Nucleotideexcisionrepairratesinrat tissues
Anastas Gospodinov, Rumen Ivanov, Boyka Anachkova and George Russev
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria
We have determined and compared nucleotide excision
repair capability of several rattissues by a method based on
restoration of the transformation activity of UV-irradiated
pBlueScript by incubation in repair-competent protein
extracts. After 3 h of incubation, plasmid DNA was isolated
and used to transform competent Escherichia coli cells.
Damaged plasmids showed low transformation efficiency
prior to incubation in repair-competent extracts. After
incubation the transformation efficiency was restored to
different extents permitting calculation of the repair capacity
of the extracts. Our results showed that rapidly proliferating
tissues such as liver, kidney and testis showed higher nuc-
leotide excisionrepair capacity than slowly proliferating tis-
sues such as heart, muscle, lung and spleen. When liver and
splenocytes were stimulated to proliferation by partial hepa-
tectomy and mitogen stimulation, their repair capability
increased in parallel with the respective proliferative rates.
Keywords: DNA repair; DNA replication; protein extract;
rat tissues; UV irradiation.
DNA repair and particularly nucleotideexcision repair
(NER) is one of the primary pathways by which mamma-
lian cells remove a wide variety of DNA lesions. In the
process of repair, the products of more than a dozen genes
are involved in damage recognition, incision, elongation and
ligation of DNA to collectively restore its original structure
[1–3]. Many other cellular factors, normally participating in
transcription and replication, also play a role in the process
of DNA repair [4–6]. The NER pathway can recognize a
broad spectrum of helix-distorting lesions, such as UV-
induced pyrimidine dimers, bulky adducts, cross-links, etc.
and remove them by excision of oligonucleotide fragments
containing the damaged site [7]. In many cases the capacity
for DNA repair changes as a result of mutation of some of
the genes related to the repair process. The cases of severe
reductions of NER capacity due to mutations in some genes
are manifested phenotypically and are diagnosed as diseases
or syndromes such as xeroderma pigmentosum, Cockain
syndrome, trichothiodistrophy, etc. All of them are charac-
terized by decreased genetic stability and increased cancer
incidence [8]. The linkage between perpetual DNA damage
and oncogenic activity suggests that such persistence of
DNA damage is a reflection of the diminished involvement
of DNA repair activities in tumorigenesis. This suggests that
DNA repair proficiency could exist as an aetiologic
correlate for the general population at risk for cancer
[9,10]. This problem has not been addressed in a systematic
way and there are only few such investigations with
contradictory results. In most of these studies, DNA repair
was measured in peripheral human lymphocytes as they
were easy to collect and handle [11–15]. However, this
approach has at least two major drawbacks: (a) lympho-
cytes are not capable of NER unless stimulated to
proliferation [12], and (b) lymphocytes could have different
repair capability than target tissues. Several studies tried to
avoid these drawbacks by directly determining expression
rates of major NER genes intissues obtained by biopsy
or other means [16,17]. These results showed considerable
differences between the expression of the same genes
in different tissues as well as between the expression of
different genes in the same tissue. However, the results
obtained could not be directly related to repair capacity as it
is difficult to assess the contribution of the different genes in
the overall lesion removing process.
In this study our aim was to determine the NER rates of
different rattissues and to determine whether these rates
correlated with the proliferative status of the tissues. To this
end repair-competent cell extracts were prepared from the
target tissues and UV-damaged plasmid DNA was incuba-
ted in them. We used UV-damaged DNA molecules to
avoid mixing up the base excisionrepair (BER) and NER
pathways as UV-induced lesions are repaired only by NER,
while many other lesions such as single-strand breaks,
abasic sites, oxidative damages, etc., could be repaired by
both BER and NER pathways [18]. The repairrates were
calculated by comparison of the transformation efficiency of
the damaged plasmids before and after incubation in the
extracts. Our results indicated that rapidly proliferating
tissues exhibited higher NER rates than slowly proliferating
tissues. Upon stimulation to proliferation the repairrates of
the tissues increased.
Materials and methods
Cells
K562 cells were obtained from ATCC and the XPA
lymphoblastoid cell line was obtained from NIGMS
Human Genetic Cell Repository. They were grown in
Correspondence to G. Russev, Institute of Molecular Biology,
Bulgarian Academy of Sciences, Academic G. Bonchev Street,
block 21, 1113 Sofia, Bulgaria. Fax/Tel.: + 359 2 72 35 07,
E-mail: grs@obzor.bio21.bas.bg
Abbreviations: BER, base excision repair; NER, nucleotide excision
repair; TdR, thymine desoxyriboside.
Note: A web site is available at http://www.bio21.bas.bg/imb/
(Received 19 November 2002, revised 15 January 2003,
accepted 21 January 2003)
Eur. J. Biochem. 270, 1000–1005 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03473.x
suspension in RPMI 1640 medium, containing 4.5 mgÆmL
)1
glucose in the case of K562. The media were supplemented
with 10% foetal bovine serum and antibiotics and cells were
grown in an atmosphere of 95% air/5% CO
2
at 37 °C.
Splenocytes were prepared from freshly obtained rat spleens
by squeezing the tissue through a metal mesh under sterile
conditions. The cell suspension was overlayered onto 3 mL
of Lymphoprep (Sigma) and centrifuged at 180 g for
10 min. Splenocytes were collected from the interphase,
washed with NaCl/P
i
, suspended in RPMI 1640 supple-
mented with 10% fetal bovine serum and stimulated to
proliferation with 5 lgÆmL
)1
each phytohemagglutinin,
concanavalin A and lipopolysaccharide (Sigma-Aldrich).
[
3
H]Thymine desoxyriboside (TdR) was added at different
intervals after stimulation and 1 h later splenocytes were
used to prepare repair-competent extracts. DNA was
isolated from the high-speed pellet obtained in the course
of cell extract preparations and aliquots were counted in
Beckman scintillation counter.
Tissues
Wistar albino rats (200 g) were killed under ether anaes-
thesia by dislocation of the cervical cord and the tissues were
immediately removed and rinsed in ice cold NaCl/P
i
.Partial
hepatectomy was performed under ether anaesthesia by
removing two-thirds of the liver. At 6-h intervals after the
operation animals were injected intraperitoneally with
200 lCi [
3
H]TdR and 1 h later were sacrificed and livers
were used for preparation of repair-competent extracts and
DNA for counting.
Protein extracts
Protein extracts were prepared as described in [19] with
modifications. Cells were rinsed twice in ice-cold NaCl/P
i
,
resuspended in hypotonic buffer (10 m
M
Tris/HCl,
pH 8.0; 1 m
M
EDTA and a tablet of Complete
TM
protease inhibitor cocktail per 50 mL solution, Roche
Diagnostics GmbH) and left on ice for 20 min to swell.
Cells were then homogenized by 20–30 strokes in a
Dounce homogenizer.
When solid tissues were used 0.4–0.5 g of the material
were finely minced, twice rinsed in ice cold NaCl/P
i
and
4 mL of hypotonic buffer added to the pellet. This crude
suspension was immediately homogenized in a Teflon pestle
homogenizer.
To each cell lysate 4 mL of an ice cold solution
containing 50 m
M
Tris/HCl pH 8.0, 10 m
M
MgCl
2
,2m
M
dithiothreitol, 25% sucrose, 50% glycerol were added
slowly with stirring. One ml neutralized saturated ammo-
nium sulfate was then added with gentle mixing. The
mixture was kept on ice with occasional stirring for
30 min, and then centrifuged at 25 000 r.p.m. in a
Beckman SW41 rotor at 2 °C for 3 h. The upper two-
thirds of the supernatant were withdrawn and the protein
was precipitated by the addition of 0.33 gÆmL
)1
neutral-
ized ammonium sulfate. The precipitate was collected by
centrifugation, resuspended in a minimum volume of
buffer containing 25 m
M
Hepes/KOH pH 7.9, 0.1
M
KCl,
12 m
M
MgCl
2
,1m
M
EDTA, 2 m
M
dithiothreitol, 17%
glycerol. The extract was desalted using a Sephadex G-25
coarse column and the total protein concentration deter-
mined by the Bradford reaction.
Plasmids and irradiation
Plasmids pBlueScript SK+ (Stratagene) and pEGFP-N1
(Clontech) were propagated in Escherichia coli strain XL-1
Blue.
The UV irradiation of pBlueScript and pEGFP-N1 was
carried out with a germicidal mercury lamp as described by
Rougev and Russev [20]. Plasmid DNA was dissolved in
Tris/EDTA buffer to a final concentration of 100 lgÆmL
)1
.
It was poured into Petri dishes to form 1–2-mm thick layer,
placed on ice and irradiated from a 10-cm distance for 1, 2, 4
and 10 min. With these irradiation conditions the exposure
rate was 8.3 JÆm
)2
Æs
)1
as measured with a UV power energy
meter (Scientech 362) and plasmid DNA received doses of
0.5, 1, 2 and 5 kJÆm
)2
, respectively.
Plasmid pEGFP-N1 irradiated with different doses of UV
light was linearized by EcoRI digestion and used as a
template to amplify a 1.05-kb fragment (nt 440–1490) using
the primers: 5¢-CCATTGACGTCAATGGGA-3¢ and
5¢-AGGTTCAGGGGGAGGT-3¢. Twelve PCR cycles were
run using the following parameters: denaturation at 95 °C
for 60 s, annealing at 59 °C for 45 s and extension at 72 °C
for 60 s. Amplified products were run on a 1% agarose gel,
stained with ethidium bromide and quantified with Gel Pro
Analyzer v.3 software for Windows (Media Cybernetics).
DNA repair reactions
A50-lL reaction containing 300 ng UV irradiated pBlue-
Script repair substrate, 300 ng unirradiated pEGFP internal
control, 45 m
M
Hepes/KOH pH 7.8, 70 m
M
KCl, 7.4 m
M
MgCl
2
,0.9m
M
dithiothreitol, 0.4 m
M
EDTA, 2 m
M
ATP,
25 l
M
each of dGTP, dATP, dTTP and dCTP, 40 m
M
phosphocreatine, 2.5 lg creatine phosphokinase, 18 lg
bovine serum albumin, 100 lg cell-extract protein were
incubated at 30 °C for the specified intervals. Repair was
stopped by the addition of equal volume of 40 m
M
EDTA,
100 m
M
Tris/HCl pH 8.0. Following incubation with
100 lgÆmL
)1
RNaseAfor15min,SDSwasaddedto
0.5% and proteinase K to 200 lgÆmL
)1
and proteins were
digested for 1 h at 37 °C. The mixture was extracted with
phenol/chloroform (1 : 1) once, and DNA from the aque-
ous phase precipitated with 0.8 vols isopropanol and
0.1 vol. 3.3
M
ammonium acetate. Plasmids were dissolved
in Tris/EDTA, electrophoresed in a 1% agarose gel, stained
with 0.5 lgÆmL
)1
ethidium bromide and their concentration
was adjusted.
Bacteria and transformation
E. coli strains XL-1 Blue and the UV-sensitive strain
SOLR-UvrC were obtained from the Bulgarian Cell Type
Collection. Transformation was carried out by adding 20 ng
plasmid DNA to 200 lL competent E. coli. After 10 min
on ice, bacteria were heat-shocked at 42 °C for 30 s and
placed on ice for additional 10 min. One mL unselective
medium was added to each transformation and incubated
for 30 min at 37 °C. Bacteria were plated on Petri dishes
with solid Luria–Bertani medium (10 g Bacto tryptone, 5 g
Ó FEBS 2003 Nucleotideexcisionrepairratesinrattissues (Eur. J. Biochem. 270) 1001
yeast extract, 10 g NaCl, 15 g Bacto agar per litre)
containing 100 lgÆmL
)1
ampicillin or 50 lgÆmL
)1
kana-
mycin and grown for 12 h at 37 °C.
Results and discussion
Validation of the repair assay
To assess the repair capacity of cells and tissues we prepared
repair-competent extracts and monitored the repair of UV-
irradiated BlueScript plasmid DNA incubated in them. We
normalized the repairrates against the total amount of
protein in the extracts rather than against any specific
housekeeping protein as the expression of individual
proteins varies in different tissues or in the same tissue in
different physiological states. The main products of UV
irradiation are pyrimidine dimers and pyrimidine (6–4)-
photoproducts. Both types of lesions are effective blocks of
Taq polymerase in PCR [21,22] and both are repaired by the
NER pathway [23–25]. To calibrate the lesions, pEGFP-
N1 was irradiated with 0.5, 1, 2 and 5 kJÆm
)2
and after
linearization with EcoRI was used as template for quanti-
tative PCR to amplify the 1-kb fragment containing the
EGFP gene. By using the Poisson formulae which gives
the relationship between the average number of lesions and
the proportion of the undamaged fragment, we were able to
calculate that under our conditions irradiation with
1kJÆm
)2
produces, on average, 0.5–1 lesions per kb in the
plasmid DNA. This figure was in agreement with other
recent data showing that the rate of cyclobutan pyrimidine
dimer and pyrimidine (6–4) pyrimidone product [(6–4)PP]
formation upon irradiation with UV light was within the
range 0.3–1.2 lesions per kb per 1 kJÆm
)2
[26,27].
There are data showing that plasmids carrying UV
lesions are seldom replicated in the absence of SOS
induction of E. coli, and do not support colony formation
on selective medium [28,29]. This permits the establishment
of a direct link between the number of colonies obtained
after transformation of E. coli cells with irradiated plasmid
and the amount of native plasmid molecules in the sample,
and opens the possibility to calculate the repair capacity of
the extracts. We used irradiated BlueScript and control
EGFP-N1 plasmids with and without incubation in K562
extracts to transform competent E. coli cells (Fig. 1). The
transformation efficiency of the UV irradiated pBlueScript
was expressed relative to the transformation efficiency of the
control undamaged pEGFP assuming the ratio of pBlue-
Script (ampicillin resistant) colonies to pEGFP (kanamycin
resistant) colonies in the case when cells were transformed
with mixture of undamaged pEGFP and undamaged
pBlueScript in a ratio of 1 : 1 (w/w) was 100%. As shown
in Fig. 1 the K562 extract repaired plasmid DNA at an
almost constant rate up to the third hour, after which it
slowed considerably. To demonstrate the direct relationship
between colony formation potential of the irradiated
plasmids and the number of DNA lesions, we carried out
two control experiments. First we incubated the irradiated
pBlueScript in an extract from repair-deficient XPA cells
and then used the plasmid DNA for transformation of
E. coli. In this case the pBlueScript irradiated with 1 kJÆm
)2
showed 5–10% transformation efficiency in comparison
with the undamaged plasmid and this did not change with
the time of the incubation (Fig. 1). Using the Poisson
equation we were able to calculate that this plasmid
contained 0.9 UV lesionsÆkb
)1
, which is in reasonable
agreement with the figures obtained with the PCR assay,
and with data in the literature [26,27]. This experiment
showed that the increase in the number of colonies
obtained after incubation of the irradiated plasmid in
repair-competent cell extracts was a result of DNA repair
that had taken place in the extract.
Second, we transformed in parallel the E. coli XL-1 Blue
strain and the UvrC-deficient SOLR E. coli strain, which is
unable to repair UV lesions, with irradiated plasmids. There
were no differences in the colony formation efficiency with
the repair-competent XL1 Blue and repair deficient SOLR
E. coli strains (data not shown), which showed that no
DNA repair was taking place in the host E. coli cells. This
confirmed the finding that in the absence of SOS induction
E. coli cells were not able to replicate damaged plasmid
molecules [28,29].
NER ratesinrat tissues
We next prepared repair competent extracts from several rat
tissues and determined their repairrates by using the
described protocol. To this end we determined the number
of lesions per kb of irradiated plasmid DNA prior to and
after incubation in the respective extracts for 3 h and the
Fig. 1. NER in protein extracts from K562 cells (d) and XPA cells (s).
A 1 : 1 (w/w) mixture of pBlueScript UV irradiated at 1 kJÆm
)2
and
untreated pEGFP-N1 were incubated in K562 and XPA extracts. At
different time intervals aliquots were withdrawn, plasmid DNA was
isolated and used to transform competent E. coli cells. Transformed
cells were plated on ampicillin- or kanamycin-containing agar and 12 h
later the number of colonies was counted. The number of ampicillin-
resistant colonies was expressed as percentage of the kanamycin-
resistant colonies, assuming that the ratio of the ampicillin resistant to
kanamycin resistant colonies when both plasmids were unirradiated
was 100%. The results are means of six independent experiments and
the standard deviations from the means are shown by error bars.
1002 A. Gospodinov et al. (Eur. J. Biochem. 270) Ó FEBS 2003
difference was taken as the number of lesions repaired by
the extracts (Fig. 2). The results showed that different rat
tissues differ in their ability to repair UV lesions. Kidney,
testis and liver showed higher repair capability close to the
repair capability of transformed K562 cells. Muscle, lung
and brain showed somewhat lower repair capability, heart
showed even lower repair capability and finally splenocytes
showed practically no repair capability. This arrangement
roughly corresponds to the replication activity of the
respective tissues. Thus, kidney, liver and testis are consid-
ered rapidly proliferative tissues, while muscle, heart, brain,
lung and spleen are considered slowly proliferating tissues.
Similar results have been reported by others, who also had
found that the rapidly proliferative tissues such as skin,
testis, ovary, breast, liver, kidney, colon, etc. show higher
relative expression of five NER genes than the slowly
proliferative tissues such as muscle, heart, brain, spleen,
lung, etc. [16].
Cell proliferation and NER
To investigate further the observed relationship between
proliferation activity and NER, we carried out experiments
in which we determined the repairratesin two tissues
in norma and upon stimulation to proliferation. First we
took advantage of the fact that rat liver is capable of intense
regeneration after partial hepatectomy and followed both
DNA synthesis and repair capacity of regenerating rat livers
in the course of the first 36 h after a two-thirds hepatectomy.
In agreement with the literature data, a sharp wave of DNA
synthesis was observed between 16 and 24 h of the liver
regeneration. Simultaneously an equally sharp increase of
the liver’s ability to repair UV lesions was registered
(Fig. 3). In a second experiment we isolated rat splenocytes
and determined their repair capability prior to and after
stimulation to proliferation with mixture of lectins. Here
again we observed a synchronous wave of DNA synthesis
about 24 h after the mitogen stimulation and a simulta-
neous sharp several-fold increase of the repair rate in rat
splenocytes. (Fig. 3).
It is well established that DNA replication and DNA
repair pathways share many common proteins including
DNA polymerases. The activity of the DNA polymerases is
of crucial significance in the process of DNA repair, as
without the proper polymerase the end-product of most
repair pathways, i.e. the repaired DNA, will not be
produced, even if all other repair proteins are present and
active. It is especially relevant that the gap-filling step in
NER is carried out by the replicative polymerases -d and -e,
Fig. 2. NER ratesinrat tissues. Mixtures of pBlueScript irradiated at
1kJÆm
)2
and unirradiated pEGFP-N1 were incubated in cell extracts
prepared from different rattissues for 3 h and were used to transform
XL-1 Blue E. coli cells. Repair capacity of the tissues was expressed in
number of lesions per kb of DNA removed during the incubation and
were determined from the number of colonies calculated as in Fig. 1 by
usingthePoissonformula.Tissues:1,testis;2,kidney;3,liver;4,lung;
5,muscle;6,brain;7,heart;8,splenocytes.Theresultsaremeansfrom
six animals and the standard deviations are shown by error bars.
Fig. 3. NER and DNA replication ratesin normal and regenerating rat
liver and unstimulated and stimulated rat splenocytes. Wistar albino rats
were subjected to partial hepatectomy and the excised parts of the
livers were used to prepare repair competent extract. After 24 h the
animals were sacrificed and the regenerating livers were removed and
again used to prepare repair-competent extract. To follow DNA syn-
thesis one group of animals was injected intraperitoneally with 200 lCi
[
3
H]TdR 1 h before the operation and the second group of animals was
injected with the same dose 1 h before sacrifice. DNA was isolated and
counted and the radioactivity was expressed as countsÆmg
)1
DNA.
Splenocytes were isolated from the spleens of rats and cultured in
RPMI 1460. Aliquots were labelled with 1 lCiÆmL
)1
[
3
H]TdR for 1 h
and used to prepare repair competent cell extract and to isolate DNA.
The remaining splenocytes were stimulated with mitogens as described
in Materials and methods and 24 h later were labelled with 1 lCiÆmL
)1
[
3
H]TdR for 1 h and were used to prepare repair competent extracts
and DNA. pBlueScript irradiated at 1 kJÆm
)2
and control pEGFP
were incubated in the liver and splenocyte extracts and used to
transform E. coli. Repair capacity (black columns) of liver tissue and
splenocytes were determined as in Fig. 2 and the proliferative rates are
shown by the
3
H counts incorporated in DNA (shadowed columns).
1, Normal liver; 2, regenerating liver; 3, normal splenocytes; 4, sti-
mulated splenocytes. The results are means of six independent
experiments and the standard deviations from the mean are shown
with vertical error bars.
Ó FEBS 2003 Nucleotideexcisionrepairratesinrattissues (Eur. J. Biochem. 270) 1003
which are mostly expressed in proliferating cells [6,30].
This explains why quiescent, or slowly proliferating
tissues, where the levels of these enzymes are low, are
less effective in NER, although they may express most of
the NER specific proteins [12]. On the other hand as BER
is using polymerase-b, whose expression is relatively
independent of the proliferative state of the cells, this
repair pathway is efficient in both proliferative and
quiescent tissues and this explains why lymphocytes can
remove a number of lesions repaired by it [31,32]. Apart
from the DNA polymerases, products of other genes
could be also involved in the relationship between DNA
replication and repair. Recent reports show that the
transcription of the major repair genes differ during
development and follow distinct temporal patterns that
are functionally linked to initiation of DNA replication
and progression through S phase [17,30,33]. It has also
been reported that PCNA, which is expressed only in
proliferating cells, is required for normal repair activity
[34,35].
Our results show that there is a considerable difference
in the NER rates of rattissues and that it is dependent on
the proliferative activity of the tissues. In this respect we
assume that it is not correct to deduce the NER capacity
of any tissues from that of other surrogate tissues.
However, when this has to be done, surrogate tissues
should be chosen that have the same proliferative activity
as the target tissues, which would ensure that their NER
rates would be similar.
Acknowledgements
This work was supported by the Bulgarian National Scientific Council
(grant MU-K-1002/00 to A.G) and the European Commission (grant
QLK4-1999–01629 to G.R).
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NER rates in rat tissues
We next prepared repair competent extracts from several rat
tissues and determined their repair rates by using the
described. results indicated that rapidly proliferating
tissues exhibited higher NER rates than slowly proliferating
tissues. Upon stimulation to proliferation the repair