title: The Human RecQ4 Helicase Contains a Functional RecQ C-terminal Region (RQC) That Is Essential for Activity.
abstract: RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix β-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.
authors.affiliation: From the Structural Biology Laboratory, Elettra-Sincrotrone Trieste, 34149 Basovizza, Trieste, Italy and.|the Dipartimento di Scienze della Vita, Università degli Studi di Trieste, 34127 Trieste, Italy.
authors.forename: Aditya
authors.initials: A
authors.lastname: Mojumdar
authors.affiliation: From the Structural Biology Laboratory, Elettra-Sincrotrone Trieste, 34149 Basovizza, Trieste, Italy and.
authors.forename: Matteo
authors.initials: M
authors.lastname: De March
authors.affiliation: From the Structural Biology Laboratory, Elettra-Sincrotrone Trieste, 34149 Basovizza, Trieste, Italy and.
authors.forename: Francesca
authors.initials: F
authors.lastname: Marino
authors.affiliation: From the Structural Biology Laboratory, Elettra-Sincrotrone Trieste, 34149 Basovizza, Trieste, Italy and silvia.onesti@elettra.eu.
authors.forename: Silvia
authors.identifier: 0000-0002-0612-7948
authors.initials: S
authors.lastname: Onesti
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D019295:Computational Biology; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030562:Databases, Protein; D006801:Humans; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D053484:RecQ Helicases
keywords: DNA helicase; DNA repair; mutagenesis in vitro; protein purification; protein-DNA interaction; recombinant protein expression; small angle X-ray scattering (SAXS); structural model