title: A transient α-helical molecular recognition element in the disordered N-terminus of the Sgs1 helicase is critical for chromosome stability and binding of Top3/Rmi1.
abstract: The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays show that the defects of the proline mutants are the result of impaired binding of Top3 and Rmi1 to Sgs1. Extending mutagenesis N-terminally revealed a second functionally critical region that spans residues 9-17. Depending on the position of the proline substitution in the helix functional impairment of Sgs1 function varied, gradually increasing from the C- to the N-terminus. The multiscale approach we used to interrogate structure/function relationships in the long disordered N-terminal segment of Sgs1 allowed us to precisely define a functionally critical region and should be generally applicable to other disordered proteins.
authors.affiliation: Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA, Center for Drug Discovery and Innovation, University of South Florida, Tampa, FL 33612, USA and Cancer Biology and Evolution Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Kennedy
authors.forename: Gary W
authors.initials: GW
authors.lastname: Daughdrill
authors.forename: Kristina H
authors.initials: KH
authors.lastname: Schmidt
mesh_terms: D043171:Chromosomal Instability; D004268:DNA-Binding Proteins / Q000378:metabolism*; D064267:Intrinsically Disordered Proteins / Q000737:chemistry; D016296:Mutagenesis; D011392:Proline / Q000235:genetics; D017433:Protein Structure, Secondary; D053484:RecQ Helicases / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D064267:Intrinsically Disordered Proteins; C502206:Rmi1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C502374:TOP3 protein, S cerevisiae; D011392:Proline; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases
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