Chromatin Assembly Factor 1 (CAF-1) is the only known replication dependant histone chaperone, responsible for the deposition of the histone H3/H4 tetramer onto DNA. Found in all eukaryotes, CAF-1 consists of three subunits, p150, p60 and p48. Since its identification work on CAF-1 has mainly focused on in vivo studies due to the lack of a reliable method to produce large quantities of recombinant protein for biochemical studies. Herein the cloning, production and purification of the three subunits of recombinant CAF-1 is described. The proteins were expressed as complexes and individually in insect cells and Escherichia coli, optimised protocols are described for maximum protein recovery and purity. Constructs of p150 and p60 were also produced and used to analyse the binding regions and modes of both the p48 and p60 proteins to p150. It is shown that the two smaller subunits of CAF-1 do not interact in the absence of p150 and that the p150 subunit of CAF-1 acts as a scaffold for assembly of the complex, binding directly to both p48 and p60. The stoichiometry of the CAF-1 complex was also investigated and a basis for further work, including structural studies, discussed.

Une détérioration des ARNm, le vecteur de l’information génétique, ou une altération des mécanismes régulant leur synthèse, leur traduction, ou leur dégradation peut être responsable de l’apparition d’une maladie. Ainsi, il est important de comprendre les mécanismes affectant les ARNm et notamment leur dégradation. Cette dernière est initiée par la réaction de désadénylation caractérisée par le raccourcissement progressif de la queue poly(A) de l’ARNm. Un complexe responsable de la désadénylation est CCR4-NOT. Les protéines BTG/Tob lient la désadénylase CAF1 de CCR4-NOT via leur domaine APRO. Dans cette thèse, l’interaction de BTG2 avec PABPC1, la protéine liant la queue poly(A), a été étudiée. Cette association augmente l’activité désadénylase de CAF1 in vitro et in cellulo. Le rôle de cette liaison sur les propriétés antiprolifératives de BTG2 et l’impact de BTG2 sur la traduction ont aussi été analysés. Un modèle expliquant l’activation de la désadénylation par BTG2 est proposé. / Damaging mRNA, the molecules carrying the genetic information, or altering the mechanisms responsible for their synthesis, their translation or their degradation can be responsible for initiating diseases. It is thus important to understand the mechanisms impacting mRNA, especially their degradation. The latter is initiated by the deadenylation reaction characterized by the progressive shortening of the mRNA 3’ poly(A) tail. One of the complexes responsible for deadenylation is CCR4-NOT. BTG/Tob proteins are able to bind the CAF1 deadenylase of CCR4-NOT via their APRO domain. In this thesis, interaction of BTG2 with PABPC1, the factor binding poly(A) tail, was analyzed. This binding stimulates CAF1 deadenylase activity in vitro and in cellulo. The role of this interaction on the antiproliferative properties of BTG2 and the impact of BTG2 on translation were also investigated. A model explaining deadenylation activation by BTG2 is proposed.

Cette thèse traite du rôle de la chaperonne d'histone ASF1 dans la déposition des histones au cours de la réplication. / That thesis is about the role of ASF1 in histone deposition during replication.