Genetic Instability and Cancer, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse Cedex, France
* Authors for correspondence (e-mail: jseb{at}ipbs.fr; cazaux@ipbs.fr)
Accepted 5 September 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Pol, DNA replication, DNA mutagenesis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A current working hypothesis suggests that when replicative complexes are
stopped at DNA lesions these errorprone DNA polymerases interfere with the
replication machinery to facilitate lesion bypass by acting sequentially,
perhaps by replacing for the replicative polymerases. For instance Pol ,
which is active in the accurate bypass of UV lesions
(Johnson et al., 1999
),
associates with PCNA foci in UV irradiated cells
(Kannouche et al., 2001
) and
interacts with the proliferating cell nuclear antigen (PCNA)
(Haracska et al., 2001a
), a
ring-shaped protein forming a clamp at primer-template junctions of the
replication machinery (Kornberg and Baker,
1992
). Pol
, which incorporates nucleotides opposite abasic
sites or the 3'T of the (6,4) T-T photoproduct
(Johnson et al., 2000b
), also
binds the replication machinery via an interaction with PCNA
(Haracska et al., 2001b
).
The recently discovered (Johnson et
al., 2000a) human DNA polymerase
, (Pol
, previously
named Pol
), is a homologue of the Escherichia coli DinB (Pol
IV) protein, a SOS protein involved in untargeted UV-induced mutagenesis of
bacteriophage
(Brotcorne-Lannoya
and Maenhaut-Michel, 1986
) and whose activity is stimulated in
vitro by PCNA (Haracska et al.,
2002
). In vitro, Pol
can bypass certain DNA lesions
including 1,N6-ethenodeoxyadenosine or acetylaminofluorene-derived
DNA adducts by generating base substitutions and deletions
(Gerlach et al., 1999
;
Levine et al., 2001
;
Ohashi et al., 2000b
;
Suzuki et al., 2001
),
8-oxo-7,8-dihydrodeoxyguanosine by incorporating dAMP opposite the lesion
(Zhang et al., 2000
) and
(-)-trans-anti-benzo[a]pyrene-N2-dG in an error-free manner
(Zhang et al., 2000
). In
contrast, Pol
is unable to perform translesion synthesis opposite
either a cisplatin adduct (Ohashi et al.,
2000b
), a cis-syn TT dimer or a TT (6-4) photoproduct
(Johnson et al., 2000a
;
Ohashi et al., 2000b
;
Zhang et al., 2000
), although
it may extend from a G opposite the 3'T of a TT dimer
(Washington et al., 2002
).
Moreover Pol
was shown to be unable
(Johnson et al., 2000a
) or
inefficient (Ohashi et al.,
2000b
; Suzuki et al.,
2001
) in bypassing abasic sites. Here we examine whether
Pol
can associate with the replication machinery by determining its
cellular localisation in the presence or absence of antireplicative agents. We
have used PCNA as a cellular marker in order to identify replisome-containing
foci. Our data indicate that Pol
is present at replication forks in
some human MRC5 fibroblasts cells in the absence of blocking lesions as well
as in most MRC5 cells when replication forks are stalled. These findings thus
suggest that Pol
is involved in the replication machinery of untreated
cells and, as already shown for Pol
and Pol
, could be recruited
into replisomes at replication arrests.
Upregulation of Pol has been recently found in lung tumours in
comparison with adjacent nontumorous tissues
(O-Wang et al., 2001
), and has
been observed by Serial Analysis of Gene Expression (SAGE)
(Velculescu et al., 1995
) in
human ovarian and prostate cancers
(http://www2.ncbi.nlm.nih.gov/SAGE/,
Cancer Genome Anatomy Project from NCBI), indicative of a role in tumor
development. In view of the potential presence of Pol
at replication
forks, we investigated in the present study the role of the enzyme in
mutagenesis. Our findings are discussed with reference to the mutagenic impact
of high levels of hPol
on cell proliferation and carcinogenesis
processes.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell lines and DNA transfection
MRC5 (ATCC) and XP12ROSV40 (provided by B. Salles, France) cells were grown
in MEM alpha (Gibco) medium supplemented with 10% foetal calf serum,
penicillin and streptomycin. Tranfection of plasmids was performed using
Lipofectamin Plus reagent (Gibco).
Irradiation and drug treatment
Cells in PBS were irradiated using a 254 nm UV lamp. For drug treatment,
cisplatin was diluted in complete medium to a final concentration of 30 µM
and added onto the transfected cells for 1 hour. After 1 hour the cells were
washed with PBS and incubated in complete medium. For hydroxyurea treatment,
the cells were incubated for 12 hours with complete medium containing 1.5 mM
hydroxyurea, washed with PBS and incubated in drug free complete medium for 8
hours.
Immunofluorescence microscopy
For the localisation of eGFP or eGFP-pol, cells were grown on
coverslips, washed in PBS and fixed in 4% formaldehyde for 30 minutes at
4°C, then rinsed three times in PBS and mounted in Mowiol. To visualise
the eGFP-pol
and PCNA proteins at the same time, cells were fixed in 4%
formaldehyde for 30 minutes at 4°C and rinsed three times in PBS.
Permeabilisation was then performed by incubating the cells for 10 minutes at
-20°C in the presence of cold methanol. They were then washed before
treatment with 0.25% Triton X-100 for 5 minutes at room temperature. After
washing three times with PBS, the cells were blocked for 10 minutes at room
temperature in FCS 1% in PBS and incubated for 1 hour with primary antibody
against PCNA diluted 1:200 with FCS 1% in PBS (PC10 monoclonal anti-PCNA,
Dako). Finally they were washed three times over a 3 minutes period in FCS 1%
in PBS, incubated for 30 minutes with TRITC conjugated goat anti-mouse IgG
(Sigma), and washed three times more in PBS before mounting. More than 1000
fluorescent cells were examined in each experiment.
Cell cycle analysis
Cells were grown to 80% confluency and stained with propidium iodide. DNA
content was evaluated using a FACScan flow cytometer (Beckson Dickinson).
Histograms were analysed using ModFit cell cycle analysis software.
Selection of Pol-overexpressing MRC5 cells
The Tet-Off gene expression system (Clontech) was used to obtain regulated,
high-level overexpression of Pol. Human pol
cDNA was
PCR-amplified from the pHSE2 plasmid using Pfu polymerase and the
primers, 5'-CCggatccTCAGATAAGTTTATA-3' and
5'-CCatcgatATGATAAAATGTTCA-3'. The PCR product was then digested
using BamHI and ClaI before insertion into the
BamHI-ClaI sites of the pTRE2 (Clontech), which carries the
Tet operator sequence and the CMV minimal promoter, to obtain the
pTRE2-pol
plasmid. MRC5 cells were transfected according to the
manufacturer's protocol (Lipofectamin, Gibco) with the pTet-Off vector which
carries the regulatory protein TetR/HSV-VP16. A stable transfectant clone
displaying the highest level of Doxycyclin (Dox)-dependent induction was
screened out by transient transfections of more than forty clones using an
inducible pTRE2-luc vector carrying the luciferase gene. The selected clone
was then stably transfected with pTRE2-pol
plasmid to select for
Pol
-overexpressing MRC5 cells.
Quantitative RT-PCR analysis
RNA was extracted from cells using TriReagent (GibcoBRL). 120 ng RNA were
incubated with 0.2 mM dNTPs, 1 mM MgSO4, 0.25 u AMV reverse
transcriptase, 0.25 u Tfl ADN polymerase and specific primers for
pol and hprt. The RT-PCR reaction was performed at
48°C for 45 minutes, 94°C for 2 minutes and then at 94°C for 30
seconds, 52°C for 1 minute, and 68°C for 45 seconds for 30 cycles.
After the first 94°C incubation, [
32P]dGTP was added and
12 µl aliquots were removed after 10, 20 and 30 cycles. Gel electrophoresis
was then carried out in 2% agarose. The gel was dried and scanned in a
phosphoimager (Storm 840, Molecular Dynamics).
Mutagenesis experiments
Experiments were carried out as described
(Canitrot et al., 1998) and
mutant frequencies were corrected for plating efficiency.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Antireplicative treatments increase the local concentration of eGFP-
Pol
When the replicative forks were arrested following treatment with
hydroxyurea (HU), an agent which halts the biosynthesis of dNTPs from rNTPs,
we observed a dramatic increase in the number of cells containing fluorescent
Pol nuclear spots. Twelve hours after treatment, the proportion of
cells containing spots increased from 20% for untreated controls to 70% for
cells treated with HU. Moreover, these foci colocalised with PCNA in the
presence of the antimetabolic drug (Fig.
2Ab,c). When treated cells were rinsed to remove HU we found that
the number of cells with eGFP-pol
foci dropped to 10%
(Fig. 2Ba) and that 90% of the
remaining cells did not display GFP and PCNA foci
(Fig. 2Bb,c). Taken together,
these results indicate that eGFP-pol
foci are associated with
HU-mediated stalled replication forks.
|
To further demonstrate the relation between replicative arrests and
hPol-containing foci, the pol
-transfected cells were exposed to
UV irradiation (Fig. 3A) or
treated with cisplatin (Fig.
3B), both of which one known to induce bulky DNA adducts not
bypassed in vitro by Pol
. The proportion of transfected cells
containing foci increased following both genotoxic treatments reaching maximal
respective values of 75% and 65% six hours after 10 J/m2 UV
irradiation (Fig. 3Aa) or one
hour incubation in the presence of cisplatin
(Fig. 3Ba). These foci all
colocalised with PCNA in both cases (Fig.
3A,Bb,c), further supporting the view that the eGFP-Pol
protein is locally concentrated near PCNA-containing replicative complexes
arrested by UV or cisplatin damage.
|
We then conducted experiments with the XPA-deficient XP12R0 cells, that are
unable to excise UV lesions. In such cells, the number of non-coding UV
adducts is increased in comparison with control MRC5 cells where many lesions
are repaired. We thus observed that the number of foci-containing cells was
higher for the XP12R0 cells as compared to the MRC5 cells following UV
irradiation (Fig. 4). For
example, at 2 J/m2, we observed a 2-fold increase in the number of
foci in XP cells. In the XP12R0 cells, all the eGFP-Pol foci
colocalised with the PCNA spots (data not shown). These data further indicate
that hPol
colocalises near replication forks when the replisomes are
stopped and does not seem to be present in complexes controlling repair
synthesis following the excision of UV or cisPt adducts.
|
Excess hPol induces spontaneous mutagenesis
In view of the error-prone characteristics of Pol in vitro and its
potential involvement at replication forks in MRC5 human cells, it is
reasonable to suppose that its overexpression might affect the fidelity of DNA
replication in these cells. We first selected a single clone stably
overexpressing hPol
mRNA by 2.1-fold, as estimated
semi-quantitatively by RT-PCR (Fig.
5A). Cells from this selected clone were then incubated in the
absence of doxycycline (Dox) in order to maintain high intracellular levels of
Pol
. It should be noted that the Pol
levels remained constant
during the incubation (data not shown). 6-thioguanine was then added and
hprt mutants counted a week later. A 7-day and 52-day incubation of
Pol
-overexpressing cells led to 4.6- and 16.1-fold respective increases
in the number of hprt mutants
(Fig. 5B), compared to control
8-4 cells incubated in the presence of doxycycline. In an additional control
experiment, mutagenesis rates were examined in 8-TRE2 cells that are similar
to 8-4 cells but which contain both pTet-Off and an empty pTRE2 plasmid.
Fig. 5B shows that these cells
are not mutated in the presence or absence of doxycycline as compared with
parental MRC5 cells, indicating that ectopic Pol
expression is specific
in its impact. The data suggest that excess Pol
induces spontaneous
mutagenesis.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By analysing the intracellular localisation of a eGFP-Pol fusion
protein in human fibroblasts, we found, in cells probably in S-phase, that the
tagged Pol
is present in nuclear foci containing the essential
replicative cofactor PCNA. We demonstrated that under conditions in which
replication is arrested, the number of cells harbouring eGFP-hPol
foci
increases. These findings suggest that Pol
might be spontaneously part
of the replication machinery although we cannot rule out the possibility that
only specialised replicative forks, such as those replicating heterochromatin,
or secondary DNA structures, require Pol
. They also suggest that
Pol
could be recruited when DNA replication is halted by certain
lesions.
It has been shown that the yeast DNA polymerase Pol is an efficient
extender of base-pair mismatches in undamaged DNA or blocking lesions in
damaged DNA (Johnson et al.,
2000b
). Human Pol
has also been shown to possess similar
properties (Washington et al.,
2002
). It can extend mispaired termini on untreated DNA with high
frequency (10-1-10-2) and can also extend from a G base
opposite the 3'T of a cis-syn T-T dimer
(Washington et al., 2002
). The
role of Pol
in replicative foci may therefore be to extend mispairs or
to promote full bypass replication following the action of other DNA
polymerases such as Pol
or Pol
in the case of UV lesions.
PCNA endows polymerase with high processivity
(Kornberg and Baker, 1992
) and
seems also to be a Pol
cofactor late in the S phase
(Fuss and Linn, 2002
). As has
been previously suggested for Pol
and Pol
(Haracska et al., 2001a
;
Haracska et al., 2001b
), our
data are consistent with the possibility that PCNA could target Pol
to
replication machinery stalled at some lesion sites, thereby allowing
replication to resume. This hypothesis is supported by recent work showing
that purified Pol
and PCNA proteins are able to form a complex in
vitro, PCNA stimulating the Pol
-mediated DNA synthesis
(Haracska et al., 2002
).
Pol makes about nine errors when replicating one kilobase of
undamaged DNA in vitro (Ohashi et al.,
2000a
). We hypothesised that the involvement of an error-prone DNA
polymerase in replication may affect accuracy when overexpressed in vivo. Ogi
et al. have reported that transient expression of mouse pol
cDNA in untreated murine cells results in increased mutagenesis
(Ogi et al., 1999
). Here we
provide evidence that the controlled and stable expression of human Pol
in human cells also induces spontaneous mutagenesis. Previously we have
published similar data for another error-prone DNA polymerase, Polß
(Canitrot et al., 1998
) and we
have also demonstrated that excess Polß interferes with the replication
machinery (Servant et al.,
2002
). In the light of the data presented here, it is reasonable
to assume that excess Pol
might similarly interfere with replisomes and
affect their capacity to correctly copy DNA.
The intracellular balance between error-free and error-prone DNA
polymerases appears to be of great importance within the context of genetic
integrity. Because of its high mutagenic incidence, misregulated expression of
an error-prone DNA polymerase may generate variant cells able to rapidly
proliferate and reduce therapeutic efficacy. For example, deficiency in
Pol, which is involved in the accurate bypass of UV lesions, causes skin
cancers because of the inability of the cell to properly copy pyrimidine
dimers (Johnson et al., 1999
;
Masutani et al., 1999
). We
have shown that Polß, which is over produced in some human cancer tissues
(Srivastava et al., 1999
), is
also associated with cell proliferation and tumour progression
(Louat et al., 2001
). In the
case of Pol
, which was found to be upregulated in lung tumors relative
to physically adjacent non tumorous tissue
[(O-Wang et al., 2001
),
unpublished data from our group], and in human ovarian and prostate tumour
cell lines (Cancer Genome Anatomy Project), its error-prone features may also
favour cancer progression. It has been suggested that an x-fold
increase in an in vivo mutation rate could increase cancer incidence by a
factor of xn, where n is the number of mutations
required to develop a tumour (Yao et al.,
1999
). The 7-9-fold increase in the number of mutations that we
observed here as a result of a limited 2-fold overexpression of Pol
shows how a weak misregulation might exert a significant influence on the
progression of cancer.
DNA repair genes are currently considered as tumour suppressors since their
deficiency is often related to cancer susceptibility. The root sources of the
loss of genomic integrity are however multiple. Alterations in DNA replication
genes, which are represented in human cells as DNA repair genes [Online
Mendelian Inheritance in Man, OMIMTM (2000) McKusick-Nathans Institute
for Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National
Center for Biotechnology Information, National Library of Medicine (Bethesda,
MD)
(http://www.ncbi.nlm.nih.gov/omim)],
or misregulation of their protein expression, could also contribute to tumour
genomic heterogeneity. Further studies are however required to determine which
human cancers might be associated with an upregulation of error-prone DNA
polymerases such as Pol or the expression of hyperactive mutants. An
understanding of why error-prone polymerases are abnormally expressed in some
cancer cells, and whether the protein is induced by endogenous and/or
exogenous stress will be the next challenge.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brotcorne-Lannoya, A. and Maenhaut-Michel, G. (1986). Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene. Proc. Natl. Acad. Sci. USA 83,3904 -3908.[Abstract]
Canitrot, Y., Cazaux, C., Frechet, M., Bouayadi, K., Lesca, C.,
Salles, B. and Hoffmann, J. S. (1998). Overexpression of DNA
polymerase beta in cell results in a mutator phenotype and a decreased
sensitivity to anticancer drugs. Proc. Natl. Acad. Sci.
USA 95,12586
-12590.
Friedberg, E. C., Feaver, W. J. and Gerlach, V. L.
(2000). The many faces of DNA polymerases: strategies for
mutagenesis and for mutational avoidance. Proc. Natl. Acad. Sci.
USA 97,5681
-5683.
Fuss, J. and Linn, S. (2002). Human DNA
polymerase epsilon colocalizes with PCNA and DNA replication late, but not
early in S phase. J. Biol. Chem.
277,8658
-8666.
Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin,
E. V. and Friedberg, E. C. (1999). Human and mouse homologs
of E. coli DinB members of the UmuC/DinB superfamily. Proc. Natl.
Acad. Sci. USA 96,11922
-11927.
Haracska, L., Johnson, R. E., Unk, I., Phillips, B., Hurwitz,
J., Prakash, L. and Prakash, S. (2001a). Physical and
functional interactions of human DNA polymerase eta with PCNA. Mol.
Cell. Biol. 21,7199
-7206.
Haracska, L., Johnson, R. E., Unk, I., Phillips, B. B., Hurwitz,
J., Prakash, L. and Prakash, S. (2001b). Targeting of human
DNA polymerase iota to the replication machinery via interaction with PCNA.
Proc. Natl. Acad. Sci. USA
98,14256
-14261.
Haracska, L., Unk, I., Johnson, R. E., Philips, B. B., Hurwitz,
J., Prakash, L. and Prakash, S. (2002). Stimulation of DNA
synthesis activity of human DNA polymerase kappa by PCNA. Mol.
Cell. Biol. 22,784
-791.
Johnson, R. E., Prakash, S. and Prakash, L.
(1999). Efficient bypass of a thymine-thymine dimer by yeast DNA
polymerase Poleta. Science
283,1001
-1004.
Johnson, R. E., Prakash, S. and Prakash, L.
(2000a). The human DinB1 gene encodes the DNA polymerase Polq.
Proc. Natl. Acad. Sci. USA
97,3838
-3843.
Johnson, R. E., Washington, M., Haracska, L., Prakash, S. and Prakash, L. (2000b). Eukaryotic polymerases iota and zeta act sequentially to bypass DNA lesions. Nature 406,1015 -1019.[CrossRef][Medline]
Kannouche, P., Broughton, B. C., Volker, M., Hanaoka, F.,
Mullenders, L. H. F. and Lehman, A. R. (2001). Domain
structure, localization, and function of DNA polymerase eta, defective in
xeroderma pigmentosum variant cells. Genes Dev.
15,158
-172.
Kornberg, A. and Baker, T. A. (1992).DNA replication . New York.
Levine, R. L., Miller, H., Grollman, A. P., Ohashi, E., Ohmori,
H., Masutani, C., Hanaoka, F. and Moriya, M. (2001).
Translesion DNA synthesis catalysed by human Pol eta and Pol kappa across
1,N6-Ethenodeoxyadenosine. J. Biol. Chem.
276,18717
-18721.
Louat, T., Servant, L., Rols, M. P., Teissie, J., Hoffmann, J.
S. and Cazaux, C. (2001). Antitumour activity of 2',
3'-dideoxycytidine nucleotide analogue against tumours up-regulating DNA
polymerase beta. Mol. Pharmacol.
60,553
-558.
Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K. and Hanaoka, F. (1999). The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature 399,700 -704.[CrossRef][Medline]
Ogi, T., Kato, T., Kato, T. and Ohmori, H.
(1999). Mutation enhancement by DinB1, a mammalian homologue of
the Escherichia coli mutagenesis protein DinB. Genes
Cells 4,607
-618.
Ohashi, E., Feaver, W. J., Bebenek, K., Matsuda, T., Gerlach, V.
L., Friedberg, E. C., Ohmori, H. and Kunkel, T. A. (2000a).
Fidelity and processivity of DNA synthesis by DNA polymerase kappa, the
product of the human DINB1 gene. J. Biol.
Chem. 275,39678
-39684.
Ohashi, E., Ogi, T., Kusumoto, R., Iwai, S., Masutani, C.,
Hanaoka, F. and Ohmori, H. (2000b). Error-prone bypass of
certain DNA lesions by the human DNA polymerase kappa. Genes
Dev. 14,1589
-1594.
O-Wang, J., Kawamura, K., Tada, Y., Ohmori, H., Kimura, H.,
Sakiyama, S. and Tagawa, M. (2001). DNA polymerase kappa,
implicated in spontaneous and DNA-damage-induced mutagenesis, is overexpressed
in lung cancer. Cancer Res.
61,5366
-5369.
Radman, M. (1999). Enzymes of evolutionary change. Nature 401,866 -868.[CrossRef][Medline]
Servant, L., Bieth, A., Cazaux, C. and Hoffmann, J. S. (2002). Involvement of DNA polymerase beta in DNA replication and mutagenic consequences. J. Mol. Biol. 315,1039 -1047.[CrossRef][Medline]
Srivastava, D. K., Husain, I., Arteaga, C. L. and Wilson, S.
H. (1999). DNA polymerase beta expression differences in
selected human tumors and cell lines. Carcinogenesis
20,1049
-1054.
Suzuki, N., Ohashi, E., Hayashi, K., Ohmori, H., Grollman, A. P. and Shibutani, S. (2001). Translesional synthesis past acetylaminofluorene-derived DNA adducts catalysed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV. Biochemistry 40,15176 -15183.[CrossRef][Medline]
Velculescu, V. E., Zhang, L., Vogelstein, B. and Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270,484 -487.[Abstract]
Washington, M. T., Johnson, R. E., Prakash, L. and Prakash, S. (2002). Human DinB1-encoded DNA polymerase kappa is a promiscuous extender of mispaired primer termini. Proc. Natl. Acad. Sci. USA 19,1910 -1914.
Yao, X., Buernmeyer, A. B., Narayanan, L., Tran, D., Baker, S.
M., Prolla, T. A., Glazer, P. M., Liskay, R. M. and Arnheim, N.
(1999). Different mutator phenotypes in Mlh1- versus Pms2-
deficient mice. Proc. Natl. Acad. Sci. USA
96,6850
-6855.
Zhang, Y., Yuan, F., Wu, X., Wang, M., Rechkoblit, O., Taylor,
J. S., Geacintov, N. E. and Wang, Z. (2000). Error-free and
error-prone lesion by-pass by human DNA polymerase kappa in vitro.
Nucleic Acids Res. 28,4138
-4146.