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Structural Motifs Critical for In Vivo Function and Stability of the RecQ-Mediated Genome Instability Protein Rmi1.

Kennedy JA, Syed S, Schmidt KH - PLoS ONE (2015)

Bottom Line: Deletion of RMI1 results in a severe growth defect resembling that of top3Δ.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Molecular Biology, and Microbiology, University of South Florida, Tampa, Florida, 33620, United States of America.

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.

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Proposed structure-prediction-based alignment of yeast and human Rmi1.Residues to which confirmed domains in the human Rmi1 crystal structure (4CGY) map are indicated above the alignment in black. Proposed location of conserved domains and motifs in yeast Rmi1 are indicated in red. Conserved residues whose mutation to proline abolished Rmi1 function in vivo are indicated in the alignment with the proposed corresponding residue in human Rmi1 (F63/F53; Y281/Y201). Compared to human Rmi1, the N-terminus (DUF1767) of yeast Rmi1 appears to be extended by approximately 18 residues. The predicted size and location of the topoisomerase-binding loop (residues 116–145) differs from that previously proposed (residues 87–145) for yeast Rmi1 [19].
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pone.0145466.g005: Proposed structure-prediction-based alignment of yeast and human Rmi1.Residues to which confirmed domains in the human Rmi1 crystal structure (4CGY) map are indicated above the alignment in black. Proposed location of conserved domains and motifs in yeast Rmi1 are indicated in red. Conserved residues whose mutation to proline abolished Rmi1 function in vivo are indicated in the alignment with the proposed corresponding residue in human Rmi1 (F63/F53; Y281/Y201). Compared to human Rmi1, the N-terminus (DUF1767) of yeast Rmi1 appears to be extended by approximately 18 residues. The predicted size and location of the topoisomerase-binding loop (residues 116–145) differs from that previously proposed (residues 87–145) for yeast Rmi1 [19].

Mentions: Performing the same structural prediction analysis for human Rmi1 suggests that the two N- and C-terminal α-helices and the spacing between them, where the core of an OB-fold has been confirmed in human Rmi1, are conserved (Fig 5). In human Rmi1, a loop that maps to residues 98–134 emerges from the OB-fold between strands β1 and β2 and inserts itself into the Topo IIIα gate; its deletion eliminates complex formation of Rmi1 with BLM and Topo IIIα [20]. Based on a sequence alignment Bocquet and colleagues [19] suggested that the equivalent loop for Top3 binding in yeast Rmi1 maps to residues 87–146 and showed that replacement of this region with a scrambled version of equal chemistry still mediated binding to Sgs1 and Top3, but failed to stimulate Top3 catalytic activity and dHJ dissolution. The structural alignment in our study, however, which we explored because of the poor sequence conservation of only 18% between yeast and human Rmi1, suggests that residues 87–116 of yeast Rmi1 contain the β1 strand of the OB-fold and the disordered linker that connects β1 to the N-terminal helical region (Fig 5). The insertion loop in yeast Rmi1, therefore, may be significantly shorter, mapping to residues 116–145. As in human Rmi1, this insertion loop contains two segments of increased helical propensity (Figs 1B and 6E), but proline mutagenesis of these segments (A128P, A139P) suggests that the adoption of helical structure is not required for Rmi1’s function. Since both regions adopt short helices, if any, and the prolines replaced alanines that are likely to be in the first turn, it is also possible that proline substitution is not structurally disruptive in this disordered loop.


Structural Motifs Critical for In Vivo Function and Stability of the RecQ-Mediated Genome Instability Protein Rmi1.

Kennedy JA, Syed S, Schmidt KH - PLoS ONE (2015)

Proposed structure-prediction-based alignment of yeast and human Rmi1.Residues to which confirmed domains in the human Rmi1 crystal structure (4CGY) map are indicated above the alignment in black. Proposed location of conserved domains and motifs in yeast Rmi1 are indicated in red. Conserved residues whose mutation to proline abolished Rmi1 function in vivo are indicated in the alignment with the proposed corresponding residue in human Rmi1 (F63/F53; Y281/Y201). Compared to human Rmi1, the N-terminus (DUF1767) of yeast Rmi1 appears to be extended by approximately 18 residues. The predicted size and location of the topoisomerase-binding loop (residues 116–145) differs from that previously proposed (residues 87–145) for yeast Rmi1 [19].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696737&req=5

pone.0145466.g005: Proposed structure-prediction-based alignment of yeast and human Rmi1.Residues to which confirmed domains in the human Rmi1 crystal structure (4CGY) map are indicated above the alignment in black. Proposed location of conserved domains and motifs in yeast Rmi1 are indicated in red. Conserved residues whose mutation to proline abolished Rmi1 function in vivo are indicated in the alignment with the proposed corresponding residue in human Rmi1 (F63/F53; Y281/Y201). Compared to human Rmi1, the N-terminus (DUF1767) of yeast Rmi1 appears to be extended by approximately 18 residues. The predicted size and location of the topoisomerase-binding loop (residues 116–145) differs from that previously proposed (residues 87–145) for yeast Rmi1 [19].
Mentions: Performing the same structural prediction analysis for human Rmi1 suggests that the two N- and C-terminal α-helices and the spacing between them, where the core of an OB-fold has been confirmed in human Rmi1, are conserved (Fig 5). In human Rmi1, a loop that maps to residues 98–134 emerges from the OB-fold between strands β1 and β2 and inserts itself into the Topo IIIα gate; its deletion eliminates complex formation of Rmi1 with BLM and Topo IIIα [20]. Based on a sequence alignment Bocquet and colleagues [19] suggested that the equivalent loop for Top3 binding in yeast Rmi1 maps to residues 87–146 and showed that replacement of this region with a scrambled version of equal chemistry still mediated binding to Sgs1 and Top3, but failed to stimulate Top3 catalytic activity and dHJ dissolution. The structural alignment in our study, however, which we explored because of the poor sequence conservation of only 18% between yeast and human Rmi1, suggests that residues 87–116 of yeast Rmi1 contain the β1 strand of the OB-fold and the disordered linker that connects β1 to the N-terminal helical region (Fig 5). The insertion loop in yeast Rmi1, therefore, may be significantly shorter, mapping to residues 116–145. As in human Rmi1, this insertion loop contains two segments of increased helical propensity (Figs 1B and 6E), but proline mutagenesis of these segments (A128P, A139P) suggests that the adoption of helical structure is not required for Rmi1’s function. Since both regions adopt short helices, if any, and the prolines replaced alanines that are likely to be in the first turn, it is also possible that proline substitution is not structurally disruptive in this disordered loop.

Bottom Line: Deletion of RMI1 results in a severe growth defect resembling that of top3Δ.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Molecular Biology, and Microbiology, University of South Florida, Tampa, Florida, 33620, United States of America.

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.

Show MeSH
Related in: MedlinePlus