Thèse de doctorat en Science de la vie
Sous la direction de Éric Gilson.
Soutenue en 2009
à École normale supérieure (Lyon) , en partenariat avec Laboratoire de biologie moléculaire de la cellule. LBMC (laboratoire) .
Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes to maintain the integrity of chromosome ends. In human cells, telomeres are protected by a complex of six proteins bound to telomeric DNA, named shelterin. As a crucial component of shelterin, the human telomeric capping protein TRF2 is required for telomere stabilization and function. Recently, TRF2 was shown to form a complex with Apollo, a 5' -exonuclease of the metallo-beta-lactamase family. In this thesis, biochjemical, genetic, and cellular approaches were combined to understand the mechanism of 5' -exonuclease of Apollo in the telomere protection. First, in vitro evidence shows that TRF2 plays a dual role in controlling Apollo nuclease activity. TRF2 stimulates the nuclease activity of Apollo on TRF2-free DNA ends and inhibits it when it is bound proximately to the 5' end. This stimulatory effect of Apollo nuclease depends on the basic domain of TRF2. Second, the specific coupling between the Apollo nuclease activity and the dynamics of TRF2 DNA binding shows in vivo that TRF2 and Apollo are not only involved in the protection of the very-terminal structure of telomeres but also of the inner part of telomere repeat tracts, which is required for telomere integrity during S-phase. Moreover TRF2 and Apollo protect from DNA damage telomere repeat tracts inserted far away from chromosome ends, furthze indicating that TRF2 couples the exonuclease activity of Apollo to protect telomere during fork progression. Third, exonuclease activity of Apollo, which is required for the telomere protection, is directly regulated by TRF2. These data are consistent with a new model of telomere protection in with TRF2 couples the exonuclease activity of Apollo to replication in order to eliminate clastogenic and recombinogenic structures. The work in my thesis suggest that replicative senescence in human cells may not only be caused by loss of the terminal cap integrity but also by replication-linked DNA breakages and recombinations within the inner part of telomeres.