Mutagenesis Advance Access originally published online on March 8, 2006
Mutagenesis 2006 21(2):125-130; doi:10.1093/mutage/gel006
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Endonuclease IV and Exonuclease III are involved in the repair and mutagenesis of DNA lesions induced by UVB in Escherichia coli
Laboratório de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho–IBCCF and 1LAMIAG, Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro-UFRJ, Brazil
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Abstract |
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Exonuclease III (Exo III) and endonuclease IV (Endo IV) play a critical role in the base excision repair (BER) of Escherichia coli. Both are endowed with AP endonucleolytic activity, cleaving the 5' phosphodiester bond adjacent to spontaneous or induced abasic sites in DNA. Although mutants defective in Exo III (xthA) are usually hypersensitive to oxidative agents such as hydrogen peroxide, near-UV-light and X-rays, mutants defective in Endo IV (nfo) are not as sensitive as the xthA strain. To further investigate the roles of these AP endonucleases in DNA repair, we evaluated the sensitivity and mutagenesis of xthA and nfo strains after UVB and compared with UVC light. Our results revealed that xthA but not nfo strain was hypersensitive to UVB. The use of Fe+2 ion chelator (dipyridyl), prior to irradiation, completely protected the xthA mutant against UVB lethal lesions, suggesting the generation of toxic oxidative lesions mediated by transition metal reactions. The nfo strain displayed increased UVB-induced mutagenesis, which was significantly suppressed by pre-treatment with dipyridyl. Although xthA strain did not display increased mutagenesis after UVC and UVB treatments, this phenotype was not related to xthA mutation, but rather to an unknown secondary mutation specifying an antimutator phenotype. After UVB irradiation, the base substitution spectra of nfo strain revealed a bias towards AT
GC transitions and GCCG transversions, which were also suppressed by previous treatment with the iron chelator. Overall, on the basis of the differential sensitivities and mutational spectra displayed after UVC and UVB treatments, we propose a role for Endo IV and Exo III to counteract DNA damage induced by the oxidative counterpart of UVB in E.coli. |
Introduction |
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In DNA, spontaneous or induced apurinic/apyrimidinic (AP) sites are extremely toxic to cells and are believed to be incompatible with life if not repaired (1
). AP sites can be spontaneously generated in DNA during normal aerobic metabolism or by exogenous agents, such as ionizing radiation, UV light or H2O2 (2). In order to circumvent AP site toxicity, pro- and eukaryotic cells have evolved efficient enzymatic mechanisms to repair damaged DNA (3). The exonuclease III (xthA) protein accounts for 
90% of the AP endonucleolytic activity found in Escherichia coli. This enzyme is a crucial member of the base excision repair (BER), a key repair mechanism to neutralize DNA oxidative stress (4). Exonuclease III (Exo III) recognizes and cleaves the phosphodiester bond 5' to an AP site, leaving a 3'-hydroxyl terminus. The remaining 5'deoxyribose 5'-phosphate can be further cleaved by a deoxyribophosphodiesterase (dRPase) to generate a single gap in DNA (5). This gap can be efficiently repaired by DNA polymerase I and DNA ligase (6). In addition, during DNA repair, the elimination of oxidized bases by DNA N-glycosylases also constitutes a direct source of AP sites, which are also substrates of Exo III.
The E.coli xthA mutants possess a residual AP endonucleolytic activity encoded by the nfo gene, the endonuclease IV (Endo IV) protein (7). The Endo IV recognizes and cleaves DNA in the same manner as Exo III (8). Both enzymes endowed with this overlapping activity (AP endonucleolytic) are expected to act as mutual back-ups. However, they do not have identical activities. Exo III is also endowed with enzymatic activities absent in Endo IV: an efficient 3'5'exonuclease activity for which it is named, a 3'-phosphomonoesterase activity and an endonuclease activity at urea residues in DNA (9). In its turn, Endo IV is able to recognize a set of oxidized pyrimidines cleaving the phosphodiester bond 5' to the damaged base, while Exo III is not. (10).
Strains deficient in nfo or xthA are usually sensitive to oxidizing agents (11). Both Exo III and Endo IV have been implicated in the protection of E.coli cells against lethal and mutagenic effects of near-UV. Indeed, UV light is able to generate oxidative damage in cellular DNA via production of reactive oxygen species (ROS). UVB is a potent inducer of ROS, including superoxide radical (
) (12), hydrogen peroxide (H2O2) and hydroxyl radical (OH.), which have been implicated in cutaneous aging as well as cancer and various inflammatory disorders (13).
The DNA damages induced by the different UV wavelengths appear to be distinct (14). Cyclobutane pyrimidine dimmers (CPD) and pyrimidine (6–4) pyrimidone photoproducts are the most abundant UVC-induced lesions in a cell genome through direct DNA absorption (15). Oxidative base modifications, strand breaks and base loss (AP site) have also been identified after UVB irradiation (16,17), but seem to play a minor role on cell genotoxicity.
Herein, we examined the relative importance of oxidative damage in the genotoxicity induced by UVB in xthA and nfo deficient strains of E.coli. Our results suggest that Exo III and Endo IV play relevant roles against DNA damage induced by oxidative counterpart of UVB radiation.
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Materials and methods |
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Bacterial strains, media, plasmid and growth conditions
The bacterial strains used in this study were derived from E.coli K-12 and are listed in Table I. Cells were grown at 37°C with shaking in Luria–Bertani (LB) medium (18
). Overnight cultures were diluted 1 : 40 in LB and grown until the exponential phase (2 x 108 cells/ml) for all experiments. Congenic sets of mutant strains were constructed by P1-mediated transduction as described previously (18).
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Sensitivity to UVB and UVC
UVB treatment Bacterial cultures (10 ml) were grown to
2 x 108 cells/ml, washed with M9 buffer (0.6% Na2HPO4, 0.3% KH2PO4, 0.1% NH4Cl and 0.05% NaCl) and resuspended in the same buffer (18). Aliquots of cell suspension (2 ml) were exposed to increasing UVB doses in a Nunc Petri plate (2.0 cm diameter) with lid. The plate lid filters out light below 300 nm (data not shown). The cultures were irradiated with a 15 Watt Vilber Lourmart Ultraviolet lamp (broad spectrum 290–320 nm). Doses were determined with a VL–215 LM radiometer (UVB photocell). After each dose, the cell suspension was properly diluted in M9 buffer, plated in LB-agar and incubated at 37°C overnight. UVC treatment Bacterial cultures were exposed to increasing UVC doses as above, but in plates without the respective lid. The cultures were irradiated with a germicidal lamp GE G15T8 (254 nm). Doses were determined with the same radiometer as above, with a proper UVC photocell.
Pre-treatment with dipyridyl. Cultures in exponential growth phase were incubated with 1.0 mM iron chelator dipyridyl (2,2'-bipyridine, Sigma, 366-18-7) for 20 min in M9 buffer. Treatment with the metal iron chelator does not affect cell viability and spontaneous mutation frequencies (19 and this study, data not shown). The use of iron chelator was based on previous reports examining the role of transition metals on oxidative DNA damage and mutagenesis induced by H2O2 (20–22).
All survival experiments were performed independently at least three times and expressed as averages with standard errors.
UVB and UVC mutagenesis
Mutagenesis was evaluated scoring rifampicin-resistant mutants (RifR) and Lac+ revertants after each treatment (23,24). Cells (108 cells/ml) were centrifuged to eliminate LB medium, washed twice with M9 buffer, resuspended in the same volume and irradiated with UVC or UVB. After that a culture aliquot was plated on LB-agar to determine survival and another (106 viable cells) was directly inoculated in liquid LB (2 ml) for 48 h at 37°C to allow the appearance of rifampicin resistance and Lac+ reversion. Then the cell density was measured by plating appropriate dilutions in M9 buffer containing glucose (for CC strains) or LB, and counting the colonies after 24 h at 37°C. The number of RifR was determined after platting aliquots (109 viable cells) on selective LB-rifampicin (100 µg/ml) medium and incubation at 37°C for 24 h. For Lac+ revertants, aliquots (109 viable cells) were plated on selective minimal medium containing 0.4% lactose for 72 h at 37°C (23). Mutation frequency was calculated in terms of cell density and normalized to 108 cells. In the case of Lac– to Lac+ reversion assay, a set of E.coli strains with different mutations at the same coding position in the lacZ gene was used. These strains have been designed so that each reverts via one of the six possible base substitutions (23). We have screened base changes after UVB and UVC using the wild-type (23) and a respective set of isogenic nfo mutant strains (Table I).
All mutagenesis experiments were performed independently at least six times and expressed as averages with standard errors.
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Results |
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Endo IV (nfo) and Exo III (xthA) are key elements of the BER pathway of E.coli, as they ensure clean 3'OH termini to polymerases after the action of DNA-N-glycosylases on oxidized bases (25
). Reports on the sensitivity of E.coli cells after irradiation UVC have demonstrated that mutants deficient in AP endonuclease activity (Endo IV or Exo III) are as sensitive as their wild-type strains (4). However, this observation is not similar to the results observed after UVB treatment of nfo and xthA deficient strains. While nfo, xthA and the wild-type strains are equally sensitive to UVC (Figure 1A), the xthA mutant is particularly hypersensitive to UVB when compared with nfo and wild-type strains (Figure 1B). This indicates that xthA gene product has a positive role counteracting lethal lesions induced by UVB (Figure 1C), while nfo gene should be of less, or of negligible, importance.
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Since the xthA mutant displayed differential sensitivity to UVB and UVC, we wonder if the increased sensitivity to UVB could be due to DNA lesions generated by the oxidative counterpart of UVB radiation. In this way, as the generation of ROS in vivo often takes place via the Fenton reaction using transition metals (26
), we decided to investigate if the chelation of Fe+2 would produce any effect on xthA survival upon UVB irradiation. Indeed, the pre-treatment with dipyridyl completely protected xthA cells from UVB lethal lesions (Figure 2). This suggests that xthA gene product is involved in the repair of lethal DNA lesions generated by UVB in a transition metal-mediated reaction. It is important to stress that dipyridyl had no protective effect when cells were irradiated with UVC (data not shown).
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The analysis of UVC- and UVB-induced mutagenesis also revealed interesting features. After treatment with UVC (LD10 for all strains), WT and nfo mutant displayed high RifR induction over their respective spontaneous levels, while xthA single mutant was slightly susceptible to UVC mutagenesis (only 2.0-fold over its spontaneous level). Since, neither nfo nor xthA mutant displayed RifR induction levels higher than that of the WT, the inactivation of Exo III or Endo IV does not seem to compromise the repair of mutagenic lesions induced by UVC treatment. Actually, the xthA mutant exhibited a particular antimutator phenotype. Spontaneous and induced (UVC as UVB) mutagenesis were drastically lower than that of the WT strain. Even when complemented with plasmid pSGR1 harbouring xthA gene (27
), the xthA strain still displays reduced UV mutagenesis (39.0 ± 8.1 and 105 ± 16 RifR/107 for UVC and UVB, respectively) compared with that of the WT (Figure 3). Apparently, the harsh chemical treatment of AB1157 used to obtain strain BW9091 (28) may have introduced another mutation, conferring to this strain an antimutator phenotype.
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Upon UVB treatment, nfo mutant displayed a marked increase in RifR induction over that of the WT strain, indicating that Endo IV has a determinant role avoiding mutagenic UVB-induced lesions (Figure 3).
The comparison between UVB mutation spectra of WT and nfo strains revealed a specific increase of Lac+ revertants in the nfo mutant, corresponding to ATGC and CG GC substitutions (Figure 4). Indeed, these base substitutions are specific to UVB, since they were not observed when cells (WT and nfo strain) were treated with UVC light (Figure 5).
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Finally, while dipyridyl had no effect on the induction of Lac+ revertants after UVB in WT strains, the pre-treatment with this iron chelator was able to partially decrease the GC
CG and drastically suppress ATGC substitutions induced by UVB in the nfo mutants (Figure 6).
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Discussion |
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In this study, we assessed the relative importance of oxidative damage in the genotoxicity induced by UVB light using a set of E.coli strains deficient in the two major endonucleases involved in BER. In order to discriminate UVB oxidative effects from classical UVC-induced DNA damage (CPD), UVB experiments were performed in parallel with UVC as a control. In addition, the use of iron chelator dipyridyl provided indirect evidence suggesting the participation of oxidative species in the generation of genotoxic DNA damage.
Previous reports have demonstrated that UVB light was able to generate oxidative species and damage DNA (29). However, these oxidative products in DNA are believed to have a minor role, both in mutagenesis as well as in cell toxicity (30,31).
In our study, we demonstrated that xthA mutant is rapidly inactivated by UVB but not by UVC light when compared with the respective WT strain. In fact, in xthA mutant, most of UVB toxicity does not seem to be due to classical CPD lesions in DNA, since xthA sensitivity could be fully abolished by the depletion of iron ions with dipyridyl (Figure 2). It is worthy to remark that the survivals of the WT, WT + dipyridyl and xthA + dipyridyl are not significantly different (P > 0.05). These data suggest that the generation of toxic oxidative species by UVB may occur through a Fenton reaction (32). Indeed, a previous report pointed up xthA mutant especially sensitive to various oxidative agents (33), which is in agreement with our evidence implicating the oxidative counterpart of UVB in high inactivation levels of xthA mutant (Figure 2). Although complementation of xthA mutant with wild-type xthA gene (Figure 1C) completely rescued the sensitivity to UVB, this was not the case for spontaneous or induced mutagenesis. This observation may reside on the fact that xthA mutant was produced by harsh chemical mutagenesis and may have introduced another mutation rendering this strain refractory to induced mutagenesis, with an antimutator phenotype (28).
While Exo III has a crucial role in the repair of UVB oxidative-lethal lesions, it does not seem to be the case of Endo IV (Figure 1). Two possible reasons may underline this observation: (i) Endo IV activity accounts only for 5–10% of total AP endonuclease activity or (ii) Endo IV is not required to repair lethal lesions induced by UVB. In fact, the second assumption seems more plausible, as Endo IV appears to be implicated in the avoidance of UVB-induced mutagenesis (Figure 3). Besides, the nfo mutant displays a specific bias towards two base substitution events after UVB, which are absent in WT strain (Figure 4). Moreover, this mutagenic profile is specific of UVB, since it was not found when we examined UVC-induced base substitutions in these same strains (Figure 5). It is important to remark that our UVB source does not comprise UVB short wavelengths, since our filter cuts off <300 nm. The elimination of short wavelengths of UVB spectrum may interfere with the overall CPD production and may explain why WT strains CC102 and CC106 do not display high induction levels after UVB treatment. Indeed, UVC wavelengths are about 100 times more efficient than UVB in terms of CPD production (10). After UVC, both CC102 (data not shown) and CC106 (Figure 5) presented high induction levels of Lac+ revertants.
The increase of CGGC transversions can be attributed to 5-hydroxycytosine, which has been already identified in DNA after UVB radiation (34). This lesion can be originated from cytosine glycol, which is highly unstable and can dehydrate to form 5-hydroxycytosine (5-OHC). In effect, 5-OHC is among the major stable free radical-damaged cytosine products (34). After UVB, these CGGC transversions are already increased in WT strain (25 Lac+/108 cells) and about 4-fold over WT levels in the nfo mutant. This specific increase in nfo mutant can be ascribed to another Endo IV activity, different from its AP endonuclease. Endo IV is also able to recognize and cleave DNA at sites containing oxidized pyrimidines, including 5-hydroxycytosine, in a mechanism named nucleotide incision repair (NIR) (10).
Although not to the same extent, the partial suppression of CGGC transversions by dipyridyl, compared with that of ATGC, may reside on the fact that cytosine glycol can also be generated with copper ions in a site-specific manner, which would not be drastically altered by the iron chelator (35). These UVB-induced transversions, partially suppressed upon cell pre-treatment with dipyridyl, suggest oxidized DNA damage involving a Fenton reaction.
Interestingly, the augmentation of ATGC was quite intriguing, since no evident mechanism or DNA lesion would explain such marked transition event after UVB irradiation. If we admit that the DNA lesion leading to ATGC is not substrate of Exo III enzyme, since UVB mutagenesis is not enhanced in xthA strain, one should discard the AP endonuclease activity of Endo IV as the one responsible for ATGC avoidance. Actually, besides its action on oxidized pyrimidines (10), Endo IV is able to recognize and cleave DNA at sites containing 
-deoxyadenosine (-dA), also via NIR (36). This lesion was first identified after DNA treatment with gamma rays in anoxic conditions, which is believed to occur because of the abstraction of the sugar anomeric hydrogen atom at C1' by OH. radical (37). In fact, to our knowledge, among several enzymes involved in BER, only Endo IV is able to recognize -dA in DNA (37,38). Therefore, -dA may be at the origin of the ATGC transitions induced by UVB in nfo cells.
However, instead of ATGC transitions, it has been demonstrated that -dA in DNA leads to –1 deletions (38). In the suggested mechanism underlying this type of mutation, the first step predicts the misincorporation of cytosine in face of -dA during DNA polymerization by replicative polymerase (36). The second step is directly dependent on the DNA sequence context. In Shimizu et al. (37), the DNA sequence of the shuttle-vector possessed a favourable environment to generate –1 deletions when -dA mutagenic potential was assessed. The shuttle-vector possesses a guanine immediately adjacent to -dA, in the same DNA strand of the lesion. According to the authors, the mispair -dA:C is unstable and would promote local denaturation to form a canonical pair between the incorporated cytosine and the guanine adjacent to the -dA. This local denaturation, followed by the G:C pairing, provokes a DNA strand slippage and would support the –1 deletion events in DNA containing the -dA lesion.
In our case, the lacZ gene sequence context in CC strains does not allow this kind of base pair, since there is no guanine immediately adjacent to the putative -dA lesion (38,39). In CC106, the target sequence is in the following context: 5'-GGGAATAAATCA-3'. The expected transition in codon 461 is AAA to GAA (40). Therefore, –1 deletions should not be favoured, but –4, –5 or –6 deletions should be favoured, since the nearest guanine is 4 bp away from the target sequence. If –1 deletions are not supposed to occur in our sequence context, and –4 deletions requires denaturation of at least 3 bp, one could admit that the prevalence of cytosine paired with -dA would produce a high incidence of ATGC transitions.
Interestingly, in our experiments, the use of dipyridyl drastically reduced the ATGC transition levels induced by UVB, corroborating the participation of OH. radicals in the generation of mutagenic lesions in nfo cells, and also supporting -dA as a putative lesion at the origin of this base substitution.
Overall, our results point out new features concerning the repair of DNA lesions induced by UVB light. The oxidative counterpart of UVB light has relevant consequences in terms of lethal and mutagenic effects in vivo, implicating BER (41) mediated by Exo III enzyme for lethal damage and possibly NIR mediated by Endo IV (10) for mutagenic lesions.
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Acknowledgments |
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We thank J.S. Cardoso for her expert technical support. L.L.S. was supported by a long-term fellowship from CAPES. I.R.E. was supported by a short-term fellowship from CNPq. This work was supported by CNPq, CAPES and FAPERJ.
Conflict of Interest Statement: None declared.
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Notes |
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* To whom correspondence should be addressed. Tel: +00 55 21 25626578; Fax: +00 55 21 22 80 81 93; Email: acleitao{at}biof.ufrj.br
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References |
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Received on September 19, 2005; revised on December 12, 2005; accepted on January 12, 2006.
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