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Originally published In Press as doi:10.1074/jbc.M207101200 on October 17, 2002

J. Biol. Chem., Vol. 277, Issue 51, 50046-50053, December 20, 2002
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A Role for DNA Polymerase beta  in Mutagenic UV Lesion Bypass*

Laurence ServantDagger §, Christophe CazauxDagger , Anne BiethDagger , Shigenori Iwai, Fumio Hanaoka||**, and Jean-Sébastien HoffmannDagger DaggerDagger

From the Dagger  Group "Genetic instability and cancer" at the Institut de Pharmacologie et Biologie Structurale, UMR CNRS 5089, 31077 Toulouse cédex 4, France, the  Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan, and the || Graduate School of Frontier Biosciences, Osaka University and Core Research for Engineering, Science, and Technology (CREST), Japan Science and Technology Corporation, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan

Received for publication, July 16, 2002, and in revised form, October 1, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report here that DNA polymerase beta  (pol beta ), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol beta  in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol beta . We firstly demonstrated that mammalian cells overexpressing pol beta  are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By using in vitro primer extension reactions with purified pol beta , we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol beta  mostly incorporates the correct dATP opposite the 3'-terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol beta  in UV lesion bypass and suggest that deregulated pol beta  may enhance UV-induced genetic instability.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Exposure of cells to UV light results in the formation of a variety of lesions in their DNA, the most common being cyclobutane pyrimidine dimers (CPD)1 and pyrimidine-pyrimidone (6-4) photoproducts ((6-4)PP) at adjacent pyrimidines (1). Unrepaired, these lesions can interfere with normal DNA metabolism including DNA replication, eventually resulting in mutations that lead to carcinogenesis and/or cell death. To maintain their genetic integrity, cells have evolved multiple pathways to repair various types of DNA damage, such as nucleotide excision and base excision repair pathways (1). However, all lesions on the genome cannot be repaired efficiently by these processes in time for DNA replication, and some types of lesions are repaired very inefficiently. To prevent cell death through arrested DNA replication at unrepaired lesions, cells have a mechanism, referred to as translesion synthesis, that allows DNA synthesis to proceed past lesions and employs specialized DNA polymerases for promoting continued nascent strand extension.

In human cells, recent genetic and biochemical studies suggest that translesion synthesis (TLS) past a CPD-TT or a (6-4)TT lesion could be facilitated by at least four DNA polymerases, pol eta , zeta , iota , and kappa . In the case of pol eta , this process appears to be efficient and largely accurate opposite a CPD (2), whereas it could be mutagenic and limited at the 3'T opposite a (6-4)TT (3). Overexpression of the antisense mRNA of Rev3, one of the components of pol zeta , leads to a dramatic drop in the extent of UV-induced mutagenesis (4), thereby implicating human pol zeta  as having a pivotal role in error-prone translesion replication in normal cells. Indeed, pol zeta  can catalyze an efficient extension of nucleotides inserted opposite the 3'T of both CPD and (6-4)TT lesions (3, 5). Another DNA polymerase, pol kappa , shows similar properties opposite the CPD (6). In the case of pol iota , the in vitro incorporation of nucleotides opposite the UV lesions and subsequent bypass can be highly error-prone, but its physiological role in TLS is still controversial (5, 7, 8). Presumably, all these polymerases can compete for the 3'-primer terminus at the site of a lesion, and one would predict an effect on the quantitative and qualitative mutagenesis in UV-irradiated cells expressing these enzymes differentially. For example, mutations in the POLH (XPV/RAD30A) human gene that generate a severely truncated and inactive pol eta  protein result in the xeroderma pigmentosum variant phenotype characterized by UV-induced hypermutability (9, 10) and a strong sunlight-induced skin cancer incidence (11-13).

The study reported here indicates that pol beta  can now be added to the list of enzymes that can perform unassisted UV lesion bypass. pol beta  is believed to function primarily in the repair of damaged bases in normal somatic cells (14). It is a monomeric protein of 335 amino acids (39 kDa) that lacks exonuclease activities and whose enhanced expression has been demonstrated by our laboratory to result in an increased mutation frequency (15) as well as chromosome instability and tumorigenesis (16). At the transcriptional level, pol beta  is overexpressed in many cancer cells (17). High levels of pol beta  have also been detected at the protein level in ovarian tumors (18) as well as in prostate, breast, and colon cancer tissues where the enzyme amount was respectively 11-, 286-, and 22-fold higher as compared with adjacent normal tissues (19). Furthermore, pol beta  level and activity are increased by 10-fold in blood samples from chronic myelogenous leukemia patients and in tumor biopsies from non-small cell lung tumors.2 The pol beta -dependent translesion replication that we observed here differs from that of the related DNA polymerases in the efficiency as well as in the accuracy of the reaction. These data may be relevant within the tumoral cellular context where pol beta  is up-regulated, especially in melanomas, since several analyses showed a significant positive association between cutaneous melanoma incidence and high levels of intermittent solar exposure (20-24).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Western Blotting-- Tissues from normal skin and metastasic melanoma, kindly given by Dr Voigt (ICR, Toulouse, France), were lysed. For analysis of pol beta , cell lysates (70 µg of proteins) were electrophoresed in a 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membrane (Schleicher and Schuell). Blots were blocked in Tris-buffered saline-Tween 20 (0.1% Tween) with 5% non-fat dry milk, incubated with anti-pol beta  monoclonal antibody (1/200, DNA polymerase beta  Ab-1, clone 18 S, Neomarkers, Interchim) followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG and revealed by using an enhanced chemiluminescence system (Amersham Biosciences). Equal loading was determined using monoclonal antibody to actin (1/5000) (Chemicon, Euromedex, France).

Clonogenic and Mutagenic Assays-- AA8 CHO cells were maintained in MEMalpha (Invitrogen) with 10% fetal calf serum, 4 mM glutamine, and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin) at 37 °C in a humidified 5% CO2 atmosphere. CHO cell lines overexpressing pol beta  were established previously after stable transfection of pUTPolbeta plasmid (15). Control cells and cells overexpressing pol beta  were plated in 6-well plates and allowed to attach overnight. Next, they were irradiated with a 254-nm UV-C lamp at the fluence rate of 0.5 J/m2/s. Colonies were fixed and stained after 6 days of postincubation, and those >50 cells were scored. For the 6-thioguanine (6-TG)-resistant tests, cells were first irradiated at 20 J/m2 and then exposed to 20 µM 6-TG-containing medium (106 cells/14-cm plate) to determine the number of hypoxanthine guanine phosphoribosyl transferase mutants. After 1 week, plates were stained, and colonies of >50 cells were counted. Mutant frequencies were corrected for plating efficiency and for UV cytotoxicity.

Proteins, Cells, and Substrates-- Rat pol beta  was purified in Escherichia coli as described (25). One unit of rat pol beta  corresponds to 1 pmol of dNTP incorporated into acid-insoluble materials at 37 °C in 60 min by using an activated calf thymus DNA preincubated with DNase I as a substrate. Human pol beta  was provided by Trevigen (Gaithersburg, MD) and showed a 0.68 µg/µl concentration and a 4 units/µl activity. Calf thymus pol alpha  and HIV-1 RT were purified as described previously (26, 27). AA8 CHO Sh::pol beta  cells and AA8 CHO Sh cells were obtained after stable transfection of pUTPolbeta and empty pUT687 vectors as reported previously (15). Briefly, pol beta  cDNA was fused in-frame with the bacterial Sh::ble gene conferring resistance to the broad-spectral zeocin xenobiotic of the phleomycin family. 30-mer UV-modified oligonucleotides and pBS-SV oriA/B vectors were prepared as described (28).

Primer Extension Assay-- UV-modified 30-mer oligomers 5'-CTCGTCAGCATCTTCATCATACAGTCAGTG-3' were chemically synthesized as described previously (29, 30). They were hybridized to 5'-32P-labeled 16-mer (5'-CACTGACTGTATGATG-3'), 17-mer (5'-CACTGACTGTATGATGN-3'), or 18-mer (5'-CACTGACTGTATGATGNN-3') primers at a molar ratio of 1:1 for 10 min at 70 °C in a buffer containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 10 mM MgCl2 followed by slow cooling to room temperature. Standard 15-µl reaction mixtures contained 14 nM of the 5'-32P-labeled primer-template DNA and specific buffer as follows: pol beta  buffer contained 50 mM Tris-HCl, pH 8.8, 10 mM MgCl2, 100 mM KCl, 0.4 mg/ml bovine serum albumin, 1 mM DTT, 10% glycerol; pol alpha  buffer contained 20 mM Hepes-KOH, pH 7.8, 3 mM MgCl2, 1 mM DTT; HIV-RT buffer contained 60 mM Tris-Hcl, pH 8.2, 7 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, 10 mM KCl; buffer for cell extract reaction contained 45 mM Hepes-KOH, pH 7.8, 7 mM MgCl2, 1 mM DTT, 0.4 mM EDTA, 3.4% glycerol, 65 mM mono-K-glutamic acid, 1 mg/ml bovine serum albumin. Reactions were performed at 37 °C and terminated by adding 5 µl of stopping buffer (90% formamide, 0.1% xylene cyanol, 0.1% bromphenol blue, 0.1 mM EDTA). Samples were denaturated for 10 min at 70 °C and loaded to a 20% polyacrylamide/7 M urea gel. CHO extract preparation was performed according to the previously described protocol (31). Competent replicative extracts from the melanoma cell lines were not feasible. An 11-mer unlabeled oligonucleotide (5'-ATGCTGACGAG-3') was also used and annealed to the template at a molar ratio of 2:1 to saturate the 3'-end of the template.

Two-step SV40 DNA Replication Assay-- pBS-SvoriA(CPD) or pBS-SvoriB(CPD) plasmids were generated as described (2). Replication reaction mixtures (25 µl) contained 30 mM HEPES, pH 7.8, 7 mM MgCl2, 200 µM each of CTP, GTP, and UTP, 4 mM ATP, 100 µM each of dATP, dCTP, dTTP, 10 µM dGTP, 40 mM creatine phosphate (Sigma), 100 µg/ml creatine phosphokinase (Sigma), 100 ng of pBS-SvoriA(CPD) or oriB(CPD), 0.5 µg of SV40 large T-antigen (Molecular Biology Resources), and 400 µg of Hela cell extract. After incubation at 37 °C for 4 h, 0.012 units of rat pol beta  and 1 µCi of [alpha -32P[dATP (4000 cpm/pmol; Amersham Biosciences) were added to reaction mixtures and incubated for 1 h. Reactions were quenched by adding an equal volume of "stop solution" (2% SDS, 2 mg/ml proteinase K, and 50 mM EDTA), and further incubation was done for 1 h at 55 °C. DNA (0.5 µg of pc-DNA II, Invitrogen, containing one BamHI site and multiple DpnI sites) was added to each sample as internal purification controls. Reaction products were purified by extraction with phenol-chloroform-isoamyl alcohol followed by ethanol precipitation. The DNA was resuspended in distilled water. The samples were then treated with BamHI and DpnI (New England Biolabs), and the restriction digests were separated on a 1% agarose gel. After ethidium bromide staining of the gel, internal control DNAs were quantified. The gel was then dried, and autoradiography was performed. Quantification analysis of the resolved radioactive bands on the gel was achieved by PhosphorImager Storm-system analysis using ImageQuant software.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overexpression of pol beta  in Melanoma Cells as Compared with Normal Skin Tissues-- Previously, we and others found that pol beta  was overexpressed at the protein level in many cancer tissues as compared with normal tissues (17-19). Here, we analyzed four independent melanoma protein extracts, and we compared their pol beta  content relative to normal skin tissues (Fig. 1). More than a 10-fold increase in pol beta  level was observed in all the melanomas tested, whereas a slight detection of the enzyme was discernible only after a long time exposure in normal skin (data not shown). In this work, we hypothesized that excess pol beta  in skin cells exposed to UV light may predispose these cells to initiation and/or progression into tumoral melanomas by raising the UV-induced genetic instability.


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  		Fig. 1.   Analysis of expression of pol beta  protein in normal skin and melanoma tissues. Cell extracts were analyzed by Western blot using monoclonal antibody to pol beta  protein. Actin was used as internal control for loading. Cell lysates were prepared from skin tissues of two normal patients (samples 1 and 2), ganglion metastasic melanoma cells of three patients (samples 3-5), and skin melanoma cells maintained in culture in aseptic conditions (sample 6).

Decreased Sensitivity to UV Radiation and Enhanced Induced Mutagenesis in CHO-polbeta Cells-- To investigate whether high levels of pol beta  can affect genetic stability after UV irradiation in mammalian cells, we examined UV sensitivity as well as UV-induced mutagenesis in two independent transfected CHO cell lines that overproduce the enzyme by 3.2- and 2.4-fold (AA8 pol beta 2::Sh cells and AA8 pol beta 3::Sh cells) (16). Firstly, we conducted clonogenic experiments after treatment with increasing doses of UV-C irradiation concomitantly with the isogenic control AA8 Sh cells. In at least three separate experiments performed in duplicate, we demonstrated a significant 1.5-2-fold resistance of cells up-regulating the enzyme as compared with the control cells (Fig. 2A). To compare the mutation frequency in the surviving irradiated cells, we used the conventional methodology testing the appearance of mutational events leading to a resistance phenotype at the locus encoding the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase. After irradiation, cells were allowed to grow for 1 week before plating in 6-thioguanine-supplemented medium and then grown for one additional week, and 6-TGR mutant colonies were counted. A 2.6-50-fold increase in mutagenesis for the pol beta ::Sh cells relative to the Sh cells was observed in three independent experiments after a 20 J/m2 UV dose (Fig. 2B). The lack of correlation between pol beta  expression level and UV resistance as well as hypermutability may be due to the mutator phenotype induced by pol beta  overexpression (15). It is possible that the higher expression of pol beta  may cause deleterious side effects and may affect other genes that would interfere with cell viability after UV treatment.


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  		Fig. 2.   Phenotypic comparison of AA8 Sh and pol beta ::Sh cells after UV treatment. A, sensitivity of Sh and pol beta ::Sh cell lines to UV radiation. Survival is expressed as the relative plating efficiency of treated cells to untreated cells. Results are the mean ± S.D. of at least three separate experiments performed in duplicate. B, UV-induced mutation frequency at the hypoxanthine guanine phosphoribosyl transferase locus in Sh and pol beta ::Sh cell lines. Cells were exposed at 20 J/m2, allowed to grow for 1 week before plating at 106 cells in 6-TG-supplemented medium, and grown for an additional week. Next, plates were stained, and 6-TGR mutant colonies counted. C, in vitro translesion synthesis of CPD adduct by AA8 Sh and pol beta ::Sh cell extracts. The 30-mer template was annealed to a 16-mer primer and used to perform primer extension reaction. Undamaged and damaged templates were replicated for 1 h by 5 µg of the indicated cell extracts. Arrows indicate the position of the primer (position 16), the products resulting from one nucleotide incorporation opposite the CPD (position 17), the products resulting from two nucleotides incorporation opposite the CPD (position 18), and the full-size product (position 30).

To investigate the molecular bases for these in vivo phenotypes, we tested a potential translesion ability of UV lesions of the replicative extracts from these cell lines. We performed in vitro primer extension reactions with a 30-mer template containing a CPD adduct, annealed to a 5'-32P-labeled 16-mer primer (Fig. 3A, upper part). The primer was localized at a position so that the two first nucleotides were always incorporated opposite the lesion. In the presence of replicative extracts prepared from the control cells and one pol beta ::Sh cell line, we found that the pol beta ::Sh cell extracts could replicate past the CPD more efficiently as compared with the control extracts (Fig. 2C), demonstrating that excess pol beta  facilitated the bypass process. Addition of purified pol beta  to the control extracts also increased a bypass synthesis capability of the CPD lesion (data not shown). In contrast, we did not observe any TLS of the heavy distorting (6-4)TT lesion with either cell extract (data not shown). These results suggest that bypass synthesis of CPD damage by excess pol beta  may contribute to the in vivo resistance and hypermutagenesis toward UV irradiation in the cells overexpressing pol beta .

Ability of Purified pol beta  to Bypass in Vitro CPD and (6-4)TT Adducts-- To investigate in more depth the specific ability of pol beta  to bypass UV photoproducts, we performed a kinetic study on the 30-mer template containing either CPD or (6-4)TT adduct, annealed to a 5'-32P-labeled 16-mer primer (Fig. 3A). We used purified human and rat pol beta , and we compared their behavior to pol alpha , which was reported previously as unable to incorporate nucleotides opposite the CPD or the (6-4)TT (2). As can be seen in Fig. 3B, by using amounts of enzymes allowing efficient and complete primer extension on undamaged template (Fig. 3B, right part), pol beta  was able to incorporate nucleotides opposite both the CPD and the (6-4)TT lesions (17- and 18-mer products) as well as to perform extension beyond the adducts (products with a size larger than 18-mer) in a time-dependent manner, whereas pol alpha  is not, as expected. Some discrete radioactive fragments were also observed as 24-mer products; the mechanism involved in the generation of these products will be addressed in more depth later in the manuscript when describing Fig. 4. We also reported here that the HIV-1 RT, which shares structural and inaccuracy features with pol beta , catalyzed efficient translesion synthesis of UV photoproducts (Fig. 3B). To better visualize and quantify the pol beta -dependent bypass process, the 30-mer template was annealed in the presence of the 16-mer-labeled primer at a ratio of 1:1 and an excess of 11-mer oligonucleotide complementary to the 3'-end of the template to generate a 3-nucleotide gapped DNA, a preferential substrate for pol beta  that offers the possibility to analyze the incorporation opposite the lesion and further extension of one nucleotide (Fig. 3A). Primer extension reactions were performed in the presence of 0.05 or 0.5 units of human pol beta , leading to a 1:1 or 10:1 molar ratio, respectively, as compared with the primer-template. We found a more efficient pol beta -mediated bypass in both a time- and dose-dependent manner on this gapped DNA as compared with the non-gapped template (Fig. 3C). The higher efficiency for nucleotide incorporation opposite the lesions may be favored by the ability of the pol beta  8-kDa domain, which binds to the downstream 5'-terminus, to promote processive extension of misinserted nucleotides on undamaged gapped DNA (32). In the presence of 0.05 and 0.5 units of pol beta , the efficiency of the bypass of CPD into the 3-nucleotide gap represented 20 and 75% extension, respectively, of the primer for 60 min of incubation time (Fig. 3C). A minor bypass product also showed full-size synthesis in the presence of higher polymerase concentration, probably resulting from the previously reported in vitro strand displacement activity by pol beta  of the 11-mer oligonucleotide (18). In the case of the (6-4)TT adduct, the presence of the 11-mer oligonucleotide allowed a 3-fold increase of nucleotide incorporation opposite the 3'T of the adduct by 0.05 units of pol beta  (Fig. 3C). A complete 3-nucleotide gap-filling reaction was achieved in the presence of 0.5 units of human pol beta , and bypass products represented more than 55% of the extended oligonucleotides after 20 min of incubation (Fig. 3C). Taken together, these results demonstrated that purified pol beta  can bypass CPD and (6-4)TT adducts during in vitro primer extension.


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  		Fig. 3.   pol beta  translesion synthesis activity on CPD and (6-4)TT containing templates. Reactions were performed as described under "Experimental Procedures" for the times noted above each track. A, primed UV-modified 30/16 and 30/16/11 templates used for the primer extension assays. B, primer extension assays with 0.012 units of rat pol beta , 1 unit of calf thymus pol alpha , and 1 unit of HIV-1 RT with the UV-modified or undamaged templates. C, primer extension assay by human pol beta  with the UV-modified 30/16 and 30/16/11 templates. oligo, oligonucleotides.


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  		Fig. 4.   Extension of primers opposite the 3'T of the CPD or (6-4)TT by human pol beta . The sequence of each primer is shown above each group of experiments. A primer containing one nucleotide opposite the 3'T of each adduct was annealed to the templates depicted above each figure. In A, reactions were performed in the presence of 0.5 units of human pol beta  and 4 dNTP (200 µM) for 1 h. In B and C, reactions were performed in the presence of 0.5 units of human pol beta  for 1 h. 0, A, T, C, and G indicate reactions in the absence of nucleotides or in the presence of dATP, dTTP, dCTP, or dGTP, respectively.

Specificity of pol beta -dependent Incorporation Opposite the CPD and (6-4)TT-- A steady-state "single hit" gel kinetic assay (33) was performed using primed unmodified or UV-modified 30-mer DNA templates to quantitatively determine the specificity of nucleotide incorporation opposite the 3'T of CPD and (6-4)TT. For damaged templates, the concentration of incoming dNTP varied from 5 to 1000 µM, and incubation time was 1 h in the presence of 0.5 units of pol beta . Regarding the undamaged templates, the concentration of incoming dNTP varied from 1 to 500 µM, and incubation time was 15 min in the presence of 0.001 units of pol beta  when using dATP and 30 min with 0.5 units of pol beta  when using dCTP or dGTP. All the data we obtained are summarized in Table I, and these data revealed that the dATP represented 55 and 71% of the inserted nucleotides opposite the CPD and the (6-4)TT lesions, respectively, leading to an error-free insertion. Insertion of dCTP opposite the 3'T of the lesion represented the major error-prone insertion with 32 and 26% of the inserted nucleotides opposite the CPD and the (6-4)TT lesions, respectively. Some dGTP residues can be inserted opposite the 5'T but at a lesser extent.

                              
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Table I
Steady-state kinetic parameters of incorporation opposite the 3'T of the CPD or (6-4)TT by human Pol beta

Interestingly, when comparing kinetic parameters, we found that the ability of pol beta  to insert the incorrect dGTP nucleotide opposite the 3'T of an undamaged template was only 4-16-fold higher as compared with the pol beta  efficiency to misinsert dATP or dCTP opposite the 3'T of the CPD or the (6-4)TT, signifying the high capacity of pol beta  to incorporate nucleotide opposite distorting lesions. Finally, pol beta  inserted dATP opposite the 3'T of the CPD or the (6-4)TT lesion with an efficiency 3500-8000 less as compared with the insertion of dATP opposite the 3'T of the undamaged template.

pol beta -dependent Efficiency of Extending Primers with One Base Opposite the CPD and the (6-4)TT-- As it could be of biological significance to determine whether the incorporated nucleotide can be extended, 17-mer primers in which each of the four bases was paired to the 3'T of each adduct were annealed to damaged templates. Extension of these primers was assayed after a 1-h reaction in the presence of 0.5 units of pol beta  and either all four dNTPs (200 µM) (Fig. 4A) or a unique dNTP (Fig. 4, B and C). The most efficient extension to the full-size product of a primer annealed to the CPD-containing template occurred with the correctly paired dATP in the presence of all 4 dNTPs (Fig. 4A). We analyzed the 5'T incorporation specificity at the AT primer and found that, although dATP was mostly incorporated, all the other dNTPs could be also incorporated with a slightly lower efficiency (Fig. 4B). Indeed, there was a significant misincorporation of dTTP, dCTP, and dGTP opposite the 5'T of the CPD, and after dGTP incorporation, an incorporation opposite the adjacent undamaged dCTP in the template occurred, leading to a complete lesion bypass. With the GT primer in the presence of all 4 dNTPs, the obtaining of full-size product was as efficient as compared with the correctly paired AT primer (Fig. 4A), probably initiated by dATP incorporation (Fig. 4B). In contrast, polbeta -dependent extension of CT and TT mispairs was inefficient since it was aborted after incorporation of one nucleotide (Fig. 4A). Extension reactions of primers annealed to the (6-4)TT-containing template revealed 1-nucleotide incorporation but no further extension, as shown in Fig. 4, A and C. Extension of AT, CT, and GT mispairs occurred only in the presence of dTTP, rendering this weak process highly mutagenic (Fig. 4C). A discrete radioactive fragment product migrating as a 24-mer product was observed when the CT mispair was extended on both the CPD and the (6-4)TT templates (Fig. 4A). This seems likely to correspond to a 6-nucleotide synthesis resulting from an annealing event of the microsequence ATGC at the 3'-terminus of the 17-mer primer that can pair to a homologous sequence TACG at positions 20-23 of the template, generating a template loop. Such a misalignment incorporation mechanism facilitated by pol beta  has already been described during TLS of an abasic site (34, 35), an 8-oxo-deoxyguanosine (36), and propano-deoxyguanosine lesions (37). This specific ability to catalyze template misalignment by searching microhomology sequence is shared by pol µ, another member of the DNA polymerase X family (38).

pol beta -dependent Extension of Primers with Two Bases Opposite the CPD or the (6-4)TT-- To investigate whether pol beta -dependent incorporated nucleotides opposite the two damaged bases could be extended, we used 18-mer primers whose termini were located directly opposite the CPD or (6-4)TT (Fig. 5). We focused on a set of 11 primers, 8 primers representing the best incorporations opposite the dimers (primers ending with AA, AG, AT, AC, GA, GT, CA, CT for the CPD; primers ending with AT, CT, GT for the (6-4)TT), and 3 primers randomly chosen (primers ending with GG, CG, GC). pol beta  was able to extend efficiently a primer with two dA residues opposite the CPD, generating a full-size product. Interestingly, efficient extension was also observed with the AG, GA, AC, and CA primers with a decreasing efficiency (AG>AC>GA>CA). None of the primers randomly chosen were extended by pol beta  opposite the CPD. Taken together, this shows that the best efficiencies were obtained with the nucleotides specifically incorporated opposite the CPD by pol beta . Discrete radioactive fragments were also observed as 21- and 24-mer products when we used the AC and CT primers opposite either lesion, probably reflecting a misalignment incorporation mechanism facilitated by pol beta  between TGAC or ATGCT at the 3-terminus of the 18-mer primers and the homologous sequence ACTG (position 24-27) or TACGA (position 20-24) of the 30-mer template, respectively. In the case of the (6-4)TT-containing template, we did not detect any significant primer extension with all the primers tested (Fig. 5, right part). Taken together, these results suggest that pol beta  is able to extend efficiently mutagenic as well as correct nucleotides incorporated opposite the CPD.


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  		Fig. 5.   Extension of primers with various dinucleotides opposite the CPD or the (6-4)TT by human pol beta . The 3'-dinucleotide sequence of each primer is given above each panel. Each reaction was performed for 1 h in the presence of 0.5 units of pol beta  and all 4 dNTPs. The arrow shows the starting position of the 18-mer primer.

Recruitment of Excess pol beta  during in Vitro SV40 Replication to Bypass the CPD-- To investigate whether excess pol beta  could interfere with the replicative machinery during replication of UV-damaged duplex DNA, we performed a two-step in vitro SV40 replication assay. This assay can be used to observe CPD bypass as demonstrated for pol eta  in HeLa cell extracts (2). We used two covalently closed circular templates containing the SV40 origin of DNA replication with a single CPD located on each side of the SV40 origin (Fig. 6A). Replication forks encounter the lesion during lagging strand synthesis in the case of pBS-SvoriA(CPD) and in the course of the leading strand synthesis in the case of pBS-SvoriB(CPD). These plasmids were first incubated for 4 h with 400 µg of Hela extracts in the reaction buffer, then [alpha -32P]dATP and purified pol beta  were added for an additional hour. During the first incubation period, DNA replication machinery stalled at the lesion on the damaged strand, and during the shorter period of the second incubation in the presence of radioactive dATP and purified pol beta , radioactivity will be incorporated preferentially into products of damage bypass replication. Then, DNA was purified, linearized by BamH1 and DpnI, and subjected to electrophoresis onto a 1% agarose gel. Ethidium bromide staining and autoradiography of the gel are shown in Fig. 6B. DpnI digestion was done to visualize only the DNA population that was replicated once. Additionally, we verified that addition of up to 0.024 units of pol beta  in reaction mixtures replicating undamaged DNA did not result in an increase of the radioactive replication signal (39). As observed in Fig. 6B, radioactivity incorporation during DNA replication is lower with the pBS-SVoriA DNA as compared with the pBS-SVoriB DNA. As suggested in a previous report, SV40 replication of a UV lesion-containing plasmid could be synchronous between the two parental strands in the case of pBS-SVoriA(CPD) and asynchroneous in the case of pBS-SVoriB(CPD) (2); during lagging strand synthesis, the replication fork moves past the lesion, and reinitiation occurs at the next Okasaki fragment, leaving a small single-stranded gap; during the leading strand replication, the progression of the fork is inhibited, and uncoupling of leading and lagging strand occurs; the replication machinery continues to synthesize the lagging strand (40, 41).


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  		Fig. 6.   Excess pol beta -mediated translesion synthesis of CPD during SV40 replication. A, possible implications of excess pol beta  in the bypass of the CPD adduct during the two-step SV40 replication assay. SV40-DNA constructs are shown here during bidirectional semiconservative SV40 replication that began at the origin (Ori A or Ori B for pBS-SV(CPD) oriA or oriB, respectively). In B, 100 ng pBS-SV oriA and oriB were replicated by 400 µg of cell-free extracts from human Hela cells in a T-antigen-dependent manner in the presence or absence of 0.012 units of pol beta , and then they were linearized by BamHI (one unique site) or digested by BamHI and DpnI (multiple sites). The two-step SV40 DNA replication and analysis of the products are described under "Experimental Procedures."

We found that, in the presence of 0.012 units of rat pol beta , DpnI-resistant products increased by 4-fold with pBS-SVoriA(CPD) and by 2-fold with pBS-SVoriB(CPD) as compared with the control reactions without pol beta  (Fig. 6B, right tracks). For the global replication products (without digestion by DpnI; Fig. 6B, left tracks), a pol beta -dependent increase was also detected. When pol beta  and the radioactive nucleotide were added at the beginning of the reaction (one-step reaction), a 2-, 3.5-, and 5-fold signal increase was observed with pBS-SVoriB(CPD) in the presence of 0.0048, 0.012, and 0.024 units of rat pol beta , respectively (data not shown). Taken together, these results suggest that when DNA synthesis during replication of duplex DNA is stopped by a CPD, excess pol beta  can be notably recruited to overcome the lesion.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We showed here that pol beta , an enzyme required in somatic cells for the base excision repair pathway (14), can facilitate translesion replication of a CPD as well as, to a lesser extent, a thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct ((6-4)PP). Such a result was obtained by using the well calibrated primer extension assay using site-specific UV-modified oligonucleotides as well as the SV40 replication assay, which reconstitutes the mammalian DNA replication fork, using CPD-modified duplex DNA. pol beta  mostly incorporated the correct dATP opposite the 3'T of the CPD and the (6-4)PP but could also misinsert dCTP. For the CPD, we found that the 5'T incorporation specificity by pol beta  at the AT and CT primers was highly mutagenic. Whether the nucleotides were correctly or incorrectly inserted opposite the CPD, some of them were efficiently extended by pol beta , and this extension is highly error-prone, supporting the possibility that pol beta  could compete with pol zeta  (5) or pol kappa  (6) to extend nucleotides incorporated opposite the 3'T of the CPD adduct. Opposite the (6-4)TT lesion, the incorporation by pol beta  opposite the 3'T, essentially the dATP-like pol iota , is poorly efficient and is most of the time aborted, probably because of the strong distortion of DNA. This low extension capability of pol beta  is shared with pol eta  (3) and pol iota  (5, 7). It has been proposed that pol zeta  is responsible for the subsequent extension of the nucleotide incorporated opposite the 3'T of the (6-4)TT damage (3, 5). This suggests that in vivo, an efficient, mostly accurate, but potentially error-prone TLS of the (6-4)TT lesion may result from the combined activities of pol beta  and pol zeta .

To date, among the 12 eukaryotic DNA polymerases that have been identified, only pol eta , pol zeta , pol kappa , and pol iota  have been shown to exhibit such potential involvement in CPD and (6-4)TT photoproducts bypass activity (2, 5-7). In normal somatic cells, the majority of translesion replication is normally pol eta -dependent since in xeroderma pigmentosum variant cell extracts, in which pol eta  is inactive, only 10% of the lesion bypass activity of normal cell extracts is observed (2, 42). So what could be the biological significance of such a pol beta -dependent bypass? Analysis of the mutagenic spectra observed after exposing human cells to UV light suggests that most mutations are, in fact, targeted to the 3'-site of a di-pyrimidine containing a dC (at CC and TC) (1 ,43). However, some minor mutations T right-arrow A and T right-arrow C targeted to the 5'-site of TT can also be observed (10, 44), and these match to the pol beta -dependent mutations that we observed here in vitro, suggesting a role of pol beta  in some of the UV-induced mutations in somatic cells. The frequency of this kind of mutation increases strongly up to 45% in xeroderma pigmentosum variant cells (1 ,10, 45), supporting that pol beta , like pol iota , may be involved in the TLS process at the TT sites in the absence of pol eta .

Moreover, situations in which the imbalance of pol beta  expression in cells occurs may be of interest in such translesion process of UV lesions. Interestingly, we observed in this work that high levels of pol beta  can be found in various melanomas tumors, which are known to be associated with UV radiation exposure. We recently showed that pol beta  can interfere in vitro with duplex DNA replication when up-represented, rendering the process inaccurate (39). The data presented here suggest strongly that interference of excess pol beta  at the replication forks not only can affect the accuracy of the process but can also modulate the genotoxicity of UV lesions when present on the genomic DNA. Although the mutagenic translesion replication experiments reported here were performed entirely in vitro, we believe that they shed light on the mutagenic process in vivo in melanoma cells and that excess pol beta  may enhance CPD translesion in a mutagenic manner by competing with pol eta . By using isogenic CHO cells, we found that the sole pol beta  overexpression event resulted in a resistant phenotype toward UV treatment and can dramatically enhance the induced mutagenesis. Both phenotypes may result from the TLS catalyzed by pol beta  during the elongation of the replication forks. Overexpression of pol beta  could be therefore identified as a host risk factor that may potentiate the genetic instability in cells exposed to UV and may consequently affect melanoma risk.

    ACKNOWLEDGEMENT

We thank Dr. T. Kunkel for the pBS-SV oriA and oriB vectors.

    FOOTNOTES

* This work was exclusively supported financially by "La Ligue Nationale contre le Cancer" (Equipe labelisée).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ An ARC fellowship recipient.

** To whom correspondence may be addressed. E-mail: fhanaoka@imcb.osaka-u.ac.jp.

Dagger Dagger To whom correspondence may be addressed. E-mail: jseb@ipbs.fr.

Published, JBC Papers in Press, October 17, 2002, DOI 10.1074/jbc.M207101200

2 Y. Canitrot, C. Cazaux, and J.-S. Hoffmann, unpublished data.

    ABBREVIATIONS

The abbreviations used are: CPD, cis-syn cyclobutane pyrimidine dimer; (6-4)PP, (6-4) photoproduct; (6-4)TT, (6-4) photoproduct at TT site; 3'T, 3' thymine of the UV(TT) lesion; 5'T, 5' thymine of the UV(TT) lesion; pol, DNA polymerase; TLS, translesion synthesis; CHO, Chinese hamster ovary; 6-TG, 6-thioguanine; HIV-1 RT, human immunodeficiency virus-1 reverse transcriptase; DTT, dithiothreitol; pol, DNA polymerase; TLS, translesion synthesis; CHO, Chinese hamster ovary; 6-TG, 6-thioguanine; HIV-1 RT, human immunodeficiency virus-1 reverse transcriptase; DTT, dithiothreitol.

    REFERENCES
TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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