Caractérisation des ADN polymérases de Pyrococcus abyssi en présence de matrices ADN endommagées

Hyperthermophilic archaea cope with harsh conditions such as high temperatures, high pressures, pH shifts and ionising radiations. Such environments favour the apparition of DNA lesions like apurinic (AP) sites or oxidized DNA bases (8-oxo-dG and 8-oxo-dA). Here, Pyrococcus abyssi (Pab) was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. The first part aimed at detecting DNA damages in the genome of Pab: -Endogenous AP sites and the oxidized DNA base 8-oxo-dG persist at a slightly higher level in Pab genome compared with Escherichia coli. -Under oxydative stress conditions performed in a gas-lift bioreactor for the first time, the 8-oxo-dG increasing rate is correlated to the important cell survival diminution, assessing the genotoxic effects of this stress on Pab. The second objective was to unravel the behaviour of the two replicative DNA polymerases from Pab, PabpolB (Family B) and PabpolD (Family D), in the presence of damaged DNA. The significant results are the following: -Both Pabpols are able to bypass 8-oxo-dG and 8-oxo-dA whereas they are strongly blocked by the AP site. -Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site independently of the nucleotidic sequence context, albeit inefficiently. Their exonuclease function seemed to endow with an important role in the protection against mutagenesis since it acts as a kinetic barrier, preventing from miscoding insertion in front of an AP site. -The preferential incorporation of the dAMP is also favoured in front of 8-oxo-dG by both Pabpols. However, PabpolD bypass 8-oxo-dA by inserting a dAMP or a dTMP opposite the lesion while PabpolB could not insert a dNTP. -Pabpols can also incorporate the oxidized dGTP, 8-oxo-dGTP, opposite template cytosine. The third part focused on biotechnological applications of the Pabpols and showed that both Pabpols are able to amplify damaged DNA by PCR. In the future, these enzymes could be used as powerful tools in order to amplify, for example, forensic or ancient DNA, DNA from hydrothermal vents or clinical specimens damaged DNA...