Structural and Functional Analysis of the Human RecQ4 Helicase

From the simplest to most complex life forms, DNA serves as the universal blueprint for life, underpinning essential biological processes. Ensuring the structural and functional integrity of the genome is therefore fundamental to cellular survival. Among the many molecular guardians of the genome stability, the RecQ helicases have emerged as crucial players. These enzymes participate in diverse aspect of the nucleic acid metabolism, including DNA replication, repair, recombination, transcription and telomere maintenance. In humans, five paralogues (RecQ1, BLM, WRN, RecQ4, RecQ5) function both independently and synergistically to safeguard genomic integrity. Mutations in these enzymes are linked to distinct heritable syndromes and cancer predisposition, highlighting the critical role of these enzymes. However, RecQ helicases also represent a double-edged sword: while they suppress tumorigenesis in normal cells, their upregulation in cancer cells can confer a survival advantage, promoting resistance to replication stress and chemotherapeutic agents. This PhD thesis focuses on the structural and functional characterisation of the human RecQ4 helicase. RecQ4 is a unique and multifunctional member of the RecQ family distinguished by its essential roles in DNA replication initiation and its dual DNA-RNA substrate specificity; in addition to the canonical RecA helicase core, the protein includes a number of unique domains, connected by flexible links. To investigate RecQ4 biochemical properties, the contribution of different domains and suitability for structural studies, multiple constructs were designed and expressed in both bacterial and eukaryotic systems. Building and extending the previous results obtained in our laboratory, a variety of fragments corresponding to a combination of the various domains were expressed and purified. In particular we designed new constructs, exploiting the novel AI protein prediction tools that became available. The corresponding proteins were expressed in both bacterial and baculovirus-infected insect cells. Extensive biochemical assays were carried out with a variety of DNA and RNA nucleic acid substrates, including forked duplexes and displacement loops: bacterially expressed RecQ4s efficiently and preferentially binds and unwinds different substrates, with complete helicase activity observed at nanomolar concentrations. However different constructs showed diverse binding and unwinding preferences suggesting that substrate recognition and unwinding efficiency may be uncoupled. These findings point to a potential requirement for the full N-terminal domain of RecQ4 (absent in these fragments) for optimal engagement and processing of RNA-containing structures. In parallel with biochemical studies, a structural investigation was conducted using both X-ray crystallography, SAXS and single-particle Cryo-Electron Microscopy. Although crystallisation screens yielded some crystals, no diffraction was detected possibly due to crystal disorder or sub-optimal freezing conditions. Nonetheless, SAXS analysis was used to reconstruct a low- resolution 3-D map of RecQ4 encompassing the full C-terminal domain. The data confirmed that all the constructs are mostly monomeric in solution, and we could explain the experimental scattering for our fragments, based on the available crystal structure and a SAXS-based modelling of the additional regions. Preliminary Cryo-EM experiments produced a 5.3 Å resolution map of apo RecQ4. Ongoing experiments aim to increase the Cryo-EM resolution and resolve substrate-bound complexes to provide a detailed mechanistic understanding of this helicase multifunctional roles. The structural analysis is still in its early stages and additional experiments are required and will be performed in the near future to try improving the results obtained so far.