 |
INTRODUCTION |
Eukaryotic cells generally possess multiple pathways as a defense
against UV-induced DNA damage. Among them, nucleotide excision repair
(NER)1 is distributed most
widely and its core mechanism is considered to have been conserved
throughout evolution (1). NER is driven by the coordinated action of
more than a dozen proteins. These proteins are thought to form either a
huge repairsome complex (2) or several subcomplexes, which act
cooperatively on DNA damage (3). While NER is able to repair a wide
variety of DNA damage in addition to UV-induced damage, most organisms
also have simpler, UV damage-specific repair systems. Photoreactivation is one of those alternatives to NER, in which a single enzyme called
photolyase catalyzes complete repair of UV-induced DNA damage without
scissions of the phosphodiester bonds in DNA strands. Two types of
photolyase that differ in substrate specificity have been described,
one exclusively acting on cyclobutane pyrimidine dimers (CPDs) (4) and
the other acting on (6-4) photoproducts (5, 6).
The fission yeast, Schizosaccharomyces pombe, and the
filamentous mold Neurospora crassa have another type of UV
damage-specific repair system called UV-damaged DNA endonuclease
(UVDE)-dependent excision repair (UVER) (7-9). In this
system the UVDE protein catalyzes a specific cleavage of a
phosphodiester bond 5' adjacent to a CPD or a (6-4) photoproduct.
After nicking of damaged DNA, repair is thought to be completed by a
base excision repair (BER)-like process (10). In contrast to
photolyase, a single type of UVDE can recognize both CPDs and (6-4)
photoproducts, which have been considered to have significantly
different conformation.
The reason for the existence of multiple repair pathways for UV damage
is not fully understood. One might think that UV-specific pathways such
as photoreactivation or UVER are only a backup to NER and functionally
redundant, or that they are just remnants of primitive repair systems
that existed when UV irradiation was much stronger than today.
However, it is difficult to imagine that such a completely
redundant pathway would have been maintained for long during evolution.
The most likely possibility is that these multiple pathways have
partially overlapping but also differentiated functions.
Multiple organelles harboring their own genomes exist in most
eukaryotes. These organelles have fundamental roles in respiration or
photosynthesis, and the gene products encoded by their genomes are
essential for these biochemical events. Therefore, these genomes should
be protected by DNA repair systems, as are nuclear genomes, from
harmful DNA damage. Photoreactivation has been reported to function in
mitochondria or in chloroplasts in some species. In the green alga
Euglena gracilis, UV irradiation induces the appearance of
white color colonies consisting of cells lacking chloroplasts in
addition to cell killing. This "bleached" colony induction is known
to be photoreactivable (11). UV irradiation also causes induction of
petite colonies lacking mitochondria with remarkably high efficiency in
budding yeast Saccharomyces cerevisiae. Petite induction is
also alleviated by photoreactivation of UV damage in mitochondrial DNA
(12, 13). Our previous results showed that the N-terminal protruding
sequence of S. cerevisiae photolyase is crucial for
its mitochondrial function (14). In both cases, however, NER cannot
repair UV damage in organelle genomes. Although cells lacking
chloroplasts or mitochondria are viable under laboratory condition,
they would be expected to suffer from great disadvantage compared with
wild type cells in nature.
Interestingly, S. pombe totally lacks photolyase activity
(15) and instead possesses UVDE. In the current study, we examined the
possible function of UVER in mitochondria of S. pombe. We demonstrate that UVER works efficiently in mitochondria as well as in
the nucleus by means of a repair kinetics assay and cytological observation of UVDE protein.
| |
EXPERIMENTAL PROCEDURES |
Strains, Media, and Transformation of S. pombe--
Strains used
in this study are listed in Table I. All
strains were grown in either YES or MM at 30 °C (16). Transformation of S. pombe cells was performed by electroporation with a
ECM600 Electro Cell Manipulator (BTX) following the manufacturer's
protocol. Double mutants were made by crossing each of the single
mutants.
UV Survival Experiment--
Exponentially growing cells
(1-2 × 107/ml) in YES liquid medium were
appropriately diluted with distilled water and 500 or 10,000 cells/plate were seeded on YES agar plates. 254-nm UV was administered
using a set of germicidal lamps (GL-10, Toshiba, Japan) at a dose rate
of 0.4-3.3 J/m2 s. After incubation at 30 °C for 3 days, colonies were counted.
Strand-specific CPD Assay--
Exponentially growing cells in
YES medium were washed twice with distilled water and resuspended in
phosphate-buffered saline at 5 × 106 cells/ml.
Approximately 30 ml of cell suspension was poured into a 15-cm plastic
dish (Falcon) and irradiated with 100 J/m2 254-nm UV.
During the washing and UV irradiation processes cells were kept on ice
and all materials were pre-chilled. For each strain, a total of 300 ml
of UV irradiated cell suspension was prepared and divided into 10 aliquots. Each aliquot was centrifuged, and the cells were resuspended
in 30 ml of pre-warmed YES medium and incubated at 30 °C for 0, 20, 40, 60, or 120 min (two aliquots for each time point). The cells were
pelleted again, frozen with liquid nitrogen, and stored at 
80 °C
until DNA preparation. Genomic DNA was isolated as described previously
(16) with a slight modification. After digestion with
HindIII, DNA was treated with T4 endonuclease or
mock-treated and electrophoresed under alkaline condition (17). DNA was
transferred to Hybond-N+ nylon membrane (Amersham Pharmachia Biotech).
Blots were prepared in duplicate with the same DNA sample, and one was
hybridized with a sense strand-specific myo2 or
coI probe the other with an antisense specific probe (see
below) using ExpressHyb hybridization solution (CLONTECH) following the manufacturer's protocol.
The blots were analyzed using a FLA-2000 fluoroimage analyzer (Fuji,
Tokyo, Japan). The number of CPDs in the target fragment was calculated
assuming Poisson distribution. After the first hybridization and
analysis, the blots were incubated at 100 °C in 0.5% SDS for 5 min
to remove probes and rehybridized reciprocally with the opposite
strand-specific probes.
Generation of myo2 and coI Strand-specific Probes--
A part of
the myo2 gene (Ref. 18, nucleotides 1496-2302 of GenBank
Accession U75357) was amplified by PCR with primers SY78
(5'-CCCCCGGATCCCAAAGCTACTTTATTGGTATTTT-3') and SY79
(5'-CCCCCGGATCCATTATCTCATATCTGACTCTAAA-3') using Y4 strain genomic DNA
as a template, and cloned into pBluescriptII SK+ (Stratagene).
Strand-specific probes were generated by asymmetric PCR using the
cloned fragment as a template and either SY78 or SY79 as a primer (19).
After the PCR reaction, the probe was purified by passing through G-50.
Approximately 3 × 106 cpm/ml was used for
hybridization. For coI strand-specific probes, a part of the
gene (nucleotides 4901-5405 of GenBank accession no. X54421) was
PCR-amplified with primers SY61
(5'-CCCCCGGATCCACTTATGTTAATAGATGGATATTCT-3') and SY62
(5'-CCCCCGGATCCAAGTTAGTAATCTTTTTACTAGT-3') and similarly labeled.
Computer Analysis of UVDE Sequence--
Subcelluar localization
of UVDE was predicted by PSORTII, which is available on the Internet
(20).
Site-directed in Vitro Mutagenesis of uvde Gene--
The
uvde-M1 mutation was introduced into the cloned genomic
fragment with a Mutan-Express Km kit (Takara, Tokyo, Japan) and SY96
(5'-CGTTTTCATTTTTTAAAGCTTAGGCTATTG-3'). This mutation disrupts the
first methionine codon of the uvde and creates a
HindIII site. The uvde-M56AM64A mutation, which
converts the second and the third methionine codons to alanine codons,
was introduced by PCR-based method with SY95
(5'-TGCCCACCTCCTCGAGATGAGATTG-3'), SY101
(5'-GTTTACTTCCAGCGAGCTCAAAAACCACACTCTCAGCGTTACCGCAAG-3'), SY102
(5'-CTTGTCGATATCTCTTTTCACTTAC-3'), and SY103
(5'-TGAGCTCGCTGGAAGTAAACTTTTAAGGGT-3'). Sequences were verified using a
DSQ-1000 DNA sequencer (Shimadzu, Kyoto, Japan).
Expression of UVDE-GFP Fusion Protein--
The EGFP
gene from a pEGFP-N1 (CLONTECH) vector together
with its original multicloning site was isolated and inserted into the
XhoI and SmaI sites of pREP4X (21). This plasmid
was named pSY8. Wild type, 5'-truncated, or point mutated versions of
uvde were cloned into the SacII and
BamHI sites of pSY8 so that C-terminal GFP fusion protein
was expressed from a nmt1 full-strength promoter (see Fig.
2b, pSY57-59, pSY74). When PCR was used in plasmid
construction, sequences of the amplified region were verified. S. pombe transformants harboring either of these constructs were
maintained in MM + thiamine (5 µg/ml) medium to minimize the toxicity
due to overexpression of the fusion protein. Prior to microscopic
observation, exponentially growing transformants were washed once with
water, diluted 50-100-fold into thiamine-free MM and cultured at
30 °C for 16 h. The subcellular localization of UVDE-GFP fusion
protein was observed by using Leica DM LB microscope (Leica, Germany)
with an appropriate filter for GFP.
Gene Replacement of uvde Gene--
Gene replacement was
conducted by a pop-in/pop-out method (16) using
ura4+ as the selectable marker. The mutated
uvde gene and ura4+ marker are
subcloned next to each other into a ars-less vector. The
plasmid was integrated into the uvde locus and targeted
integration was confirmed by Southern hybridization. This strain was
cultured in YES medium for several generations and plated on MM + 5-FOA plates for selection against ura4 marker. The pop-out event
was checked by Southern hybridization and the nucleotide sequences of
replaced region were confirmed by direct sequencing of the PCR-amplified fragment.
| |
RESULTS |
UVER in Mitochondria--
It has been reported that photolyase in
S. cerevisiae functions in mitochondria as well as in the
nucleus and that it actually contributes to the reduction of the petite
induction rate by UV irradiation (12-14). While S. cerevisiae cells do not possess UVER, S. pombe lacks
any detectable photoreactivation of UV damage. Each repair system
depends on a single protein, photolyase or UVDE. This complementary
relationship between two repair pathways in the two yeast species
prompted us to examine whether UVER functions in the mitochondria of
S. pombe cells.
We first looked at CPD removal from the myo2 locus on the
nuclear genome and the coI locus on the mitochondrial genome
by using a T4 endonuclease-based strand-specific damage assay. These loci encode a type II myosin heavy chain and a cytochrome oxidase subunit I, respectively. When digested with HindIII, these
loci give rise to a 4.1- or a 4.3-kilobase pair signal in Southern hybridization experiments with the appropriate probes (see
"Experimental Procedures"). Wild type cells were able to repair
CPDs in both loci quite efficiently (Fig.
1, WT). The repair kinetics
were not remarkably affected when functional NER was eliminated by the
disruption of rad13, XPG/RAD2 homolog of S. pombe (22), suggesting that UVER operates both in the nucleus and
mitochondria (Fig. 1, rad13
). When UVER was eliminated,
CPDs in the myo2 locus were removed with a clear strand bias
(Fig. 1, uvde, left column) Transcription-coupled repair by NER in S. pombe was
described elsewhere (23). In contrast, NER did not repair damage at the coI locus during the 120 min incubation time (Fig. 1,
uvde, right column) indicating that NER does not function
in mitochondria. Strains lacking both types of excision repair did not
show any ability to repair damage at either locus (Fig. 1,
rad13 uvde).
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
Repair kinetics of CPDs at the
myo2 and coI loci by two excision
repair systems, NER and UVER. Data for repair of myo2
are cited from our independent manuscript with modification (23). Cells
with or without functional NER or UVER were irradiated with 100 J/m2 254-nm UV and allowed for damage repair for 0-120 min
at 30 °C. Each point stands for a mean value calculated from four
hybridizations with two independent UV irradiations and DNA isolations.
 , transcribed strand;  , nontranscribed strand.
|
|
Subcellular Localization of UVDE--
UVER is initiated by the
nicking of damaged DNA by UVDE (7, 8). In order for UVER to repair
damage on the mitochondrial genome, UVDE itself should exist in
mitochondria. Therefore, we next examined the subcellular localization
of UVDE. The uvde+ nucleotide sequence possesses
three putative initiation methionine codons, Met-1, Met-56, and Met-64
at its N terminus (Fig. 2a). Scanning of the full-length UVDE amino acid sequence (599 amino acids)
with PSORTII program (20) picked up features of a mitochondrial protein
between the first and the second methionines, as well as motifs for
nuclear localization after the third methionine (Fig. 2a).
The full-length protein was predicted to be mitochondrial with a score
of 56.5%, while the N-terminal truncated version in which the sequence
preceding the second methionine was removed (544 amino acids) was
predicted to be nuclear with a score of 65.2%. These sequence features
suggest that alternative usage of two or three methionine codons for
translation initiation may target UVDE to two different organelles.
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
a, sequence motifs of UVDE for
subcellular sorting detected by PSORTII (20). Nuclear localization
signals are shown double underlined. Putative
initiation methionines were boxed in black.
Although PSORTII does not predict the exact sequence, the first 20 amino acids at N terminus shown with a box possess strong
features for transport to mitochondria. b, subcellular
localization of ectopically expressed UVDE. UVDE was expressed from
wild type (pSY57) or mutant (pSY58, pSY59, and pSY74) genes as
C-terminal GFP fusion protein and fluorescence from GFP was observed
microscopically.
|
|
We then observed the localization of ectopically expressed UVDE-GFP
fusion protein microscopically. Typical mitochondria in S. pombe appear as a stringlike structure, as has been shown (24, 25). Wild type UVDE-GFP was localized to both nucleus and mitochondria (Fig. 2b, pSY57), which is consistent with the
repair kinetics result (Fig. 1, WT). In contrast, UVDE-GFP
expressed from the second or the third methionine codons was observed
exclusively in nucleus (Fig. 2b, pSY58 and
pSY59). We also expressed the mutant version of UVDE-GFP,
where both the second and the third methionine codons were replaced
with alanine codons. This mutation would repress the translation
initiation from these two sites and consequently repress targeting to
the nucleus. As expected, the mutant UVDE-GFP was localized only to
mitochondria (Fig. 2b, pSY74). All of the wild
type and mutant versions of UVDE-GFP fusion proteins were functional as
UVDE in a repair-deficient Escherichia coli-based survival
assay (7) (data not shown).
Effect of UVDE Mutation on Repair Kinetics--
Cytological
observation of ectopically expressed UVDE-GFP suggested an alternative
usage of two or three methionine initiation codons for targeting of
this protein to multiple organelles. We next introduced the same
mutations into the chromosomal copy of the uvde gene and
studied its effect on repair kinetics of myo2 and
coI loci in NER-deficient (rad13) background.
When the first methionine was disrupted, UVER repaired damage only at
the myo2 locus (Fig. 3,
rad13 uvde-M1). When the second and the third methionine codons were replaced with alanine codons, UVER operated only
on the coI locus (Fig. 3, rad13
uvde-M56AM64A) as anticipated.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of uvde-M1 and
uvde-M56AM64A mutations on nuclear and mitochondrial
function of UVER in NER-deficient
(rad13) background. Repair kinetics
of CPDs on myo2 and coI loci was examined as in
Fig. 1.
|
|
Contribution of Nuclear and Mitochondrial UVER to UV
Survival--
As shown above, UVER is the only excision repair system
for UV damage in the mitochondrial genome of S. pombe. To
evaluate its relative significance in damage repair in nucleus and
mitochondria, we examined the UV survival of the strains whose nuclear
or mitochondrial UVER was selectively inactivated.
When mitochondrial UVER was eliminated, UV survival was apparently
unchanged (Fig. 4, a (uvde-M1)
and b (rad13 uvde-M1)). On the other hand,
inactivation of nuclear UVER (uvde-M56AM64A) rendered the
cells sensitive to UV almost to the same degree as complete
inactivation of UVER (Fig. 4, a (uvde-M56AM64A)
and b (rad13 uvde-M56AM64A)). The result was
the same whether or not NER was functional. The surviving colonies of
the mitochondrial UVER-deficient strains did not show any retardation
growth, and therefore were not considered to have respiration defect.
Although UVER efficiently works on UV damage in the mitochondrial
genome, its mitochondrial function may not contribute to UV resistance of S. pombe cells.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of uvde-M1 and
uvde-M56AM64A mutations on UV survival of S. pombe with (a) or without functional NER
(b). Panel a: , wild type;  ,
uvde-M1;  , uvde-M56AM64A; ,
uvde. Panel b: , rad13; ,
rad13 uvde-M1; , rad13
uvde-M56AM64A; , rad13 uvde.
500 or 10000 cells were plated and irradiated with an appropriate dose
of UV. Colonies were counted after 3-day incubation at 30 °C. A
typical result from a single experiment was shown.
|
|
| |
DISCUSSION |
There are many examples of products of a single gene localized to
several different intracellular compartments (26). A variety of
mechanisms are responsible for this multiple localization, such as
variable pre-mRNA processing (including alternative splicing), transcription initiation at multiple sites, translation initiation at
multiple sites, or variable post-translational modification, depending
on each case. The S. pombe uvde+ gene possesses
three putative initiation methionine codons at its N terminus and the
amino acid sequence preceding the third methionine is dispensable for
its enzymatic activity (8). Although it remains to be confirmed by
direct examination of N-terminal sequence of the protein, our repair
kinetics and cytological data in the present study strongly suggest
that multiple subcellular localization of UVDE is accomplished by
translation initiation at multiple methionine start codons. According
to Zhang and Marr (27), the most conserved nucleotide around
translation initiation sites in S. pombe is A at 3
position. None of the three methionine codons at the N terminus of UVDE
meet this criterion (T for the first and the third methionine codons,
and C for the second methionine codon). The absence of any strongly
preferred translation initiation sites may enable the usage of multiple sites.
In the present study, we demonstrated that UVER efficiently removes UV
damage on the mitochondrial genome as well as on the nuclear genome.
This means that, after nicking of damaged DNA by UVDE, two independent
but analogous mechanisms may complete the repair reaction in the
respective organelles. We previously showed that at least a part of
UVER is dependent on Rad2p (10), a FEN-1 homolog of S. pombe, which has also been implicated in a proliferating cell
nuclear antigen-dependent subpathway of BER (28). This
suggests the possibility that UVER and BER share their later steps in
nuclei. On the other hand, recent several works unequivocally showed
the existence of BER in mitochondria probably involving DNA polymerase
and mitochondrial DNA ligase (29-31). Although Rad2p itself is
unlikely to be involved in mitochondrial BER (and UVER) judging from
its amino acid sequence, it is highly possible that UVER and BER share
some steps also in mitochondria.
As mentioned above, the lack of mitochondrial DNA repair increases the
rate of petite induction by UV in S. cerevisiae (12, 13). We
expected that inactivation of mitochondrial DNA repair would lead to an
increased sensitivity of S. pombe to UV, since this species
has been long regarded as a petite-negative yeast and loss of the
mitochondrial genome was thought to result in cell death (but also see
Refs. 32 and 33). However, our results demonstrated that the defect in
mitochondrial UVER does not affect the UV survival.
The first question is why apparent absence of DNA repair in
mitochondria of the mutant S. pombe strains does not lead to
an increased UV sensitivity. Two explanations for this will be
possible. One is that any types of DNA damage tolerance systems in
mitochondria are irrelevant to UV resistance of S. pombe
cells. The other is that other type(s) of damage tolerance mechanisms
in mitochondria (which should not be damage repair in a true sense,
since we did not observe any damage removal in the absence of UVER)
mask the effect of UVER. Since the size of the nuclear genome is much
larger and the copy number is much lower than those of the
mitochondrial genome, one can easily imagine that the nuclear genome
would be much more sensitive to the same number of damage per unit
length of DNA than the mitochondrial genome. Under such conditions,
cells will die before the effect of the damage tolerance system in the mitochondria is realized. This way of argument supports the former explanation. In our experimental condition, however, 100 J/m2 UV induced about 10 CPDs in a single copy of S. pombe mitochondrial genome in average (data not shown). Taking
into account that the copy number of mitochondrial genome is several
hundred at most (34), this means that virtually any DNA molecules of
mitochondrial genome bear at least a few CPDs. At this UV dose, about
50% of wild type and mitochondrial UVER-deficient cells are still
survived (Fig. 4a). We also know from earlier work that CPDs
are a strong barrier to DNA replication and transcription in the
absence of any damage tolerance systems (35-38). This argues that the
former explanation is very unlikely, and suggests the possible
existence of other types of damage tolerance systems in the
mitochondria of S. pombe. Ling et al. (39)
described how, in S. cerevisiae mitochondrial DNA,
recombination has a important role in damage tolerance. If a similar
mechanism operates in the mitochondria of S. pombe, it would
be a good candidate for major mitochondrial damage tolerance. This
should be examined in a future study.
In the Introduction, we argued that completely redundant mechanisms
cannot be maintained during evolution. Following this argument,
mitochondrial UVER is expected to give an advantage at least under some
condition. The second question is what conditions would these be? In
contrast to UV, reactive oxygen species generated by respiration may
cause the damage preferentially in mitochondrial DNA. In this case
mitochondrial DNA repair would have a greater significance than for UV
damage. Recently we identified an apurinic/apyrimidinic endonuclease
activity of UVDE (40). This may suggest a possible involvement of UVER
in repair of oxidative damage on mitochondrial DNA and its contribution
to cell survival. Although we have not found any increased sensitivity
of uvde-disrupted cells against oxidative damage, this
possibility remains to be elucidated.
The copy number of the mitochondrial genome is known to fluctuate
depending on culture conditions. In S. cerevisiae, a well known phenomenon, glucose repression, also includes a decrease in
mitochondrial DNA copy number (41). This is quite reasonable since, if
a carbon source is abundant, energy generation does not have to depend
solely on respiration. Additionally, earlier work on S. pombe showed that the mitochondrial DNA content in exponentially
growing cell is much lower than that in stationary phase cells (34). As
discussed above, when the mitochondrial DNA copy number is lower,
mitochondrial DNA would have a greater significance. Mitochondrial UVER
might be crucial under such conditions. At this moment, we do not have
any positive evidence for this.
and
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Div. of Radiation Life
Science, Research Reactor Inst., Kyoto University, 1010 Ooaza-Noda,
Kumatori-Cho, Sennan-Gun, Osaka 590-0494, Japan. Tel.: 81-724-51-2392;
Fax: 81-724-51-2628; E-mail: shinji@rri.kyoto-u.ac.jp.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
