Previous studies have identified Cockayne Syndrome B (CSB) as a helicase that can resolve non-canonical DNA structures, called G-quadruplexes (G4s). The aim of this study is to investigate the properties of CSB as a G4-binder and -resolvase, and examine the correlation between the G4-helicase activity of CSB and premature ageing phenotype observed in CSB-deficient cells. Accordingly, the recombinant CSB full-length protein (FL) and its helicase- “like" domain (HD) were respectively expressed from insect and bacterial cells, and their resolvase and binding activities were tested over a large panel of DNA substrates. Native gel analysis and biophysical characterisations revealed that ribosomal DNA (rDNA) sequences, that typically act as CSB substrate, can form intermolecular G4s. We discovered that intermolecular G4s were strongly bound by CSB with picomolar affinity, whilst negligible binding to intramolecular G4s was observed. In vitro and cellular data demonstrated that G4-ligands can compete with CSB for binding to intermolecular rDNA G4, which results in CSB being displaced off the nucleoli of cells upon treatment with G4-ligands. Immunostaining with the selective G4-antibody BG4 revealed a lack of BG4-staining in the nucleoli of CSB-deficient cells after exogenous expression of recombinant CSB, further corroborating the hypothesis that CSB can bind intermolecular rDNA G4s in the nucleoli and compete with BG4 for the binding of such DNA-substrate. The work presented in this thesis allowed us to observe that (I) intermolecular G4s are likely to form from long-range distant rDNA sequences within the nucleoli of cells, and (II) CSB specifically binds and resolves these structures. Our results provide the first evidence of an endogenous protein that specifically interacts with intermolecular G4s, suggesting potential biological significance of these structures. The biological relevance of intermolecular rDNA G4s could be key in rare genetic disorders like Cockayne Syndrome, where senescence and premature ageing is observed when CSB is functionally mutated.Open Acces
