Molecular mimicry of SUMO promotes DNA repair - PubMed (original) (raw)

Molecular mimicry of SUMO promotes DNA repair

John Prudden et al. Nat Struct Mol Biol. 2009 May.

Abstract

Rad60 family members contain functionally enigmatic, integral SUMO-like domains (SLDs). We show here that despite their divergence from SUMO, each Rad60 SLD interacts with a subset of SUMO pathway enzymes: SLD2 specifically binds the SUMO E2 conjugating enzyme (Ubc9), whereas SLD1 binds the SUMO E1 (Fub2, also called Uba2) activating and E3 (Pli1, also called Siz1 and Siz2) specificity enzymes. The molecular basis of this selectivity is revealed by our 0.97-A resolution crystal structure of Rad60 SLD2, which shows that apart from the conserved non-substrate SUMO:Ubc9 interface, the surface features of SLD2 are distinct from those of SUMO. Abrogation of the SLD2:Ubc9 FEG motif-dependent interaction results in hypersensitivity to genotoxic stress and an increase in spontaneous recombination associated with aberrant replication forks. Our results provide a mechanistic basis for the near-synonymous roles of Rad60 and SUMO in survival of genotoxic stress and suggest unprecedented DNA-damage-response functions for SLDs in regulating sumoylation.

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Figures

Figure 1. Rad60 SLDs interact with distinct components of the sumoylation machinery

(a) Ectopically expressed GST-Rad60 SLD2, GST-Rad60 SLD1+2, GST-Rad60 N-terminus+SLD1 (or GST alone) were induced in the Ubc9-TAP strain, and subjected to GST pull-down. Inputs and pull-downs were immunoblotted with antisera for TAP (anti-PAP) or GST (anti-GST). Ubc9 interacts specifically with GST-fusion proteins that contain Rad60 SLD2. (b) Ectopically expressed GST-Rad60 SLD2, GST-Rad60 SLD1+2, GST-Rad60 N-terminus+SLD1 (or GST alone) were induced in a Fub2-TAP background, and subjected to GST pull-down. Inputs and pull-downs were immunoblotted with antisera for PAP or GST. Unlike Ubc9, Fub2 interacts specifically with GST-fusion proteins containing Rad60 SLD1. (c) Ectopically expressed GST-Rad60 SLD2, GST-Rad60 SLD1+2, GST-Rad60 N-terminus+SLD1 (or GST alone) were induced in a Pli1-TAP strain, and subjected to GST pull-down. Inputs and pull-downs were immunoblotted with antisera for PAP or GST. As seen with Fub2, Pli1 also interacts only with GST-fusion proteins that contain Rad60 SLD1. (d) Ectopically expressed GST-Rad60 SLD2, GST-Rad60 SLD1+2, GST-Rad60 N-terminus+SLD1 (or GST alone) were induced in an Slx8-myc strain, and subjected to GST pull-down. Inputs and pull-downs were immunoblotted with antisera for myc or GST. As seen with Fub2 and Pli1, Slx8 interacts only with GST-fusion proteins that contain Rad60 SLD1. (e) Schematic depicting the interaction of Rad60 SLD1 and Rad60 SLD2 with components of the sumoylation pathway and STUbL complex.

Figure 2. Rad60 SLD2 Crystal Structure

(a) The 0.97 Å crystal structure of Rad60 SLD2 forms a β-GRASP fold that is composed of 5 β-strands, an α-helix and a 310helix. A Ca2+ ion, yellow sphere, is bound by two backbone oxygen’s and the Asp344 side chain carboxylate (in addition to two water molecules and the Glu373 side chain carboxylate from a symmetry related molecule). (b) Structural superimposition of Rad60 SLD2, depicted in dark blue, and SUMO-1 depicted in cyan (SUMO crystal structure taken from PDB code 2UYZ). A conserved non-covalent Ubc9 binding face is observed in Rad60 SLD2, with well-conserved residues depicted as sticks, and labeled for Rad60. These residues include the β3/β4-loop FEG motif, residues 379-381, where the equivalent acid side chains make water mediated interaction with Ubc9 in the human and budding yeast structures. Also well-conserved are carboxylate residues in the 310-helix β-strand 5 region, including residues Glu396 and Asp399, whose equivalents make direct hydrogen bond contacts with Ubc9 surface residues in the human and budding yeast SUMO:Ubc9 complex structures. (c) Electrostatic surface diagram for Rad60 SLD2 that is depicted partially transparent and beneath which is the SLD2 secondary structure, shown in dark grey. The α-helix 1 β-strand 2 region shows a net negative charge and the SIM-binding pocket highlighted on this structure, which occurs in this region of SUMO, is absent (red, -3.0 KTe-1, blue +3.0 KTe-1).

Figure 3. Analyzing the Rad60 SLD2:Ubc9 interface

(a) Ectopically expressed GST-Rad60 SLD2, GST-Rad60 SLD2E380R or GST alone were induced in the Ubc9-TAP strain, and subjected to GST pull-down. Inputs and pull-downs were immunoblotted with antisera for PAP or GST. Despite similar levels of protein induction, GST-Rad60 SLD2E380R did not pull down Ubc9. (b) Bacterially expressed GST-Ubc9, 6His-Rad60 SLD2 or 6His-Rad60 SLD2E380R were combined as indicated and subjected to GST pull-down, followed by immunoblotting with antisera for His or GST. Wild-type 6His-Rad60 SLD2 interacted robustly with GST-Ubc9 in vitro, whereas 6His-Rad60 SLD2E380R did not, even when loaded in excess of wild-type SLD2. (c) Immunoblot of the indicated strains with anti-GFP. (d) GST, GST-Rad60 SLD2 or GST-Rad60 SLD2E380R were expressed in an slx8-1 mutant and immunoblotted with antisera for GFP or GST. Induction of wild-type GST-Rad60 SLD2 inhibited chain formation, whilst overexpression of GST-Rad60 SLD2E380R had no effect on the slx8-1 mutant phenotype. (e) The indicated strains were serially diluted onto selective media, which either repressed (+B1), or induced (-B1) ectopic expression of GST alone, GST-Rad60 SLD2 or GST-Rad60 SLD2E380R. All plates were incubated at 32 °C. (f) Total lysates from the indicated strains were immunoblotted with antisera for GST or Cdc2 (loading control). Despite similar levels of protein induction to wild-type GST-Rad60 SLD2, GST-Rad60 SLD2E380R overexpression was not toxic in an rqh1Δ mutant.

Figure 4. The _rad60_E380R mutant is sensitive to genotoxic stress

(a) Serial dilutions of the indicated strains were either untreated (No drug), or treated with the indicated concentrations of hydroxyurea (HU), methylmethane sulfonate (MMS), camptothecin (CPT), or ultra-violet (UV) irradiated. All plates were incubated at 32 °C. The rad60E380R mutant is hypersensitive to all of the genotoxic agents shown. (b) Serial dilutions of the indicated strains were either untreated, or treated with the indicated concentrations of hydroxyurea (HU), or ultra-violet (UV) irradiated, and incubated at 32 °C. An additional untreated plate was incubated at 36 °C. Combining the rad60E380R and rad60-3 mutations suppresses the phenotypes observed in either single mutant.

Figure 5. Genetic interactions between rad60E380R and mutants in the HRR, or sumoylation pathways

Representative tetrad dissections are shown when rad60E380R was mated against: (a) rqh1Δ, (b) mus81Δ, (c) rhp51Δ, (d) nse6Δ, (e) pli1Δ, (f) nse2-SA, (g) slx8-1 and (h) ulp2Δ. For each cross a key depicts the genotypes present within each tetrad, which are denoted by various shaped white lines placed around each colony. Combining rad60E380R with these mutants results in extreme synthetic sickness, or is a lethal event. (i) Schematic of the lethal (solid black lines) and synthetic-sick (dashed grey lines) interactions involving rad60E380R and the indicated mutants.

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Molecular mimicry of SUMO promotes DNA repair - PubMed (original) (raw)