Evelyne Sage

Distribution and repair of photolesions in DNA : genetic consequences and the role of sequence context

Photolesions are induced in DNA following direct absorption of ultraviolet radiation, or through the action of photosensitizers. The best-characterized photolesions are those induced by UVC (190-280 nm),* especially at 254 nm, which is very close to the absorption maximum of DNA. The biological effects of sunlight, i.e., 0.3% UVB (280-320 nm), 5.1% UVA (320-400 nm), 62.7% visible light, and 31.9% infrared, are much less well defined. The UVC component of solar light, which is also the most harmful to the genome, is absorbed by the atmosphere. Thus, a recent source of concern results from the depletion of the ozone layer, which is shifting the intensity and wavelength spectrum of solar UV to which mankind is exposed. This would inevitably result in an increased incidence of skin cancer in human populations, apparently as a consequence of the rise in UVC-like carcinogenic DNA damage.’ Among DNA photoreactive compounds, furocoumarins have received much attention due to their widespread use in the photochemotherapy of skin diseases, cutaneous T-cell lymphoma, and of some viral infections.z Other photosensitizers include potential phototherapeutic agents such as hematoporphyrins, phthalocyanins, promazine, as well as dyes such as methylene blue or pr0flavin.j Ultraviolet radiation or photosensitizers damage DNA by direct interaction via a photodynamic, oxygen-dependent mechanism, or both. For example, UVCand UVB-induced cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts ([6-41 photoproducts) are formed by direct absorption of UV radiation by DNA. Some photosensitizers, like furocoumarins and their derivatives, photoreact directly with DNA via their singlet or triplet state to form covalent bonds. Alternatively, oxygen-mediated photolesions result from the production of activated oxygen species or radicals by charge, electron, or energy transfer during ifradiation of photosensitizers. Also, UVA, UVB, and, to a lesser extent, UVC are able to induce this class of DNA photodamage, likely by involvement of endogenous cellular

Wavelength dependence of ultraviolet-induced DNA damage distribution: involvement of direct or indirect mechanisms and possible artefacts.

DNA damage profiles have been established in plasmid DNA using purified DNA repair enzymes and a plasmid relaxation assay, following exposure to UVC, UVB, UVA or simulated sunlight (SSL). Cyclobutane pyrimidine dimers (CPDs) are revealed as T4 endonuclease V-sensitive sites, oxidation products at purine and pyrimidine as Fpg- and Nth-sensitive sites, and abasic sites are detected by Nfo protein from Escherichia coli. CPDs are readily detected after UVA exposure, though produced 10(3) and 10(5) times less efficiently than by UVB or UVC, respectively. We demonstrate that CPDs are induced by UVA radiation and not by contaminating UVB wavelengths. Furthermore, they are produced at doses compatible with human exposure and are likely to contribute to the mutagenic specificity of UVA [E. Sage et al., Proc. Natl. Acad. Sci. USA 93 (1996) 176-180]. Oxidative damage is induced with a linear dose dependence, for each region of the solar spectrum, with the exception of oxidized pyrimidine and abasic sites, which are not detectable after UVB irradiation. The distribution of the different classes of photolesions varies markedly, depending on wavelengths. However, the unexpectedly high yield of oxidative lesions, as compared to CPDs, by UVA and SSL led us to investigate their production mechanism. An artificial formation of hydroxyl radicals is observed, which depends on the material of the sample holder used for UVA irradiation and is specific for long UV wavelengths. Our study sheds light on a possible artefact in the production of oxidative damage by UVA radiation. Meanwhile, after eliminating some potential sources of the artefact ratios of CPDs to oxidized purine of three and five upon irradiation with UVA and SSL, respectively, are still observed, whereas these ratios are about 140 and 200 after UVC and UVB irradiation.

Clustered DNA lesion repair in eukaryotes: Relevance to mutagenesis and cell survival

A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1-2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed.

Oxidation of guanine in cellular DNA by solar UV radiation: biological role.

Abstract. The formation of cyclobutane pyrimidine dimers (CPD) and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3‐2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8‐oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 102 and 102 more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8‐oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8‐oxodGuo were produced between 290–320 nm and 320–340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8‐oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8‐oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8‐oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation

Processing of a complex multiply damaged DNA site by human cell extracts and purified repair proteins

Clustered DNA lesions, possibly induced by ionizing radiation, constitute a trial for repair processes. Indeed, recent studies suggest that repair of such lesions may be compromised, potentially leading to the formation of lethal double-strand breaks (DSBs). A complex multiply damaged site (MDS) composed of 8-oxoguanine and 8-oxoadenine on one strand, 5-hydroxyuracil, 5-formyluracil and a 1 nt gap on the other strand, within 17 bp was built and used to challenge several steps of base excision repair (BER) pathway with human whole-cell extracts and purified repair enzymes as well. We show a hierarchy in the processing of lesions within the MDS, in particular at the base excision step. In the present configuration, efficient excision of 5-hydroxyuracil and low cleavage at 8-oxoguanine prevent DSB formation and generate a short single-stranded region carrying the 8-oxoguanine. On the other hand, rejoining of the 1 nt gap occurs by the short-patch BER pathway, but is slightly retarded by the presence of the oxidized bases. Taken together, our results suggest a hierarchy in the processing of the lesions within the MDS, which prevents the formation of DSB, but would dramatically enhance mutagenesis. They also indicate that the mutagenic (or lethal) consequences of a complex MDS will largely depend on the first event in the processing of the MDS.

The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells

It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3–8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells.

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions

Purpose: This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions – hadrontherapy – with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. Materials and methods: Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 μM PtTC, and then irradiated by fast ions C6+ and He2+, with Linear Energy Transfer (LET) within range 2–70 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). Results: For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. Conclusion: This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

Antibodies to DNA modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene

Several studies have shown that the carcinogen N-acetoxy-N-2-acetylaminofluorene (AAAF) reacts in vivo and in vitro with native DNA and that the DNA contains a major (80%) adduct N(deoxyguanosin8-yl)-acetylaminofluorene (dGuo8-AAF) and a minor (20%) adduct 3{deoxyguanosin-N2-yl)-acetylaminofluorene (dGuo-N2-AAF) (reviewed [1,2]). It has been shown that the major alteration induced by the fluorene ring is the creation of locally disorganized regions inside the double helical structure [3-81 . Methods sensitive enough to assay the regions of DNAmodified by a carcinogen at the levels of modification occuring in the ‘in vivo’ carcinogenesis experiments would be of great value. The immunological method has already been shown to be able to detect small modifications in DNA (see e.g. [9,10]). On the other hand, the study of the specificity of the antibodies can bring some knowledge on the conformation of the antigen. For these reasons, we have undertaken a study of the immunogenicity of native DNA after reaction with AAAF. In this paper, we show that native DNA slightly modified by AAAF can induce in rabbits the synthesis of specific antibodies which selectively recognize AAF-substituted DNA. A methods of purification of these antibodies is described. Also, the association constants for the binding of the antibodies and several ligands are reported.

UVA-induced damage to DNA and proteins: direct versus indirect photochemical processes

UVA has long been known for generating an oxidative stress in cells. In this paper we review the different types of DNA damage induced by UVA, i.e. strand breaks, bipyrimidine photoproducts, and oxidatively damaged bases. Emphasis is given to the mechanism of formation that is further illustrated by the presentation of new in vitro data. Examples of oxidation of proteins involved in DNA metabolism are also given.

Oxidative Stress in Mammalian Cells Impinges on the Cysteines Redox State of Human XRCC3 Protein and on Its Cellular Localization

In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.