Ma contribution à ce travail été par la réalisation de la totalité des travaux expérimentaux, la rédaction et la soumission de l’article. Dr. Pierre Cloutier nous a aidé à analyser nos résultas expérimentaux à l’aide des modèles mathématique.
Résumé:
Les cassures doubles brins (CDBs) sont généralement connues comme les lésions toxiques les plus fréquemment induites par le rayonnement ionisant. Afin de déterminer l’effet de CDBs et d’autres types de lésions créées par l'effet indirect de la radiation ionisante sur la fonctionnalité (‘‘viabilité’’) du plasmide, nous avons utilisé un modèle simple, soit E. coli transformée avec un plasmide (pGEM-3Zf (-)) préalablement irradié en solution aqueuse avec des rayonnements gamma (137Cs). Comme prévu, nous avons constaté que l'efficacité de transformation diminue avec l'augmentation de la dose de rayonnement, mais cette diminution ne peut pas être expliquée par la formation des CDBs. Par exemple, pour une dose de 500 Gy, l'efficacité de transformation relative a diminué de 100% à 53%, alors que seulement 5.7% des plasmides contiennent des CDBs. Malgré le fait que les CDBs sont clairement toxiques, leur nombre pour une dose donnée, est insuffisant pour expliquer la perte de la viabilité du plasmide. La perte de la ‘‘viabilité’’ du plasmide peut être expliquée par une lésion (s) formée à une fréquence environ 8 fois plus grande que celle des CDBs. Ces lésions peuvent être des sites multiples localement endommagés (LMDS) (incluant un pontage inter-brins), de sorte que l'information est
perdue sur les deux brins d’ADN. Elles semblent être réparables, car la courbe de survie présente un épaulement assez remarquable à des doses de rayonnements ionisants faibles. Le rendement des LMDS (non-DSB cluster damage), tel que révélé par les enzymes de réparation Fpg et Nth, est 31 fois plus élevé que celui des CDBs franches. De plus, à l’aide de modèles mathématiques, nous avons estimé qu’il faut au moins trois lésions toxiques pour inactiver les plasmides irradiés.
RÉSULTATS: PREMIER ARTICLE
ABSTRACT
The majority of studies on lethal radiobiological damage have focused on double- strand breaks (DSBs), a type of clustered DNA damage and the evaluation of their toxicity, while other types of clustered DNA damage have received much less attention. The main purpose of this study is to evaluate the contribution of different lesions induced by ionizing radiation to the loss of plasmid DNA functionality. We employed a simple model system comprising E. coli transformed with an irradiated plasmid [pGEM-3Zf (–)] to determine the effect of DSBs and other lesions including base damage and clustered lesions on the functionality (“viability”) of the plasmid. The yields of γ-radiation-induced single-strand breaks (SSBs) and DSBs were measured by gel electrophoresis. We found that the transformation efficiency decreases with radiation dose, but this decrease cannot be explained by the formation of DSBs. For example, at doses of 500 and 700 Gy, the relative transformation efficiency falls from 100% to 53% and 26%, respectively, while only 5.7% and 9.1% of the plasmids contain a DSB. In addition, it is also unlikely that randomly distributed base lesions could explain the loss of functionality of the plasmid, since cells can repair them efficiently. However, clustered lesions other than DSBs, which are difficult to repair and result in the loss of information on both DNA strands, have the potential to induce the loss of plasmid functionality. We therefore measured the yields of γ-radiation- induced base lesions and cluster damage, which are respectively converted into SSBs and DSBs by the base excision repair enzymes endonuclease III (Nth) and
formamidopyrimidine-DNA glycosylase (Fpg). Our data demonstrate that the yield of cluster damage (i.e., lesions that yield DSBs following digestion) is 31 times higher than that of frank DSBs. This finding suggests that frank DSBs make a relatively minor contribution to the loss of DNA functionality induced by ionizing radiation, while other toxic lesions formed at a much higher frequencies than DSBs must be responsible for the loss of plasmid functionality. These lesions may be clustered lesions/locally multiply damaged sites (LMDS), including base damage, SSBs and/or intrastrand and interstrand crosslinks, leading to the loss of vital information in the DNA. Using a mathematical model, we estimate that at least three toxic lesions are required for the inactivation of plasmid functionality, in part because even these complex lesions can be repaired.
27 RÉSULTATS: PREMIER ARTICLE
INTRODUCTION
DNA integrity can be altered by different exogenous and endogenous genotoxins (1–3), including those produced by ionizing radiation (IR). Human malignancies are often treated by ionizing radiation (4) because of its ability to kill cells within a reasonably well defined volume. Ionizing radiation induces damage to DNA by both direct energy deposition in DNA (direct effect) and by generating reactive species from the radiolysis of water and other biomolecules surrounding the DNA (indirect effect), which subsequently react with DNA (5, 6). Water radiolysis results in the formation of free radicals and reactive species such as hydroxyl radicals, hydrated electrons and hydrogen atoms that either interact with biomolecules or recombine to produce other species such as H2O2 (7).
Hydroxyl radicals are the most damaging species for DNA since they produce both base damage and DNA strand breaks (8, 9). For example, sugar oxidative degradation pathways resulting from hydroxyl radical attack can lead to DNA strand break formation (10, 11).
Ionizing-radiation induces SSBs, modified (oxidized) bases, abasic sites and additional products such as DNA intra and interstrand crosslinks, DNA-protein crosslinks and most importantly, DSBs and clustered lesions, which are also termed locally multiply damaged sites (LMDS) (12). Base damage and DNA SSBs, which occur much more frequently than DSBs, can be repaired rapidly with high fidelity (12). However, it is generally accepted that DNA DSBs are the most frequent toxic DNA lesion induced by IR (13, 14). A DSB is a type of clustered DNA damage in which two SSBs are formed in close proximity on opposite DNA strands, by either a one or two hit damage process or during DNA repair. Unrepaired DSBs can cause large-scale deletions and cell death (15, 16), whereas misrepaired DSBs can give rise to mutations and chromosomal rearrangements, and hence cell death or cancer in multicellular organisms. In the late S and G2 phases of the
eukaryotic cell cycle, homologous recombination permits error-free DSB repair, while the error-prone non-homologous end-joining pathway operates during the rest of the cell cycle (17–19). A single unrepaired DSB in the DNA of E. coli is lethal (20–24). In addition to radiation-induced DSBs, this lesion can also be generated by enzymatic repair of other lesions in DNA such as SSBs and base damage (24–26).
LMDS were defined by Ward (12) as two or more DNA lesions including oxidative damage, formed within one or two helical turns by single radiation tracks after local energy deposition. From biophysical calculations using in vitro models and several biological systems, Goodhead and others (27–35) concluded that LMDS should be formed in cellular DNA by ionizing radiation. Moreover, both low- and high-LET radiation at different doses have been reported to induce LMDS in isolated DNA such as oligonucleotides (27, 36–39), in bacteria (40, 41), and in mammalian cells (42–45). It has been shown that LMDS, including DSBs (46), are produced by ionizing radiation in both isolated and cellular DNA, even at low doses of radiation (0.1–1 Gy) (42); LMDS represent a signature of the effects of ionizing radiation on DNA (47) as a result of the occurrence of multiple events in double-stranded DNA along the particle track. In human cells, oxidized purine and pyrimidine clusters and abasic clusters comprise the majority (70–80%) of the clustered lesions (42–45, 48). It has also been suggested that LMDS can be converted into DSB during the repair process (49). In E. coli, for example, a significant proportion of the DSBs are not produced directly by radiation, but arise in the course of enzymatic repair of other lesions in DNA, possibly SSBs and base damages (25, 50); In addition, cluster lesions including SSBs or other damages, such as base and sugar modifications, have been reported to inactivate vector DNA (51–53). It has also been suggested that most of the biological inactivation of damaged plasmid DNA is due to the enzymatic conversion of the SSBs into lethal damages in the cell (53, 54). Blaisdell and Wallace have demonstrated that E.
coli lacking three oxidative DNA glycosylase/AP lyases are more radioresistant than wild-
type cells, due to the lower yield of post-irradiation DSB formation in the mutant bacteria relative to the wild-type (41). Furthermore, post-irradiation formation of DSB has been reported to increase lethality in strains that overexpress formamidopyrimidine-DNA N- glycosylase (Fpg) (41). These findings highlight the difficulty for a cell to repair complex DNA damage and support the hypothesis that LMDS produced by ionizing radiation play an important role in cell toxicity (13, 14). Such complex lesions are probably more difficult to repair and exhibit a higher genotoxic potential leading to mutations, genomic instability, cellular transformation and cancer (13, 14) compared to isolated lesions (28). Although, DSBs and other LMDS have the potential to be lethal, the relative contribution of each lesion to the loss of functionality of DNA is not known.
29 RÉSULTATS: PREMIER ARTICLE In this study, we used pGEM-3Zf (–) plasmid DNA in a dilute aqueous solution to investigate the indirect effect of γ radiation on the transformation efficiency of the plasmid DNA into E. coli (55–57). The relative abundance of supercoiled, circular and linear DNA was quantitatively determined by gel electrophoresis followed by laser scanning. We observed that the transformation efficiency decreased with radiation dose, but only a small proportion of this decrease can be attributed to the formation of DSBs. Thus, other toxic, potentially lethal lesions formed at much higher frequencies than DSBs, must be responsible for the loss of plasmid functionality. Our analysis using DNA glycosylases to convert base damage and apurinic/apyrimidinic (AP) sites to DSBs indicates that the formation of LMDS in which information is lost across both DNA strands can account for much of the observed loss of DNA functionality.