Mutagenesis Advance Access originally published online on July 14, 2007
Mutagenesis 2007 22(5):343-351; doi:10.1093/mutage/gem024
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Transcription through 8-oxoguanine in DNA repair-proficient and Csb–/Ogg1– DNA repair-deficient mouse embryonic fibroblasts is dependent upon promoter strength and sequence context
Laboratory of Genetic Instability and Cancer, FRE 2939 CNRS, Institut Gustave Roussy, 94805 Villejuif, France 1Present address: Laboratoire MPI, Université d'Evry Val d'Essonne, Bd F. Mitterrand, 91025 Evry Cedex, France
Cells from Cockayne syndrome patients are characterized by a deficiency in transcription-coupled repair (TCR) of UV-induced lesions. These cells have also been shown to be sensitive to oxidative stress and defective in TCR of some oxidative lesions. Because some discrepancies about this pathway have been recently reported in the literature, we describe here a system that allows us to analyze the effect of a unique 8-oxoguanine (8-oxoG) lesion on gene transcription in vivo. We have constructed nonreplicative shuttle vectors containing a single 8-oxoG in the transcribed strand of the luciferase reporter gene. We have positioned this unique lesion in different sequence contexts and we have tested the effect of two promoters with different transcriptional strength on the level of transcriptional bypass/pause due to the presence of the lesion. When we transfected DNA repair-deficient mouse cell lines with these shuttle vectors, we found a 
50% decrease in relative luciferase activity in Ogg1–/– and Csb–/– embryonic mouse cell lines. In Csb–/–/Ogg1–/– cells, this decrease was even more important achieving eventually up to 90% inhibition of luciferase expression depending upon the promoter strength and the position of the lesion. These results show clearly that a unique 8-oxoG exhibits different effect on gene expression depending upon the nucleotidic sequence around it and needs the wild-type activities of Csb and Ogg1 proteins to be fully repaired.
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Introduction |
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DNA repair pathways are essential for genome caretaking in cells. Among these pathways, the nucleotide excision repair (NER) is the most versatile one, able to specifically remove bulky DNA adducts from exogenous or endogenous sources (1
,2). Transcription-coupled repair (TCR) is a sub-pathway of NER that facilitates the preferential removal of lesions located on the transcribed strand of active genes. This pathway has been initially reported for the repair of bulky adducts including UV-induced lesions (3–5). Cockayne syndrome (CS) cells have a deficiency in this pathway that can be revealed by the lack of RNA synthesis recovery following UV-irradiation even though they are able to carry out almost normal unscheduled DNA synthesis (6,7). Similarly, CS cells are sensitive to ionizing radiation and oxidizing agents suggesting a repair deficiency for oxidative lesions (8–10). Since the accumulation of oxidative damage in DNA has been related to senescence, aging and cancer (11–15), it is argued that the TCR deficiency of oxidative damage could be at the origin of the neurodegenerative process in CS patients that occurs progressively from birth to early adulthood. Indeed, in the absence of full repair, oxidative damage accumulation will probably lead to a progressive blockage/pause of transcription in nonproliferating cells such as neurons, and therefore to apoptosis.
Oxidative damage is not only produced by exogenous agents such as UVA or ionizing radiations but also by endogenous sources as normal cellular metabolism during the production of energy by oxygen consumption. Most of these lesions are repaired by the base excision repair (BER) pathway. 8-Oxoguanine (8-oxoG) is one of the most frequent oxidative DNA damage found in living cells and is frequently used as a marker of oxidative level in organisms (16,17). For this reason, we took the repair of 8-oxoG as a model system hopefully applicable to other types of oxidative lesions. 8-oxoG is repaired via BER, whose first step is the recognition of the lesion by a DNA glycosylase. In mammalian cells, this glycosylase is OGG1 when the 8-oxoG is paired with cytosine and MYH when paired with adenine. In the absence of efficient repair, 8-oxoG can lead to mutations when paired with adenine (18). In this case, 8-oxoG replication will result in G to T transversions. The absence of 8-oxoG repair in Ogg1–/– mice leads to an accumulation of abnormal levels of this lesion and a moderate increase of spontaneous mutation rate (19,20).
Previous studies have suggested that 8-oxoG can be repaired via TCR in murine cells (21). Cells from Csb–/– knockout mice show a deficiency in TCR of UV-induced lesions and these mice exhibit a predisposition to UV-induced cancer that is not observed in human CS patients (22). The mouse Csb–/– cells also show increased sensitivity to oxidative damage (23). Cells from double knockout mice for Csb and Ogg1 show a deficiency in the removal of 8-oxoG in the global genome that is more significant than that obtained for Ogg1–/– or Csb–/– mice (24).
At the molecular level, initiation of TCR has been hypothesized to be due to an irreversible stalling of RNA polymerase II (RNA pol II) when it encounters a lesion located in the transcribed strand of an active gene. This phenomenon has been well illustrated for UV-induced lesions (25,26), but the ability of oxidative lesions to block RNA pol II remains to be established. Some in vitro studies using HeLa cell extracts show efficient bypass of an 8-oxoG lesion (27), while some other groups could see either a pause or even a partial blockage using purified RNA pol II (28). Recently, Charlet-Berguerand et al. (29), have shown that 8-oxoG is bypassed by RNA pol II and that this bypass is regulated by transcription elongation factors as CSB, elongin and TFIIS. Studies in vivo on the entire genome are difficult to carry out because every oxidant drug or agent used to damage DNA can produce several types of oxidative lesions as well as single- and double-strand breaks (30,31). It is therefore very difficult to follow with accuracy the repair of 8-oxoG DNA lesions in the genomic DNA.
The best system up to now for studying the fate of a particular oxidative lesion in vivo relies upon transfection of a nonreplicative shuttle vector containing a single lesion like 8-oxoG that can be synthesized chemically and stably inserted in the plasmid genome (32). We developed a system in which the lesion is located at different sites on the transcribed strand of the luciferase reporter gene allowing us to follow the effect of this lesion on transcription by quantification of the enzyme's activity. This technology was applied to determine the role of promoter strength as well as the effect of nucleotide sequence context around the lesion.
Using mouse embryonic fibroblasts (MEF) isolated from Ogg1–/–, Csb–/– and double knockout mice, we report that Ogg1 as well as Csb modulate the expression of the reporter gene. Our results show that transcription through 8-oxoG is conditioned by the strength of the promoter driving the transcription of the luciferase gene and that the sequence context surrounding the lesion and/or the distance from the promoter can modulate the efficiency of the bypass.
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Materials and methods |
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Cell lines and culture conditions
MEF used in this study carried different genotypes: wild type and cells established from knockout mice, Ogg1–/–, Csb–/– and Csb–/–/Ogg1–/– (a gift from S. Boiteux CEA, Fontenay-aux-Roses, France, with the authorization of E. Seeberg, Center for Molecular Biology and Neuroscience, Rikshospitalet University Hospital, Oslo, Norway). Ogg1–/– cells were produced by Klungland et al. (20
). Csb–/– mice were produced by introduction of a mutation in Csb gene that reproduces the CSB human mutation in CS1AN cells where a truncated protein is expressed (22). Csb–/–/Ogg1–/– mice were produced by crossing Ogg1–/– and Csb–/– mice (24). Cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% heat-inactivated fetal calf serum, glutamine, sodium pyruvate and antibiotics at 37°C in a humidified atmosphere of 5% CO2.
Oligonucleotides
Purified oligonucleotides containing 8-oxoG are described in Table I and were obtained from Eurogentec (Seraing, Belgium). Oligonucleotides for reverse transcription-polymerase chain reaction (RT-PCR) studies, listed in Table II, were synthesized by Proligo (Paris, France).
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Construction of closed circular double-stranded plasmids containing a single 8-oxoG:C base pair
We used pGL3-derived plasmids containing the firefly luciferase reporter gene. These plasmids are able to replicate in bacteria but not in mammalian cells, where they can be repaired and transcribed. Luciferase was expressed under the control of either HSV-TK (called TK thereafter) or cytomegalovirus (CMV) promoters. pCMVGL3 is a gift from P.J. Brooks (Bethesda, MD). This plasmid was constructed replacing the SV40 promoter from pGL3 Charbonnières-les-Bains (Promega, France) with the CMV promoter obtained from pcDNA3.1 (Invitrogen, Cergy-Pontoise, France) (33
). pTKGL3 was constructed by substituting the HindIII fragment containing the CMV promoter of pCMVGL3 by the HindIII fragment of pRL-TK (Promega) containing the TK promoter. We have produced single-stranded DNA from plasmids as previously described (34). Plasmid constructions contained the oligonucleotides modified with a single 8-oxoG lesion located at different positions on the luciferase-transcribed strand (Figure 1A). Plasmids without lesion are pCMVGL3 with the CMV promoter and pTKGL3 with the TK promoter. The first oligonucleotide was hybridized to the region around the +1 from the coding sequence (8oxoluc3) with the 8-oxoG placed at –1 related to luciferase coding sequence, one nucleotide before the start ATG codon, to produce PC3 and TK3 constructions (with CMV and TK promoters, respectively). The second one was hybridized near the active site of luciferase coding sequence (8oxoluc7) with the 8-oxoG placed at nucleotide +698 of the luciferase coding sequence. It produces PC7 and TK7 constructions with CMV and TK promoters, respectively. The third one was hybridized after the stop codon of the luciferase coding region and before the polyadenylation signal with the 8-oxoG placed at +1671 from the start position of the luciferase coding sequence, produces PC9 and TK9 constructions. Plasmid construction was previously described (32) with some modifications to improve the efficiency. Briefly, we hybridized 1.5 pmol/µl of the oligonucleotide containing the 8-oxoG with 50 µg of single-stranded DNA at 75°C for 10 min and cooled down slowly to room temperature. This hybridized complex was incubated with 1 mM dNTPs, Bovine serum albumin (0.1 µg/µl), T4 DNA polymerase (0.3 U/µl), T4 DNA ligase (0.8 U/µl) and ATP together with T4 gp 32 single-strand-binding protein (Q-biogene, Illkirch, France) in a T4 DNA polymerase buffer (Ozyme, Saint Quentin-en-Yvelines, France). The mixture was incubated at 37°C for 2 h and then heat inactivated. After the second strand synthesis, the closed circular double-strand plasmid molecules were purified on a CsCl gradient as previously described (32). The presence of 8-oxoG in each construction was verified by its sensitivity to digestion with 10 ng of Fpg protein (a gift from S. Boiteux) at 37°C for 1 h. Quantification of each final construction was done by electrophoresis of the double-strand plasmids on a 0.8% agarose gel and analyzed using a Syngene Imaging system (Syngene, Cambridge, UK).
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Mutagenesis assay
Plasmid constructions were introduced into bacteria deficient for the repair of 8-oxoG, Escherichia coli PR195 strain (
lac-pro F' pro lacI lacZ, mutY::kan, fpg::kan, Tn10) cells (a generous gift from S. Boiteux). From 0.1 to 1 ng of plasmid containing or not the lesion were transformed into bacteria by a heat shock protocol. After incubation for 1 h at 37°C, they were seeded into Luria Broth plates containing ampicillin (100 µg/ml) and colonies were isolated for DNA extraction. Plasmid DNA was extracted using NucleoSpin Multi-8 Plasmid kits (Macherey-Nagel, Hoerdt, France). Next, DNA was digested with the restriction enzymes that recognize the sites where the lesions were located (Table I) for screening mutations induced during replication of plasmids in bacteria.
Luciferase assay
Mouse cells (70 000) were plated in 24-well plates and the day after were co-transfected with 10 ng of firefly luciferase reporter plasmid containing 8-oxoG and 500 ng of the renilla luciferase plasmid pRL-TK (Promega) used as an internal control of transfection efficiency and cellular basal transcriptional efficiency since transfection efficiency was dependent on cell line and plasmid transfected. After transfection of CMV-containing constructions, transfection efficiency was as follows: For the wild type (WT) cells, we observed a 1.5-fold greater luciferase expression than for the other three cell lines. For TK constructions, Ogg1–/– were transfected 8-fold more efficiently related to WT and Csb–/– and double knockout 2-fold related to WT.
Aliquots of the same mixture of plasmid constructions were used to transfect the four types of cells at the same time. All transfections were done using FuGENE 6 transfection Reagent (Roche Molecular Biochemicals Basel, Switzerland) according to manufacturer's instructions. Cells were washed with phosphate-buffered saline and lysed with 100 µl of passive lysis buffer 1X (Promega) 18 h after transfection. Luciferase activities were measured using a Dual Luciferase Assay System (Promega) following manufacturer's instructions and measured in a Sirius Luminometer (Berthold Detection Systems, Pforzheim, Germany).
For each transfection, calculation of relative luciferase activity (RLA) was done in triplicate. The firefly luciferase activity was normalized to the renilla luciferase activity and for each construction, this ratio was normalized to the relative activity obtained with the plasmid without lesion. The RLA is expressed as a percentage.
Reverse transcription-polymerase chain reaction
To analyze mRNA levels of the luciferase gene, 800 000 cells were plated in 6 cm2 dishes and were transfected the day after with 100 ng of firefly luciferase reporter vector. Eighteen hours after transfection, RNA was extracted with NucleoSpin RNA extraction kits (Macherey-Nagel) following manufacturer's instructions. Primers used for amplifications are listed in Table II. A scheme representing the pairs of primers and the amplified fragments is shown in Figure 1B. CMV5-LUCX primer pair amplifies a region of 329 bp around the 8oxoluc3 position. This fragment corresponds to a region just before the coding sequence and 80 nucleotides of the coding luciferase sequence. FLUC1–FLUC2 were used to amplify a 202-bp fragment around the lesion located in +698 nt of the coding sequence. TER3–TER4 amplify a 345-bp fragment around the lesion placed at 8oxoluc9 position. This fragment contains the last 275 nt of the coding sequence and 70 nt just before the polyadenylation signal. The amplification of a 300-bp fragment from the Gapdh housekeeping gene was used to normalize the results and this ratio was also normalized to the ratio luciferase/Gapdh obtained for the transfection of plasmids without lesion. Each RT-PCR reaction was done with 500 ng of total RNA and the amplifications were done in one step using SuperScriptTM One Step RT-PCR kit from Invitrogen (following manufacturer's instructions). After 30 min at 50°C for the synthesis of cDNA, PCR profiles consisted in denaturation at 95°C 2 min, 30 cycles of amplification (95°C 15 s, 60°C 30 s, 72°C 1 min) and a final incubation of 5 min at 72°C. Amplified fragments were electrophoresed on 1% agarose gels and quantifications were done using a Syngene Imaging System (Syngene).
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Analysis of plasmid constructions containing a unique 8-oxoG lesion
The presence of 8-oxoG in the constructs was verified by digestion with the E.coli Fpg enzyme. Fpg is a DNA glycosylase that is able to specifically remove 8-oxoG from DNA (35
,36). For each plasmid construction containing an 8-oxoG, >95% of form I (closed circular) plasmid DNA was converted into form II (relaxed conformation) after digestion by Fpg (Figure 2A, lane 4, Figure 2B, lane 4, Figure 2C, lane 5, and data not shown for the CMV-vectors). An almost 100% cleavage was obtained by increasing the amount of the Fpg enzyme in the assay (data not shown). This indicates that the 8-oxoG adduct was present in almost if not all the molecules. Fpg protein had no effect on unmodified plasmid indicating the absence of spontaneous 8-oxoG or abasic sites. Since we inserted all the lesions in restriction sites (Table I), the cleavage of the plasmid by the corresponding restriction enzyme that recognizes the site of the lesion was also tested. Cleavage was not inhibited by the lesion for NcoI or BspEI sites (Figure 2A, lane 5, Figure 2B, lane 5 and data not shown); the presence of 8-oxoG partially inhibited digestion by NgoMIV, while the cleavage was complete in plasmids without lesions (Figure 2C, lane 6 and data not shown for the CMV-vectors).
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Mutagenesis induced in bacteria by 8-oxoG in different plasmid sequence contexts
In order to check the influence of sequences surrounding the 8-oxoG site on mutagenesis, we used plasmid constructions with or without lesions to transform bacteria deficient for 8-oxoG repair (fpg–, mutY–). In these bacteria that cannot repair the lesion, replication of lesion-containing plasmids should result in the insertion of either A or C opposite to the lesion. In the case of an insertion of A, the sequence of the restriction site is modified and becomes resistant to digestion by the corresponding enzyme. Thus, an increase in cleavage resistance over the background is a marker for the presence of 8-oxoG at the correct position (34
,37). After transformation, colonies were isolated and plasmid DNA was extracted for digestion with the restriction enzyme that recognizes the original restriction site where the lesion was introduced. Results obtained, expressed as percent of the frequency of mutant colonies (not digested by the specific restriction enzyme) to the total number of colonies analyzed, are shown in Table III.
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When DNA repair-deficient bacteria were transformed with 8-oxoG plasmid constructions, between 14 and 23% of recovered plasmids were resistant to cleavage by one of the restriction enzymes tested. Sequencing of these mutants show that >95% correspond to point mutations and among them >90% are G to T transversions as already found in previous reports (18
,38,39).This result confirms the presence of the 8-oxoG at the correct position in the plasmid and is in agreement with values already published (32,37). We have pooled results obtained with both promoters, TK and CMV, since there is no transcription of the luciferase gene in bacteria, in our system. The mutation frequency in the NgoMIV (8oxoluc9) restriction site was significantly higher than that obtained for the NcoI (8oxoluc3) or the BspEI (8oxoluc7) site (P < 0.05 and P < 0.005, respectively). These data confirm that the 8-oxoG is located at the right position on the plasmids, is a mutagenic lesion and that the frequency of mutagenesis can be affected by the sequence context, at least in bacteria.
Luciferase activity in DNA repair-deficient MEF cell lines transfected with plasmids containing a single 8-oxoG
To establish reliable conditions for our assay, we analyzed the kinetics of luciferase expression following transfection with our shuttle vectors to determine the optimum time after transfection for measuring luciferase activities. Since we were using two different promoters, we determined the suitable single time for the analysis of both promoters (Figure 3). One hundred percent RLA was reached at 24 h after transfection of plasmids containing CMV promoter, but at this time RLA was of 70% when cells were transfected with TK promoter plasmids. We decided to use the 18-h time for the following experiments, since at this time luciferase activity is still increasing, but close to its maximal value, for both promoters TK and CMV. It has to be noted that after transfection of the same quantity of plasmid, the luciferase activity expressed from the plasmid without lesion containing the CMV promoter is at least 20-fold higher than that obtained for the plasmid without lesion containing the TK promoter (log scale in Figure 3), confirming that promoter strength of CMV is greater than that of TK in our system. We have also detected a similar kinetics of luciferase activity after transfection of CMV and TK plasmids for the other cell lines (data not shown).
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If the luciferase activity varies with the presence of 8-oxoG on its gene and according to the cell type, this should reflect an effect on its transcription efficiency. This may be due to variations in the repair pathway and/or in the transcriptional bypass of the lesion. For all DNA repair-deficient cell lines, we obtained at least
50–60% of RLA compared to wild-type cells (Figure 4). There is not a total blocking of transcription at the lesion because we detected some luciferase activity. Variation according to the cell type should reveal various repair efficiencies of the lesion.
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When an 8-oxoG lesion was located at positions 3 or 7, we obtained a similar decrease of luciferase activity in Ogg1–/– and Csb–/– cells: for the TK-containing constructions, the values were of 52 and 45% of control cells for Ogg1–/– and 55 and 59% for Csb–/–; for the CMV constructions, the values were 61 and 56% and 60 and 53%, respectively, of the WT cells (Figure 4). In Csb–/– cells, the influence of 8-oxoG was similar to that in Ogg1–/– cells, between 54 and 60% of RLA. In Csb–/–/Ogg1–/– cells, the decrease in RLA was slightly greater compared to Ogg1–/– and Csb–/– cells (for TK, 50 and 31% and for CMV, 45 and 22%). In general, decreases in luciferase expression were slightly more important for plasmids containing the CMV promoter and in double knockout cells compared to Ogg1–/– and Csb–/– cells (Figure 4).
When one 8-oxoG was placed at position 9 in TK-containing constructions, the RLA seems more affected in Ogg1–/–-defective cells than in the two previous positions (41% compared to 50–60%). Our results show that differences between Ogg1–/–, Csb–/– and Csb–/–/Ogg1–/– cells when the expression is under control of TK promoter are not very important. When the transcription is directed by the CMV promoter, the further the lesion in the transcription progression, the bigger effect on RLA. For example, this trend in Csb–/–/Ogg1–/– cells corresponds to 45, 22 and 11% for positions 3, 7 and 9, respectively.
Our data show that the transcriptional effect of the 8-oxoG lesion is different depending upon the promoter that drives the transcription and the sequence context surrounding the lesion. We can conclude that both BER and TCR proteins appear to be necessary to remove 8-oxoG completely from DNA during transcription.
Analysis of transcript levels of luciferase gene
In order to verify the results obtained with the luciferase assay, we analyzed the transcript levels of the luciferase mRNA normalized to the mRNA level of the housekeeping gene Gapdh. Results are represented in Figure 5. These experiments were carried out for plasmids containing the CMV promoter. For TK plasmids, transcript levels were too low to allow us determination of their transcriptional level with accuracy.
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The relative quantity of transcript is severely affected in double knockout cells for all constructions (Figure 5). These data correlate with RLA results and confirm a decrease in the transcription through 8-oxoG. We have also determined the efficiency of transcription at the position 3, containing no lesion, when the lesion was located at positions 7 or 9. Although 8-oxoG at position 7 or 9 decreases seriously the transcription efficiency at these regions, it did not show any significant transcription modification at undamaged position 3 (data not shown), indicating a normal transcription process before the blockage/pause at the lesion.
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Discussion |
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We have analyzed the effect of a unique 8-oxoG lesion located in the transcribed strand of a luciferase reporter gene after transfection in mouse cells with different repair deficiencies. The lack of BER for 8-oxoG (Ogg1–/– cells) resulted in 50–60% luciferase activity compared to wild-type cells. The only exception to this was the situation where the lesion was located at position 9 and with luciferase expression under the control of the CMV promoter. In this case, RLA was
20%. This difference may be explained by the sequence context. 8oxoluc9 sequence is the richest in GC content and this could probably render more difficult the repair of this lesion. The lack of TCR (Csb–/– cells) resulted in 40–50% reduction of luciferase expression compared to wild-type cells independently of the sequence context or the promoter that drove luciferase expression. Double knockout cells showed the same profile as the single knockout cells when the luciferase expression was driven by the TK promoter. With the CMV promoter, the 8-oxoG located at position 9 decreases by roughly 90% the luciferase expression in the double knockout cells. We could conclude from these results that the repair of 8-oxoG on a transcribed strand results in a timing competition between BER and TCR. Before the start of transcription, the 8-oxoG is located opposite C in a double-stranded DNA. It should, therefore, be normally repaired by BER. When RNA pol II approaches the lesion, the 8-oxoG is more in a single-strand DNA configuration rendering it probably immune to BER through Ogg1 but susceptible to TCR. In Ogg1–/–-deficient cells, up to 50% decrease in luciferase activity should be due to a partial blockage of transcription at the lesion. However, in this case the Csb protein should also be necessary to initiate and to complete the repair process. In Csb–/–-deficient cells, the 50% luciferase activity observed should be due to BER repair in the absence of transcription. In double mutants, depending upon the sequence and the promoter strength, an additive effect of both defects is observed. The final fate of an oxidative lesion in a wild-type background should, therefore, depend upon which pathway arrives first to the lesion, BER enzymes or RNA pol II. Because the 8-oxoG is located on an exogenous plasmid molecule, it may take longer to be repaired compared to genomic DNA. Plasmids have to be transfected to reach cell nucleus and probably the compartment where repair and transcription occurs. In genomic DNA, however, chromatin structure may also modify the speed of accessibility of repair enzymes to the lesion.
Several laboratories have studied 8-oxoG repair using different in vitro and in vivo systems. Data obtained in the last 4 years are contradictory but differences between the studied systems used for these experiments could explain the discrepancy.
We tried first to analyze the sequence context effect when an 8-oxoG is incorporated in DNA. Different results according to the sequence context analyzed among our three different sequences (Table I) have been obtained with mutagenesis and enzymatic assays. In our experiments, the most important mutation frequency after replication of plasmids in bacteria deficient for 8-oxoG repair was obtained for position 9, where the lesion is placed in a NgoMIV restriction site (23%) whose sequence is the richest in GC content (NgoMIV site, GCCGGC). Some systematic studies have been done with other adducts such as acetylaminofluorene, showing clearly that mutagenesis depends upon sequence context and is conditioned by the nucleotide types surrounding the lesion (40). The sequence context may affect the helix distortion due to the presence of the 8-oxoG. Different distortions can clearly affect DNA and RNA polymerase progression past the lesion and therefore modify the level of mutagenesis as well as the stalling of transcription complexes. The X-ray analysis of a DNA template containing one 8-oxoG (41) or molecular modeling assay (42) shows a weak distortion of the DNA double helix in the presence of the lesion or that the 8-oxoG is completely flipped out of the DNA double helix, respectively. The different sequences surrounding the lesions in those studies could explain their different results. There is growing evidence that the 8-oxoG lesion is seen differently by repair or polymerase enzymes according to the lesion context. For example, hOGG1 is unable to repair 8-oxoG located in the middle of a G6:C5 sequence where the 8-oxoG is flipped out (43). Furthermore, in the most mutagenic restriction site in our plasmid constructions, the 8-oxoG is present in a stretch of 16 G:C base pairs. This was also the case in the experiments done by Le Page et al. (21), where the 8-oxoG was located in a 12 G:C stretch corresponding to the sequence of the codons 10–14 of Ras human gene, a hot spot of mutagenesis in tumors. Similarly, according to the sequence, the 8-oxoG:A mismatch is a 9-fold better substrate than the 8-oxoG:C normal base pairing for the extension step by the Bacillus stearothermophilus DNA polymerase I (44).
To study the transcriptional effect of the presence of oxidative lesions, some laboratories have developed in vitro transcription experiments. They have failed to detect an in vitro arrest of RNA pol II when it encounters 8-oxoG (27). In these experiments, they have used whole HeLa cell extracts that contain many protein factors that could help the RNA pol II bypass. These results correlate with those obtained for T7 RNA polymerase (27,45) and the E.coli RNA polymerase (46). In a reconstituted system, Kuraoka et al. (28) have detected some pausing or some bypass of the lesion depending on the condition tested. This bypass was sometimes due to the misincorporation of adenine opposite to 8-oxoG. Larsen et al. (47) have also tested the effect of 8-oxoG in a reconstituted system and they detected a significant 5% pause/blockage of the RNA pol II using a plasmid template containing a single 8-oxoG. In both studies, plasmid templates contained AdML promoters but the sequence where the lesion was introduced was not the same. Recently, Charlet-Berguerand et al. (29) have shown a rate of 8-oxoG bypass of 48% in a reconstituted system and they have found that bypass of oxidative damages is regulated by transcription elongation factors. We have summarized all the results concerning 8-oxoG transcriptional effects in Table IV. Regarding the position of the lesion, we can see that most of the in vitro experiments place the lesion relatively close to the transcription initiation site. This position could be too close to detect an effect of 8-oxoG on transcription because promoter escape occurs 50 bp from the transcription initiation site (49). In fact, in last mentioned study, they have tested the bypass of 8-oxoG in two position, near the transcription initiation site (+105), where they found 77% of bypass and, farther in the sequence (+489), where they found 48% of bypass.
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In the in vivo experiments reported (Table IV), there are differences between sequences surrounding the lesion but also between promoters. Larsen et al. (47
) have also analyzed 8-oxoG effects when incorporated in a plasmid template in an in vivo test similar to ours and they failed to find any significant reduction in luciferase activity between Ogg1–/–, Csb–/– and double knockout cells. In their system, the 8-oxoG is not located in the middle of a G:C tract (Table IV) and the promoter driving transcription is SV40 with four GAL4-VP16-binding sites. The analysis of promoter sequence using an informatics tool (Mat Inspector V2.2) to determine putative transcription factor-binding site revealed us that the Gal4 and SV40 promoters present all fewer numbers of transcription factor-binding sites than TK and CMV promoters used in our study (50,51). Furthermore, we have tested SV40 constructions in the same conditions that our experiments and when the same quantity of plasmid was transfected, the luciferase activity obtained was weaker (data not shown). The internal control in Larsen's experiment contained a SV40 promoter as well, so a competition between both promoters could contribute to mask any 8-oxoG effect on transcription (52). We found that the choice of the promoters between the reporter plasmid and the plasmid used as internal control is very important. Competition between these promoters can occur changing dramatically the RLA and rendering impossible to see any effect of the 8-oxoG lesion (data not shown). The relevance of promoter-driving gene expression in the repair process has already been studied in mammalian cells and bacteria, showing that lesion can be repaired by different pathways depending on transcriptional strength and the transcription factors that are engaged during transcription (53–56).
In conclusion, we found that a unique 8-oxoG lesion can decrease the efficiency of transcription to a maximum of 90% both at enzymatic and mRNA levels in cells deficient for both BER and TCR. A competition between the kinetics of BER and of transcription on damaged templates can occur in single knockout MEF. The repair level and/or the transcriptional arrest by a unique 8-oxoG can vary according to the promoter strength and for a given promoter according to the nucleotidic sequence surrounding the lesion. We believe that these results confirm those we previously published (21). Recently, Spivak and Hanawalt have shown lower host cell reactivation of a plasmid containing randomly induced 8-oxoG damage in CSB and CSA-deficient human cells compared to wild type. They conclude from their data that ‘little repair of 8-oxoG can take place within transcription complexes in plasmid sequences’ in CSB cells (57). All these events may also explain the progressive neurological disorders found in CS and xeroderma pigmentosum/CS patients. Indeed, a high level of oxidative stress has been reported in brain cells (58). Some oxidative lesions (like 8-oxoG or others) may arrest transcription complex long enough to induce p53 and apoptosis. Moreover, if some oxidative lesions are bypassed by the RNA pol II, then mutagenic transcription can occur as already shown in bacteria (59). In both cases, unrepaired oxidative lesions can have deleterious effects in humans by either inducing apoptosis in nonreplicating cells (like neurons) or producing mutated proteins through transcriptional mutagenesis.
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Fundación Pedro Barrié de la Maza and Association pour la Recherche sur le Cancer to M.P.G. Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, the ‘Agence Nationale de la Recherche', the Scientific Council of Radioprotection of ‘Electricité de France', the University Paris-Sud and the ‘Fédération des Maladies Orphelines’ to A.S.
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Acknowledgments |
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We thank very much A. Klungland (Centre for Molecular Biology and Neuroscience, Oslo, Norway), the late E. Seeberg (Centre for Molecular Biology and Neuroscience, Rikshospitalet University Hospital, Oslo, Norway), P.J. Brooks (Department of Chemical and Biochemical Engineering, MD) and S. Boiteux (CEA, Fontenay-aux-Roses, France) for the gift of cells, plasmids and enzymes. We thank very much G. Spivak, A. Stary, D. Bregeon and P. Kannouche for the critical reading of this manuscript.
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* To whom correspondence should be addressed. Tel: +33 1 42 11 63 28; Fax: +33 1 42 11 50 08; Email: sarasin{at}igr.fr
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Received on January 8, 2007; revised on April 25, 2007; accepted on May 10, 2007.
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