Structural and functional analysis of RTEL1, an FeS helicase

The topic of my PhD thesis was the structural and functional characterisation of the human RTEL1, a DNA helicase with a pivotal role in telomere homeostasis and DNA replication. Based on a bioinformatic analysis of RTEL1, I cloned, expressed, and purified different fragments encompassing the main RTEL1 domains. To increase the chance of obtaining large amounts of pure, stable proteins, using the Ligand Independent Cloning (LIC) technique, I cloned the gene in a variety of expression vectors harbouring different fusion tags and screened a large number of expression conditions (cell strains, expression temperatures and times). Once the best expression conditions were found, I scaled up and optimised the purification of recombinant proteins by means of chromatographic methods. I started focussing on the full-length and the catalytic domain of human RTEL1, but despite many efforts I did not manage to obtain large amount of pure and soluble protein for structural studies. This was not totally unexpected, as FeS helicase are not easy to produce, and the difficulties in the assembly of the cluster can affect protein stability. I was more successful in obtaining soluble and stable protein samples from the C-terminal region, that contains a number of predicted domains interspersed with flexible and probably disordered linkers. I produced the whole C-terminal half of human RTEL1, and a number of deletion fragments, encompassing the various domains, including two repeats (RP1 and RP2, variously predicted to fold as TRP, HEAT or Harmonin Homology Domains) and a RING finger. After obtaining a good amount of well folded protein, I carried out a large number of crystallization screenings in order to obtain crystals suitable to be tested with synchrotron light. I have indeed obtained well ordered crystals from RP1; X-ray diffraction data were collected on the XRD2 macromolecular crystallography beamline at Elettra, and the structure determined. The protein folds as an Harmonin Homology Domain; the structure has been analyzed and compared with other similar folds, which suggest a possible modality of protein-protein interaction. In the meanwhile, a Small-angle X-ray Scattering (SAXS) analysis was used to reconstruct the low resolution three-dimensional shape of the C-terminal domains of RTEL1. SAXS data confirmed that all constructs are monomeric in solution; this information is discussed based on the current literature. A SAXS curve predicted from the crystal structure fits well the experimental scattering from both RP1 and RP2, confirming that both domains have the same fold. We are in the process of deriving 3D models from the longer constructs. To dissect the nucleic acid binding properties of the RTEL1 C-terminal domain, I performed Electrophoretic Mobility Shift Assays (EMSA) with the purified RTEL1 fragments, using a variety of nucleic acid substrates, in order to identify and characterise the DNA/RNA binding sites on the C-terminal half of RTEL1. As side project, in collaboration with clinical geneticists, I have characterised a DDX11 novel mutant found in patients with Warsaw Syndrome. I have thus carried out the biochemical characterisation of DDX11 variant c.2507T>C, leading to the substitution of Leucine 836 with a Proline. Using the Bac-to-Bac Baculovirus Expression System, I expressed in H5 insect cells DDX11 wild type and DDX11 L836P mutant. Once purified with a high grade of purity, I performed helicase assays and nucleic binding assays to understand whether and how much the substitution of Leucine 836 with a Proline affected the biochemical capability of DDX11 to bind and unwind DNA forks, and thus providing a molecular understanding of the disease.