining. Crossing over initiates
with the appearance of double-strand breaks in leptotene and is completed by
late pachytene (Mahadevaiah et al.,
2001). Thus, the stages with the highest levels of Ercc1 protein
follow rather than precede meiotic crossing over, again providing no support
for Ercc1 having a critical role in meiotic crossing over.
In Ercc1-null males germ cell depletion was observed in testes on
days 3 and 7 at a time when they are mitotically active, but prior to entry
into meiotic prophase, which occurs on and after day 8
(Bellve et al., 1977). Germ
cell loss was highly variable between individual testes, even those from the
same animal and, even in the absence of Ercc1, a very few germ cells in day 22
males were observed to have the appearance of pachytene spermatocytes.
Strikingly, the low level of transgene-derived Ercc1 expression was
sufficient to markedly increase the numbers of germ cells and allowed some to
complete meiosis and mature into spermatozoa.
Ercc1-deficient female mice were also infertile, but the oogenesis defect was less severe and, even in the complete absence of Ercc1, some germ cells were able to complete meiotic prophase, arrest in dictyate, produce signals to the granulosa cells to initiate organisation of follicles and grow. Unlike the situation in males, the same phenotype, namely reduced numbers of primary follicles that have completed prophase I and degenerating maturing oocytes, was seen in both Ercc1 nulls and transgene-positive Ercc1 nulls, suggesting that there is no ectopic Ercc1 transgene expression in the ovary.
Studies in S. cerevisiae have indicated that mismatch repair is
essential to repair mismatches and small loops generated in heteroduplex DNA
during meiotic crossing over (reviewed by
Kirkpatrick, 1999). The repair
of large loops also involves RAD1/RAD10, presumably because of its ability to
cleave 3' single-stranded tails projecting from DNA duplexes. Our
studies indicate that this role for Ercc1 is not essential for meiotic
crossing over in mouse gametogenesis. Indeed, a second large loop repair
pathway independent of RAD1, RAD10 and MSH2 has been identified in S.
cerevisiae (reviewed by Kirkpatrick,
1999
). The observed defects must be due instead to a more general
requirement for the recombination repair and/or NER functions of Ercc1. A
meiotic checkpoint that detects spermatocytes with unsynapsed chromosomes and
eliminates them by p53-independent apoptosis has been described
(Odorisio et al., 1998
).
However, the quality control system for sperm production operates at other
levels too: a sizeable fraction of germ cells (mainly diploid spermatogonia)
die and are removed during normal spermatogenesis (reviewed by
Braun, 1998
). The p53-dependent
apoptotic response to ionising radiation found in mitotic cells also operates
in this tissue (Odorisio et al.,
1998
). This classical DNA damage response would be expected to be
particularly important in such a rapidly dividing tissue. The disorganised
appearance and frequent gaps in Ercc1-deficient seminiferous tubules
indicate the excessive elimination of, presumably DNA-damaged, germ cells.
Clustered apoptoses, affecting a range of germ cell stages, were observed in
Ercc1-deficient testis whereas only rare apoptoses affecting single
germ cells were seen in control sections. Given the, presumably sporadic,
nature of the endogenous DNA damage occurring in the testis and the transient
nature of apoptosing cells, we did not expect to see the very high levels of
apoptosis observed after a defined cytotoxic insult, such as treatment with
methoxyacetic acid (Krishnamurthy et al.,
1998
). An accumulation of cytoplasmic lipid droplets in Sertoli
cells is often associated with germ cell degeneration
(Paniagua et al., 1987
). In
the present study the accumulation observed in 10-week transgene-positive
Ercc1 nulls provides testimony to the excessive germ cell elimination
and phagocytosis that has occurred following germ cell death.
The limited number of sperm that were produced by transgene-positive
Ercc1-deficient mice had a high frequency of malformations and the
animals were infertile. Spermatogenesis is unaffected (at least as judged by
normal fertility) in Xpa (de
Vries et al., 1995; Nakane et
al., 1995
) and Xpc
(Sands et al., 1995
) knockout
mice and, in man, males with XP are also fertile
(Kraemer, 1993
). This suggests
that infertility in Ercc1-deficient male mice results from the lack
of an additional Ercc1 function, rather than from an NER deficit.
Sperm from transgene-positive Ercc1-deficient mice had
significantly higher levels of DNA strand breaks than control samples. This
could be due to the lack of the recombination repair or NER functions of
Ercc1. In vitro an NER complex will form and 3' incision by Xpg can take
place in the absence of Ercc1/Xpf (Evans
et al., 1997). Strand breaks would not be predicted from the
failure of Ercc1/Xpf to cut adjacent to an interstrand crosslink in the
recombination repair model favoured by Kuraoka et al.
(Kuraoka et al., 2000a
).
However, in both of these cases strand breaks could accumulate at replication
forks stalled at unrepaired lesions. The failure of Ercc1 to act in the
single-stranded annealing pathway for double-strand break repair and
homologous recombination would lead directly to the accumulation of strand
breaks because the remaining single-strand tails would prevent ligation. Comet
assays on sperm from Xpa and Xpc knockout mice could help to
resolve the relative contributions of the recombination repair and NER
deficits to the accumulation of strand breaks in Ercc1-deficient
mice.
Ercc1, in common with other NER genes, is expressed in all tissues
examined. This is consistent with a role for NER in the repair of endogenous,
predominantly oxidative DNA damage in internal tissues as well as the key role
of repairing UV-induced DNA damage in the skin (reviewed by
Lindahl, 1993). Traditionally
BER was considered to have the key role in removing the commonest oxidised
base, 8-oxoG, which is strongly mutagenic and also acts as a block to
transcription by RNA polymerase II (Le
Page et al., 2000a
). However, the discovery of transcription
coupled repair of 8-oxoG and the observation that this process continues to
operate in Ogg1 null cells (Le
Page et al., 2000b
) has, belatedly, led to the recognition that
NER may also have an important role to play in the repair of 8-oxoG
(Kuraoka et al., 2000b
;
Le Page et al., 2000b
).
Three-fold higher levels of 8-oxoG were found in DNA extracted from
Ercc1-deficient testis than from control littermates, demonstrating
that Ercc1 is important for the repair of 8-oxoG, particularly considering
that the levels of 8-oxoG in the livers of Ogg1-null mice were only
1.7-fold higher than controls (Klungland
et al., 1999
). Most likely the increase in 8-oxoG results from the
lack of the NER function of Ercc1. The role of NER in the repair of 8-oxoG and
the significance of this damage to the phenotype in Ercc1-deficient
testis could be determined by measuring 8-oxoG levels in Xpa and
Xpc knockout testis.
We believe that the consequences of Ercc1 deficiency for
gametogenesis can be explained by a general requirement for Ercc1 to repair
DNA damage in all dividing cells, rather than a specific role in meoisis, and
by the stochastic nature of DNA damage. The more severe consequences of
Ercc1 deficiency for spermatogenesis than oogenesis reflect
differences between the processes themselves. Primordial germ cells migrate
from the hind gut to the gonad between day 8.5-10.5 p.c. During this time they
are actively proliferating in both sexes, so that starting from about 100
cells, more than 1000 are present on day 10.5 p.c.
(Godin et al., 1990). Female
primordial germ cells enter meiosis about day 12.5-13.5 p.c. and meiosis
continues during foetal development until it becomes arrested at diplotene
stage, on or about 5 days of age (p.p.). Male germ cells continue to divide
mitotically until day 16 p.c. and then become arrested in G1
(Vergouwen et al., 1991
) until
they resume mitotic activity after birth. Spermatogonial cells undergo up to 6
further mitotic divisions before they enter meiosis
(de Rooij, 2001
). Thus, in
both processes, mitotic expansion precedes entry into meiosis, but the
expansion is much more extensive in the male. The premeiotic loss of germ
cells with damaged DNA is greater in the male because rapidly cycling cells
are particularly susceptible to the deleterious effects of DNA damage on DNA
replication. DNA damage is a continuous, random process and, in both sexes,
some undamaged germ cells, or cells with non-lethal levels of damage, survive
the mitotic expansion and enter meiosis before succumbing to damage acquired
during the meiotic stages. In transgene-positive Ercc1-deficient
males small amounts of transgene-derived Ercc1 are sufficient to partially
rescue the phenotype resulting in complete spermatogenesis in some tubules and
production of mature sperm, albeit abnormal and with elevated levels of DNA
damage.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
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---|
Adair, G. M., Rolig, R. L., Moore-Faver, D., Zabelshansky, M.,
Wilson, J. H. and Nairn, R. S. (2000). Role of Ercc1 in
removal of long non-homologous tails during targeted homologous recombination.
EMBO J. 19,5552
-5561.
Alcivar, A. A., Hake, L. E. and Hecht, N. B. (1992). DNA polymerase-beta and poly(ADP)ribose polymerase mRNAs are differentially expressed during the development of male germinal cells. Biol. Reprod. 46,201 -207.[Abstract]
Baarends, W. M., van der Laan, R. and Grootegoed, J. A.
(2001). DNA repair mechanisms and gametogenesis.
Reproduction 121,31
-39.
Baker, S. M., Bronner, C. E., Zhang, L., Plug, A. W., Robatzek, M., Warren, G., Elliott, E. A., Yu, J., Ashley, T., Arnheim, N., Flavell, R. A. and Liskay, R. M. (1995). Male mice defective in the DNA mismatch repair gene Pms2 exhibit abnormal chromosome synapsis in meiosis. Cell 82,309 -319.[Medline]
Bauche, F., Fouchard, M. H. and Jegou, B. (1994). Antioxidant system in rat testicular cells. FEBS Lett. 349,392 -396.[CrossRef][Medline]
Bellve, A. R., Cavicchia, J. C., Millette, C. A., O'Brien, D.
A., Bhatnagar, Y. M. and Dym, M. (1977). Spermatogenic cells
of the prepubertal mouse, isolation and morphological characterization.
J. Cell Biol. 74,68
-85.
Biggers, J. D., Whitten, W. K. and Whittingham, D. G. (1971). The culture of mouse embryos in vitro. In Methods in Mammalian Embryology (ed, J. C. Daniel), pp. 86-116. San Francisco: Freeman.
Braun, R. E. (1998). Every sperm is sacred or is it? Nat. Genet. 18,202 -204.[Medline]
Chen, J., Tomkinson, A. E., Ramos, W., Mackey, Z. B., Danehower, S., Walter, C. A., Schultz, R. A., Besterman, J. M. and Husain, I. (1995). Mammalian DNA ligase III: Molecular cloning, chromosomal localization and expression in spermatocytes undergoing meiotic recombination. Mol. Cell. Biol. 15,5412 -5422.[Abstract]
Davies, A. A., Friedberg, E. C., Tomkinson, A. E., Wood, R. D.
and West, S. C. (1995). Role of the Rad1 and Rad10 proteins
in nucleotide excision repair and recombination. J. Biol.
Chem. 270,24638
-24641.
de Rooij, D. G. (2001). Proliferation and
differentiation of spermatogonial stem cells.
Reproduction 121,347
-354.
de Vries, A., van Oostrom, C. T. M., Hofhuis, F. M. A., Dortant, P. M., Berg, R. J. W., de Gruijl, F. R., Wester, P. W., van Kreijl, C. F., Capel, P. J. A., van Steeg, H. and Verbeek, S. J. (1995). Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision-repair gene Xpa. Nature 377,169 -173.[CrossRef][Medline]
de Vries, S. S., Baart, E. B., Dekker, M., Siezen, A., de Rooij,
D. G., de Boer, P. and te Riele, H. (1999). Mouse MutS-like
protein Msh5 is required for proper chromosome synapsis in male and female
meiosis. Genes Dev. 13,523
-531.
Edelmann, W., Cohen, P. E., Kane, M., Lau, K., Morrow, B., Bennett, S., Umar, A., Kunkel, T., Cattoretti, G., Chaganti, R., Pollard, J. W., Kolodner, R. D. and Kucherlapati, R. (1996). Meiotic pachytene arrest in MLH1-deficient mice. Cell 85,1125 -1134.[Medline]
Evans, E., Moggs, J. G., Hwang, J. R., Egly, J. M. and Wood, R.
D. (1997). Mechanism of open complex and dual incision
formation by human nucleotide excision repair factors. EMBO
J. 16,6559
-6573.
Fisher, H. M. and Aitken, R. J. (1997). Comparative analysis of the ability of precursor germ cells and epididymal spermatozoa to generate reactive oxygen metabolites. J. Exp. Zool. 277,390 -400.[CrossRef][Medline]
Fishman-Lobell, J. and Haber, J. E. (1992). Removal of nonhomologous DNA ends in double-strand break recombination: the role of the yeast ultraviolet repair gene RAD1. Science 258,480 -484.[Medline]
Friedberg, E. C., Walker, G. C. and Siede, W. (1995). DNA repair and mutagenesis. ASM Press, Washington DC.
Geschwind, D. H., Ou, J., Easterday, M. C., Dougherty, J. D., Jackson, R. L., Chen, Z., Antoine, H., Terskikh, A., Weissman, I. L., Nelson, S. F. and Kornblum, H. I. (2001). A genetic analysis of neural progenitor differentiation. Neuron 29,325 -339.[Medline]
Godin, I., Wylie, C. and Heasman, J. (1990). Genital ridges exert long-range effects on mouse primordial germ cell numbers and direction of migration in culture. Development 108,357 -363.[Abstract]
Hartung, M. and Stahl, A. (1977). Preleptotene chromosome condensation in mouse oogenesis. Cytogenet. Cell Genet. 18,309 -319.[Medline]
Kerr, J. B., Millar, M., Maddocks, S. and Sharpe, R. M. (1993). Stage-dependent changes in spermatogenesis and Sertoli cells in relation to the onset of spermatogenic failure following withdrawal and restoration of testosterone. Anat. Rec. 235,547 -559.[Medline]
Kirkpatrick, D. T. (1999). Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis. Cell. Mol. Life Sci. 55,437 -449.[CrossRef][Medline]
Klungland, A., Rosewell, I., Hollenbach, S., Larsen, E., Daly,
G., Epe, B., Seeberg, E., Lindahl, T. and Barnes, D. E.
(1999). Accumulation of premutagenic DNA lesions in mice
defective in removal of oxidative base damage. Proc. Natl. Acad.
Sci. USA 96,13300
-13305.
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.
Kraemer, K. H. (1993). Hereditary diseases with increased sensitivity to cellular injury. In Dermatology in General Medicine, Fourth edition (ed. T. B. Fitzpatrick, A. Z. Eisen, K. Wolff, I. M. Freedberg and K. F. Austen), pp.1974 -1992. New York: McGraw-Hill.
Krishnamurthy, H., Weinbauer, G. F., Aslam, H., Yeung, C. H. and
Neischlag, E. (1998). Quantification of apoptotic testicular
germ cells in normal and methoxyacetic acid-treated mice as determined by flow
cytometry. J. Androl.
19,710
-717.
Kuraoka, I., Kobertz, W. R., Ariza, R. R., Biggerstaff, M.,
Essigmann, J. M. and Wood, R. D. (2000a). Repair of an
interstrand DNA cross-link initiated by ERCC1/XPF repair/recombination
nuclease. J. Biol. Chem.
275,26632
-26636.
Kuraoka, I., Bender, C., Romieu, A., Cadet, J., Wood, R. D. and
Lindahl, T. (2000b). Removal of oxygen free-radical-induced
5', 8-purine cyclodeoxynucleosides from DNA by the nucleotide
excision-repair pathway in human cells. Proc. Natl. Acad. Sci.
USA 97,3832
-3837.
Le Page, F., Kwoh, E. E., Avrutskaya, A., Gentil, A., Leadon, S. A., Sarasin, A. and Cooper, P. K. (2000a). Transcription-coupled repair of 8-oxoGuanine: requirement for XPG, TFIIH, and CSB and implications for Cockayne syndrome. Cell 101,159 -171.[Medline]
Le Page, F., Klungland, A., Barnes, D. E., Sarasin, A. and
Boiteux, S. (2000b). Transcription coupled repair of
8-oxoguanine in murine cells: The Ogg1 protein is required for repair in
nontranscribed sequences but not in transcribed sequences. Proc.
Natl. Acad. Sci. USA 97,8397
-8402.
Li, L., Peterson, C. and Legerski, R. (1996).
Sequence of the mouse Xpc cDNA and genomic structure of the human XPC gene.
Nucl. Acids Res. 24,1026
-1028.
Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362,709 -715.[CrossRef][Medline]
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). Recombination DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27,271 -276.[CrossRef][Medline]
McWhir, J., Selfridge, J., Harrison, D. J., Squires, S. and Melton, D. W. (1993). Mice with DNA repair gene (Ercc1) deficiency have elevated levels of p53, liver nuclear abnormalities and die before weaning. Nat. Genet. 5, 217-224.[Medline]
Minty, A. J., Caravatti, M., Robert, B., Cohen, A., Daubas, P.,
Weydert, A., Gros, F. and Buckingham, M. E. (1981). Mouse
actin messenger-RNAs construction and characterization of a
recombinant plasmid molecule containing a complementary-DNA transcript of
mouse alpha-actin messenger-RNA. J. Biol. Chem.
256,1008
-1014.
Nakane, H., Takeuchi, S., Yuba, S., Saijo, M., Nakatsu, Y., Murai, H., Nakatsuru, Y., Ishikawa, T., Hirota, S., Kitamura, Y., Kato, Y., Tsunoda, Y., Miyauchi, H., Horio, T., Tokunaga, T., Matsunaga, T., Nikaido, O., Nishimune, Y., Okada, Y. and Tanaka, K. (1995). High incidence of ultraviolet-B-induced or chemical carcinogen-induced skin tumors in mice lacking the xeroderma pigmentosum group-A gene. Nature 377,165 -168.[CrossRef][Medline]
Nuñez, F., Chipchase, M. D., Clarke, A. R. and Melton, D.
W. (2000). Nucleotide excision repair gene (Ercc1)
deficiency causes G2 arrest in hepatocytes and a reduction in liver
binucleation: the role of p53 and p21. FASEB J.
14,1073
-1082.
Oakberg, E. F. (1956). Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Amer. J. Anat. 99,507 -516.
Odorisio, T., Rodriguez, T. A., Evans, E. P., Clarke, A. R. and Burgoyne, P. S. (1998). The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis. Nat. Genet. 18,257 -261.[CrossRef][Medline]
Paniagua, R., Rodriguez, M. C., Nistal, M., Fraile, B. and Amat, P. (1987). Changes in the lipid inclusion/Sertoli cell cytoplasm area ratio during the cycle of the human seminiferous epithelium. J. Reprod. Fertil. 80,335 -341.[Abstract]
Robinson, P. A., Anderton, B. H. and Loviny, T. L. (1988). Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries. J. Immunol. Methods 108,115 -122.[CrossRef][Medline]
Rosenquist, T. A., Zharkov, D. O. and Grollman, A. P.
(1997). Cloning and characterization of a mammalian 8-oxoguanine
DNA glycosylase. Proc. Natl. Acad. Sci. USA
94,7429
-7434.
Ruggiu, M., Speed, R., Taggart, M., McKay, S. J., Kilanowski, F., Saunders, P. T. K., Dorin, J. and Cooke, H. J. (1997). The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389, 73-77.[CrossRef][Medline]
Ruggiu, M., Saunders, P. T. K. and Cooke, H. J.
(2000). Dynamic subcellular distribution of the DAZL protein is
confined to primate male germ cells. J. Andrology
21,470
-477.
Sands, A. T., Abuin, A., Sanchez, A., Conti, C. J. and Bradley, A. (1995). High susceptibility to ultraviolet-induced carcinogenesis in mice lacking Xpc. Nature 377,162 -165.[CrossRef][Medline]
Sargent, R. G., Meservy, J. L., Perkins, B. D., Kilburn, A. E.,
Intody, Z., Adair, G. M., Nairn, R. S. and Wilson, J. H.
(2000). Role of the nucleotide excision repair gene
Ercc1 in formation of recombination-dependent rearrangements in
mammalian cells. Nucl. Acids Res.
28,3771
-3778.
Saunders, P. T. K., Williams, K., Macpherson, S., Urquhart, H.,
Irvine, D. S., Sharpe, R. M. and Millar, M. R. (2001).
Differential expression of oestrogen receptor alpha and beta proteins in the
testes and male reproductive system of human and non-human primates.
Mol. Human Reprod. 7,227
-236.
Schiestl, R. H. and Prakash, S. (1990). RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination. Mol. Cell. Biol. 10,2485 -2491.[Medline]
Sekelsky, J. J., McKim, K. S., Chin, G. M. and Hawley, R. S.
(1995). The Drosophila meiotic recombination gene Mei-9 encodes a
homolog of the yeast excision-repair protein Rad1.
Genetics 141,619
-627.
Selfridge, J., Hsia, K.-T., Redhead, N. J. and Melton, D. W.
(2001). Correction of liver dysfunction in DNA repair-deficient
mice with an ERCC1 transgene. Nucl. Acids
Res. 29,4541
-4550.
Shannon, M., Lamerdin, J. E., Richardson, L., McCutchen-Maloney, S. L., Hwang, M. H., Handel, M. A., Stubbs, L. and Thelen, M. P. (1999). Characterization of the mouse Xpf DNA repair gene and differential expression during spermatogenesis. Genomics 62,427 -435.[CrossRef][Medline]
Sharpe, R. M., Atanassova, N., McKinnell, C., Parte, P., Turner,
K. J., Fisher, J. S., Kerr, J. B., Groome, N. P., Macpherson, S., Millar, M.
R. and Saunders, P. T. K. (1998). Abnormalities in functional
development of the Sertoli cells in rats treated neonatally with
diethylstilbestrol: a possible role for estrogens in Sertoli cell development.
Biol. Reprod. 59,1084
-1094.
van der Spek, P. J., Visser, C. E., Hanaoka, F., Smit, B., Hagemeijer, A., Bootsma, D. and Hoeijmakers, J. H. J. (1996). Cloning, comparative mapping and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23. Genomics 31,20 -27.[CrossRef][Medline]
Vergouwen, R. P. F. A., Jacobs, S. G. P. M., Huiskamp, R., Davids, J. A. G. and de Rooij, D. G. (1991). Proliferative activity of gonocytes, Sertoli cells and interstitial cells during testicular development in mice. J. Reprod. Fertil. 93,233 -243.[Abstract]
Walter, C. A., Trolian, D. A., McFarland, M. B., Street, K. A., Gurram, G. R. and McCarrey, J. R. (1996). Xrcc-1 expression during male meiosis in the mouse. Biol. Reprod. 55,630 -635.[Abstract]
Weeda, G., Ma, L., van Ham, R. C., Bootsma, D., van der Eb, A. J. and Hoeijmakers, J. H. (1991). Characterization of the mouse homolog of the XPBC/ERCC-3 gene implicated in xeroderma pigmentosum and Cockayne's syndrome. Carcinogenesis 12,2361 -2368.[Abstract]
Weeda, G., Donker, I., de Wit, J., Morreau, H., Janssens, R., Vissers, C. J., Nigg, A., van Steeg, H., Bootsma, D. and Hoeijmakers, J. H. J. (1997). Disruption of mouse Ercc1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence. Curr. Biol. 7, 427-439.[Medline]
Wood, R. D. (1996). DNA repair in eukaryotes. Ann. Rev. Biochem. 65,135 -167.[CrossRef][Medline]