DNA-protein interaction dynamics at the Lamin B2 replication origin

The regulation of human DNA replication operates via a time-defined program of activation and deactivation of approximately 30,000 replication origins distributed along the genome. Due to the complexity of this process, each step requires a sequence of cascade checkpoints and licensing events, most of which are well conserved from yeasts to humans. A multi-protein complex assembles onto each origin causing the local unwinding of the DNA double helix and the start of two oppositely moving replicative forks. Despite the cis-acting elements necessary for origin firing are almost elucidated, the mechanism that governs the selection of a specific DNA sequence as human (and, more generally, metazoan) origin, in the course of G1 phase of the cell cycle, is still poorly understood. The lack of DNA sequence consensus between replication origins characterized so far, together with the poor binding-specificity displayed by the Origin Recognition Complex, suggest that origin selection might rather be determined by local chromatin structures and/or trans-acting factors. With regard to the latter possibility, it was interesting to find out that a DNA region specifically bound by the AP-1 proteins, is located close to the start site of the human Lamin B2 replication origin. In the study conducted during this Ph.D. program, the possible role of AP-1 transcription factors in origin specification was explored by investigating the involvement the principal moieties of this protein family, c-Fos and c-Jun, within the replicative complexes in living human cells. The data reported in this thesis provides evidence that both c-Fos and c-Jun interact with the LaminB2 origin of DNA replication and indeed participates in origin function. Participation of these proteins to origin binding is consistent with their interaction with both ORC4 and HOXC13, two members of the replicative complex, and is cell cycle defined, occurring before origin firing. Furthermore the observations point to the existence of specific and dynamic structural reorganizations of the complexes assembled at the origin region along with origin activation. In this view, AP-1 proteins could contribute to recruit and stabilize the replicative complexes onto the LaminB2 origin, in presence of specific chromatin and topological configurations.