Structural and Biochemical study of human RECQ4

RecQ helicases belong to a ubiquitous family of DNA unwinding enzymes that are essential to maintain genome stability by acting at the interface between DNA replication, recombination and repair. Humans have five different paralogues of RecQ helicases namely RecQ1, BLM, WRN, RecQ4 and RecQ5. This work focuses on the structural and biochemical study of human RecQ4. Germ-line mutations in the RECQ4 gene give rise to three distinct human genetic disorders (Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes). Despite the important roles of RecQ4 in various cellular processes, RecQ4 have never been fully characterized. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. A part of this work focuses on the structural and biochemical analysis of both the human and Xenopus RecQ4 cysteine-rich regions, and shows by NMR spectroscopy that the Xenopus fragment does indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a preference for RNA. We also investigated the effect of an additional Sld2 homologous region upstream the Zn knuckle. In both the human and Xenopus system, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability. Recently the catalytic core of RecQ4 has been predicted to include RecQ-like-C-terminal (RQC) domain at the C-terminus of the helicase domain, similar to other RecQ helicases. This domain is composed of a Zn-binding region and a winged helix (WH) domain. Another part of this thesis centers on the structural and biochemical characterization of the catalytic core of RecQ4 including the helicase and RQC domain. The results provide an insight in the Zn binding ligands present in the RQC domain that plays a role in DNA binding and unwinding activity of the protein. Also the presence of the characteristic aromatic residue at the tip of the WH ? hairpin and its role in DNA binding and unwinding has been established. Finally, it provides a low resolution SAXS model of the catalytic core of RecQ4.