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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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Related in: MedlinePlus

Structure-prediction-guided mutagenesis of S.c. Rmi1.A, prediction of order/disorder in Rmi1 by the VLXT algorithm [26,27]. A score of 1 denotes an ideal prediction of disorder and a score of 0 an ideal prediction of order with the order/disorder threshold at a score of 0.5. A domain of unknown function (DUF1767), and an OB-fold with an insertion loop are conserved in all Rmi1 species [39]. The position of DUF1767, the OB-fold, the insertion loop and a predicted flexible linker between DUF1767 and the OB-fold shown above the disorder plot are based on the VLXT order/disorder prediction. α-helices (cylinders) and β-strands (arrows) in this region of human Rmi1 are indicated below the domain map. B, prediction of four regions of increased helical in Rmi1 with residues F63, A128,A139, E220 having some of the highest helical propensity in the DUF1767 domain, the insertion loop, and the C-terminus, respectively. We predict that P88 is responsible for the sudden loss in helical propensity in the linker that connects N-terminal domain and the OB-fold. C–G, substitution of F63, A128, A139 and E220 with proline, which has the lowest helical propensity of all amino acids, is predicted to disrupt the increased helical propensity in these regions, whereas substitution of P88 with alanine, which has excellent helical propensity, is predicted to lead to a strong increase in continuous helical propensity of the linker. H, plasmid pRS415 expressing RMI1 and rmi1 mutants under control of the endogenous RMI1 promoter were transformed into Δrmi1 mutant KHSY4695 and tested for the ability to suppress the hypersensitivity of the rmi1Δ strain to hydroxyurea.
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pone.0145466.g001: Structure-prediction-guided mutagenesis of S.c. Rmi1.A, prediction of order/disorder in Rmi1 by the VLXT algorithm [26,27]. A score of 1 denotes an ideal prediction of disorder and a score of 0 an ideal prediction of order with the order/disorder threshold at a score of 0.5. A domain of unknown function (DUF1767), and an OB-fold with an insertion loop are conserved in all Rmi1 species [39]. The position of DUF1767, the OB-fold, the insertion loop and a predicted flexible linker between DUF1767 and the OB-fold shown above the disorder plot are based on the VLXT order/disorder prediction. α-helices (cylinders) and β-strands (arrows) in this region of human Rmi1 are indicated below the domain map. B, prediction of four regions of increased helical in Rmi1 with residues F63, A128,A139, E220 having some of the highest helical propensity in the DUF1767 domain, the insertion loop, and the C-terminus, respectively. We predict that P88 is responsible for the sudden loss in helical propensity in the linker that connects N-terminal domain and the OB-fold. C–G, substitution of F63, A128, A139 and E220 with proline, which has the lowest helical propensity of all amino acids, is predicted to disrupt the increased helical propensity in these regions, whereas substitution of P88 with alanine, which has excellent helical propensity, is predicted to lead to a strong increase in continuous helical propensity of the linker. H, plasmid pRS415 expressing RMI1 and rmi1 mutants under control of the endogenous RMI1 promoter were transformed into Δrmi1 mutant KHSY4695 and tested for the ability to suppress the hypersensitivity of the rmi1Δ strain to hydroxyurea.

Mentions: Determining functionally important residues in S. cerevisiae Rmi1 has been challenging as it lacks catalytic activity and a crystal structure has not been resolved. The primary sequence is only minimally conserved (~35% identical residues between yeast genera, 18% between S. cerevisiae and human Rmi1) and lengths range from 241 residues in S. cerevisiae to 625 residues in humans. The crystal structure of the N-terminal 219 residues of human Rmi1 was recently resolved [20]. It revealed an N-terminal three-helix bundle of unknown function (DUF1767) followed by an OB-fold with a largely unstructured loop inserted between strands β1 and β2 by which Rmi1 binds to Topo IIIα. In the absence of catalytic activity/domains and very limited sequence identity, we reasoned that structure prediction tools [25,32,33] could reveal functionally important motifs in yeast Rmi1. Analyzing the distribution of ordered and disordered residues, we noticed disordered N- and C-termini as well as two internal regions of increased disorder (Fig 1A). Further, we identified two regions of increased helical propensity, spanning residues 58–74 and residues 212–228 near the N- and C-terminal disordered regions, as well as two regions of lesser helical propensity between residues 125–132 and 137–145 (Fig 1B). Alignment of these two predictors would be consistent with the putative OB-fold core mapping to the central, ordered region, and the topoisomerase-binding loop to the disordered insertion with two segments of weak helical propensity. The DUF1767 domain, predicted to be present in Rmi1 of all species, is typically located N-terminally of the OB-fold core, and appears to be connected to it in yeast Rmi1 by a disordered linker (Fig 1A).


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)

Structure-prediction-guided mutagenesis of S.c. Rmi1.A, prediction of order/disorder in Rmi1 by the VLXT algorithm [26,27]. A score of 1 denotes an ideal prediction of disorder and a score of 0 an ideal prediction of order with the order/disorder threshold at a score of 0.5. A domain of unknown function (DUF1767), and an OB-fold with an insertion loop are conserved in all Rmi1 species [39]. The position of DUF1767, the OB-fold, the insertion loop and a predicted flexible linker between DUF1767 and the OB-fold shown above the disorder plot are based on the VLXT order/disorder prediction. α-helices (cylinders) and β-strands (arrows) in this region of human Rmi1 are indicated below the domain map. B, prediction of four regions of increased helical in Rmi1 with residues F63, A128,A139, E220 having some of the highest helical propensity in the DUF1767 domain, the insertion loop, and the C-terminus, respectively. We predict that P88 is responsible for the sudden loss in helical propensity in the linker that connects N-terminal domain and the OB-fold. C–G, substitution of F63, A128, A139 and E220 with proline, which has the lowest helical propensity of all amino acids, is predicted to disrupt the increased helical propensity in these regions, whereas substitution of P88 with alanine, which has excellent helical propensity, is predicted to lead to a strong increase in continuous helical propensity of the linker. H, plasmid pRS415 expressing RMI1 and rmi1 mutants under control of the endogenous RMI1 promoter were transformed into Δrmi1 mutant KHSY4695 and tested for the ability to suppress the hypersensitivity of the rmi1Δ strain to hydroxyurea.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145466.g001: Structure-prediction-guided mutagenesis of S.c. Rmi1.A, prediction of order/disorder in Rmi1 by the VLXT algorithm [26,27]. A score of 1 denotes an ideal prediction of disorder and a score of 0 an ideal prediction of order with the order/disorder threshold at a score of 0.5. A domain of unknown function (DUF1767), and an OB-fold with an insertion loop are conserved in all Rmi1 species [39]. The position of DUF1767, the OB-fold, the insertion loop and a predicted flexible linker between DUF1767 and the OB-fold shown above the disorder plot are based on the VLXT order/disorder prediction. α-helices (cylinders) and β-strands (arrows) in this region of human Rmi1 are indicated below the domain map. B, prediction of four regions of increased helical in Rmi1 with residues F63, A128,A139, E220 having some of the highest helical propensity in the DUF1767 domain, the insertion loop, and the C-terminus, respectively. We predict that P88 is responsible for the sudden loss in helical propensity in the linker that connects N-terminal domain and the OB-fold. C–G, substitution of F63, A128, A139 and E220 with proline, which has the lowest helical propensity of all amino acids, is predicted to disrupt the increased helical propensity in these regions, whereas substitution of P88 with alanine, which has excellent helical propensity, is predicted to lead to a strong increase in continuous helical propensity of the linker. H, plasmid pRS415 expressing RMI1 and rmi1 mutants under control of the endogenous RMI1 promoter were transformed into Δrmi1 mutant KHSY4695 and tested for the ability to suppress the hypersensitivity of the rmi1Δ strain to hydroxyurea.
Mentions: Determining functionally important residues in S. cerevisiae Rmi1 has been challenging as it lacks catalytic activity and a crystal structure has not been resolved. The primary sequence is only minimally conserved (~35% identical residues between yeast genera, 18% between S. cerevisiae and human Rmi1) and lengths range from 241 residues in S. cerevisiae to 625 residues in humans. The crystal structure of the N-terminal 219 residues of human Rmi1 was recently resolved [20]. It revealed an N-terminal three-helix bundle of unknown function (DUF1767) followed by an OB-fold with a largely unstructured loop inserted between strands β1 and β2 by which Rmi1 binds to Topo IIIα. In the absence of catalytic activity/domains and very limited sequence identity, we reasoned that structure prediction tools [25,32,33] could reveal functionally important motifs in yeast Rmi1. Analyzing the distribution of ordered and disordered residues, we noticed disordered N- and C-termini as well as two internal regions of increased disorder (Fig 1A). Further, we identified two regions of increased helical propensity, spanning residues 58–74 and residues 212–228 near the N- and C-terminal disordered regions, as well as two regions of lesser helical propensity between residues 125–132 and 137–145 (Fig 1B). Alignment of these two predictors would be consistent with the putative OB-fold core mapping to the central, ordered region, and the topoisomerase-binding loop to the disordered insertion with two segments of weak helical propensity. The DUF1767 domain, predicted to be present in Rmi1 of all species, is typically located N-terminally of the OB-fold core, and appears to be connected to it in yeast Rmi1 by a disordered linker (Fig 1A).

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