The APOBEC3 (Apo3) family of proteins are single stranded (ss) DNA cytosine deaminases (C → U). They are grouped into two different structural groups, the single catalytic domain Apo3 enzymes (Apo3A, Apo3C, and Apo3H) and the double catalytic domain Apo3 enzymes (Apo3B, Apo3D, Apo3F, and Apo3G). Apo3G has been implicated in protection from HIV proliferation by becoming encapsidated into budding HIV virions and subsequently mutationally inactivating the synthesized provirus. This largely occurs in the absence of HIV viral infectivity factor (Vif) which mediates the ubiquitination and degradation of Apo3G. Apo3G is a processive enzyme, able to catalyze numerous deaminations in a 5'CCC motif in a single interaction with a substrate. There is a paucity of biochemical data on other Apo3 family members. We performed basic biochemical assays that determined the relative specific activities, processivity, cytosine motif preferences, and binding affinities for DNA, of Apo3A and Apo3H using synthetic DNA substrates in deamination assays. We found Apo3A to be an enzyme with low processivity and Apo3H to be a highly processive enzyme; both of which deaminate a 5'TC motif. Using a reconstituted HIV replication assay we assessed if processivity is needed for efficient restriction of HIV. We were able to demonstrate that each, Apo3G, Apo3A, and Apo3H were able to catalyze deaminations during in vitro reverse transcription. The mutation profile of both Apo3A and Apo3H showed that the 5'TC motif preference was less effective compared to Apo3G in triggering missense and nonsense mutations in the HIV protease active site coding sequence. Nuclear DNA can become deaminated by the related Apo3 family member activation-induced deaminase (AID), when it is present in the nucleus of activated B cells. Apo3A and Apo3H are located in the nucleus but the extent of the damage they cause has only recently been investigated. Here we used an in vitro transcription assay to determine the efficiency of Apo3A and Apo3H deamination during transcription and found that, like AID, they are highly capable of causing deaminations during transcription. Taken together, the results presented here demonstrate that processivity is not necessary for an Apo3 enzyme to catalyze deaminations during HIV reverse transcription and that Apo3A and Apo3H can catalyze deaminations during DNA transcription that could damage host genomic DNA. These results imply a potential cost for maintaining nuclear deaminases.

APOBEC3A (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 A) appartient à la famille des cytidines désaminases, qui clivent les cytidines en uraciles. APOBEC3G (le modèle de la famille) est un facteur de restriction du VIH : son incorporation dans la particule virale lui permet de désaminer le génome viral néoformé, entrainant hypermutations et dégradation de l’ADN viral. A3A, au contraire, n’est pas incorporée dans la particule virale : la protéine, exprimée spécifiquement dans les cellules myéloïdes de façon inoffensive pour la cellule, agit de la même façon qu’A3G en s’attaquant au virus dès son entrée dans la cellule.Physiologiquement, dans les cellules non-myéloïdes, les APOBEC3 désaminent l’ADN cellulaire simple brin, dont les uraciles sont retirés par UNG2. Les sites abasiques sont clivés par la machinerie de réparation de l’ADN, conduisant parfois à des cassures double-brin et à la mort de la cellule.L’objectif de la thèse était de comprendre cette différence de comportement selon le type cellulaire. Pour cela, des lignées cellulaires inductibles pour A3A ont été créées en cellules HeLa et U937 (monocytaire). Les données obtenues indiquent qu’A3A, partiellement nucléaire, édite l’ADN des cellules en division, conduisant à des dommages à l’ADN, à la production d’espèces réactives de l’oxygène (ROS) et à la mort des cellules. Les cellules différenciées ne présentent pas ce type de dommages, et cela s’explique par une localisation différente de la protéine. Ces résultats permettent de faire pour la première fois le lien entre dommages à l’ADN induits par un membre de la famille des A3 et production de ROS, et donc à l’induction d’une activation immunitaire. Cette activation pourrait avoir des implications dans l’infection ainsi que dans les processus tumorigéniques. / APOBEC3A (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 A) belongs to a family of cytidine deaminases that can edit cytidines to uraciles. APOBEC3G (model protein for the family) is a restriction factor for HIV: since it’s incorporated in the viral particle, it can deaminate the newly formed viral genome leading to hypermutation and viral DNA degradation.A3A is not incorporated in the viral particle: this protein is specifically expressed in myeloid cells where it is harmless for the cell and edits the DNA of the incoming viral particle in the same way than A3G.Physiologically, in non-myeloid cells, APOBEC3s deaminate single strand cellular DNA and the resulting uraciles are cut out by UNG2. These abasic sites are cleaved by the DNA repair machinery and can generate double strand breaks that will result in cell death.The objective of the thesis was to understand this difference of behaviour between different cell types. For that purpose, A3A-inducible cell lines were created in HeLa and U937 (monocytic) cells. The results obtained indicate that partially nuclear A3A edits the genomic DNA of cycling cells, leading to DNA damage, to the production of reactive oxygen species (ROS) and to cell death. Differentiated cells do not present this type of damage and that phenotype can be explained by a different localization of the protein.These results link for the first time DNA damage induced by a member of the A3 proteins family to ROS production and to induction of an immune activation. This activation could have implications in infection as well as in tumorigenic processes.