Comparative systems-level analysis of the G1/S transition in yeast and higher eukaryotes: focusing on the Whi5/Rb network and initiation of DNA replication

Molecular systems biology, holds that biological processes are the result of complex, coordinated, dynamical, non-linear interactions that generate the corresponding function as an emergent property of the system, that therefore is not found in individual components, but only in their networking. Conversely, it has been shown that even a single, multi-domain protein may present a system-level behavior that can be described by adapting the formalism used to describe inter-molecular networks. Accordingly, a multi-scale approach is required to fully understand any given biological function. The G1/S transition and the initiation of DNA replication are major regulatory events in the eukaryotic cell cycle. In this thesis I have provided a comparative survey of the molecular events leading to the initiation of DNA replication and analyzed some structural features and the interactome profile of Saccharomyces cerevisiae Whi5, a regulator of G1/S-specific transcription in budding yeast in comparison with those with those of Rb – and members of the pocket family - that perform equivalent function(s) in mammalian cells. In eukaryotes DNA replication initiates from hundreds to thousands of replication origins in a coordinated manner, in order to efficiently duplicate the genome. The sequence of events leading to the onset of DNA replication is conventionally divided in two interdependent processes: licensing—a process during which replication origins acquire replication competence but are kept inactive— and firing—a process during which licensed origins are activated but not re-licensed. In this thesis we investigate the evolutionary conservation of the molecular machinery orchestrating DNA replication initiation both in yeast and in mammalian cells, highlighting a remarkable conservation of the general architecture of this central biological mechanism. Many steps are conserved down to molecular details and are performed by orthologous proteins with high sequence conservation, while differences in molecular structure of the performing proteins and their interactions are apparent in other steps. Tight regulation of initiation of DNA replication is achieved through protein phosphorylation, exerted mostly by Cyclin-dependent kinases in order to ensure that each chromosome is fully replicated once, and only once, during each cycle, and to avoid the formation of aberrant DNA structures and incorrect chromosomal duplication, that in mammalian cells are a prerequisite for genome instability and tumorigenesis. Among many regulatory proteins, Retinoblastoma (Rb) of higher eukaryotes and Whi5 of yeasts play a key role in the regulation of gene expression of proteins involved in DNA replication including Cyclin-dependent kinases and its partners. Rb is part of a protein family encompassing three members. Rb plays many cellular roles, mediated by several downstream effectors and transcriptional targets. The best known role of Rb is the control of cell cycle progression at the G1/S transition, mediated through its interaction with E2F transcription factors required for entrance into S phase. Rb is also involved in regulation of cellular differentiation during embryogenesis and in adult tissue, regulation of p53-dependent apoptosis and DNA repair, maintenance of permanent cell cycle arrest, as quiescence in stem cell promotion of cell cycle exit through inhibition of the E2F transcription factors and the transcriptional repression of genes encoding cell-cycle regulators. While proteins related to the retinoblastoma tumor suppressor Rb and the E2F transcription factor are conserved in most eukaryotic lineages, in yeast no hortologues of Rb or E2F have been found. Both Rb and Whi5 are disordered proteins. In Rb, that is a much larger protein than Whi5, structured domains alternate with disordered regions, whereas in Whi5 only a single structured domain is likely to exist. The family of Whi5-related proteins is only present in Saccharomycetales. The higher-than-expected conservation of sequence in disordered regions, correlates with abundance of phosphorylation sites and allows to predict conservation of a similar phosphorylation rhythm with a potentially functionally relevant role. Notably, conserved motifs 1 and 3 may act as phosphorylation-dependent seeds in Whi5 folding/unfolding. The (partial) disordered nature of both proteins allows them to act as protein hubs, able to interact with many partners. The interaction landscape of Rb and Whi5 is quite large, with more than one hundred proteins interacting either genetically or physically with either protein. Interestingly, the smaller Whi5 acts as a hierarchical hub. Comparison of Rb and Whi5 interactors (both physical and genetical ones) allows to highlight a significant core of conserved common functionalities associated with the interactors indicating that network structure and function – rather than individual proteins, are conserved during evolution. By step-wise adding interactors to existing models for Whi5 function, an improved “concept map” of Whi5 function is being constructed. Ultimately, such a map will allow to construct dynamic mathematical model(s) of increasing granularity and to design experiments to proof novel regulatory links within the Whi5 network.