title: Structural Motifs Critical for In Vivo Function and Stability of the RecQ-Mediated Genome Instability Protein Rmi1.
abstract: Rmi1 is a member of the Sgs1/Top3/Rmi1 (STR) complex of Saccharomyces cerevisiae and has been implicated in binding and catalytic enhancement of Top3 in the dissolution of double Holliday junctions. Deletion of RMI1 results in a severe growth defect resembling that of top3Δ. Despite the importance of Rmi1 for cell viability, little is known about its functional domains, particularly in Rmi1 of S. cerevisiae, which does not have a resolved crystal structure and the primary sequence is poorly conserved. Here, we rationally designed point mutations based on bioinformatics analysis of order/disorder and helical propensity to define three functionally important motifs in yeast Rmi1 outside of the proposed OB-fold core. Replacing residues F63, Y218 and E220 with proline, designed to break predicted N-terminal and C-terminal α-helices, or with lysine, designed to eliminate hydrophobic residues at positions 63 and 218, while maintaining α-helical structure, caused hypersensitivity to hydroxyurea. Further, Y218P and E220P mutations, but not F63P and F63K mutations, led to reduced Rmi1 levels compared to wild type Rmi1, suggesting a role of the C-terminal α-helix in Rmi1 stabilization, most likely by protecting the integrity of the OB-fold core. Our bioinformatics analysis also suggests the presence of a disordered linker between the N-terminal α-helix and the OB fold core; a P88A mutation, designed to increase helicity in this linker, also impaired Rmi1 function in vivo. In conclusion, we propose a model that maps all functionally important structural features for yeast Rmi1 based on biological findings in yeast and structure-prediction-based alignment with the recently established crystal structure of the N-terminus of human Rmi1.
authors.affiliation: Department of Cell Biology, Molecular Biology, and Microbiology, University of South Florida, Tampa, Florida, 33620, United States of America.|Graduate Program in Cell and Molecular Biology, H. Lee Moffitt Cancer Center, Tampa, Florida, 33612, United States of America.
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Kennedy
authors.affiliation: Department of Cell Biology, Molecular Biology, and Microbiology, University of South Florida, Tampa, Florida, 33620, United States of America.|Graduate Program in Cell and Molecular Biology, H. Lee Moffitt Cancer Center, Tampa, Florida, 33612, United States of America.
authors.forename: Salahuddin
authors.initials: S
authors.lastname: Syed
authors.affiliation: Department of Cell Biology, Molecular Biology, and Microbiology, University of South Florida, Tampa, Florida, 33620, United States of America.|Cancer Biology and Evolution Program, H. Lee Moffitt Cancer Center, Tampa, Florida, 33612, United States of America.
authors.forename: Kristina H
authors.initials: KH
authors.lastname: Schmidt
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D042822:Genomic Instability; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D053484:RecQ Helicases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C501527:RMI1 protein, human; C502206:Rmi1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D053484:RecQ Helicases