Study of an artificial G4-binding protein as a tool to define the proteome assembled around G-quadruplex forming sequences and efficient replication origins in DT40 chicken cells

Life goes on thanks to the ability of single cells to divide and generate new ones; for this process to happen, cells need to replicate their genome. DNA replication starts only in some specific points of the genome of eukaryotic cells, at origins of replication. Putative G-Quadruplex forming Sequences (PQSs), able to form G-quadruplexes (G4s), a DNA non-canonical secondary structure, are among the cis-elements necessary for the process. G4s can be useful to define the proteome at the level of origins of replication; therefore, the aim of this internship is to fine-tune an approach that relies on an artificial G4 binding protein, G4P, able to recognize structured G4s in vivo, to identify proteins involved in origin function in DT40 chicken cells. The host lab previously generated a clone expressing a fusion protein FLAG-G4P APEX2, and I performed a series of experiments to validate the induction and the use of G4P. I demonstrated that G4P is expressed upon induction and localized to the nucleus, I could see that genome wide, G4P binds often at CGIs and in correspondence of replication origins; I explored the proteome around G4s showing an enrichment of G4BPs, G4 helicases, proteins associated with replication functions and G4s.