title: Structural and biophysical characterization of murine rif1 C terminus reveals high specificity for DNA cruciform structures.
abstract: Mammalian Rif1 is a key regulator of DNA replication timing, double-stranded DNA break repair, and replication fork restart. Dissecting the molecular functions of Rif1 is essential to understand how it regulates such diverse processes. However, Rif1 is a large protein that lacks well defined functional domains and is predicted to be largely intrinsically disordered; these features have hampered recombinant expression of Rif1 and subsequent functional characterization. Here we applied ESPRIT (expression of soluble proteins by random incremental truncation), an in vitro evolution-like approach, to identify high yielding soluble fragments encompassing conserved regions I and II (CRI and CRII) at the C-terminal region of murine Rif1. NMR analysis showed CRI to be intrinsically disordered, whereas CRII is partially folded. CRII binds cruciform DNA with high selectivity and micromolar affinity and thus represents a functional DNA binding domain. Mutational analysis revealed an α-helical region of CRII to be important for cruciform DNA binding and identified critical residues. Thus, we present the first structural study of the mammalian Rif1, identifying a domain that directly links its function to DNA binding. The high specificity of Rif1 for cruciform structures is significant given the role of this key protein in regulating origin firing and DNA repair.
authors.affiliation: From the European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 France, the Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 France, the European Molecular Biology Laboratory, Monterotondo Outstation, Adriano Buzzati-Traverso Campus, Via Ramarini 32, 00015 Monterotondo, Italy.
authors.forename: Rasa
authors.initials: R
authors.lastname: Sukackaite
authors.affiliation: the University of Grenoble Alpes, Institut de Biologie Structurale (IBS), 6 rue Jules Horowitz, F-38027 Grenoble, France, CEA, DSV, IBS, 6 rue Jules Horowitz, F-38027 Grenoble, France, CNRS, IBS, 6 rue Jules Horowitz, F-38027 Grenoble, France, and.
authors.forename: Malene Ringkjøbing
authors.initials: MR
authors.lastname: Jensen
authors.affiliation: From the European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 France, the Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 France.
authors.forename: Philippe J
authors.initials: PJ
authors.lastname: Mas
authors.affiliation: the University of Grenoble Alpes, Institut de Biologie Structurale (IBS), 6 rue Jules Horowitz, F-38027 Grenoble, France, CEA, DSV, IBS, 6 rue Jules Horowitz, F-38027 Grenoble, France, CNRS, IBS, 6 rue Jules Horowitz, F-38027 Grenoble, France, and.
authors.forename: Martin
authors.initials: M
authors.lastname: Blackledge
authors.affiliation: the European Molecular Biology Laboratory, Monterotondo Outstation, Adriano Buzzati-Traverso Campus, Via Ramarini 32, 00015 Monterotondo, Italy sara.buonomo@embl.it.
authors.forename: Sara B
authors.initials: SB
authors.lastname: Buonomo
authors.affiliation: From the European Molecular Biology Laboratory (EMBL), Grenoble Outstation, 6 rue Jules Horowitz, 38042 France, the Unit for Virus Host-Cell Interactions, University of Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 France, hart@embl.fr.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Hart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D034501:Telomere-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D045566:DNA, Cruciform; D010446:Peptide Fragments; C492748:Rif1 protein, mouse; D034501:Telomere-Binding Proteins
keywords: Cell Cycle; DNA Replication; Directed Evolution; Intrinsically Disordered Proteins; NMR