Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability

Abstract

The Smc5-Smc6 holocomplex plays essential but largely enigmatic roles in chromosome segregation, and facilitates DNA repair. The Smc5-Smc6 complex contains six conserved non-SMC subunits. One of these, Nse1, contains a RING-like motif that often confers ubiquitin E3 ligase activity. We have functionally characterized the Nse1 RING-like motif, to determine its contribution to the chromosome segregation and DNA repair roles of Smc5-Smc6. Strikingly, whereas a full deletion of nse1 is lethal, the Nse1 RING-like motif is not essential for cellular viability. However, Nse1 RING mutant cells are hypersensitive to a broad spectrum of genotoxic stresses, indicating that the Nse1 RING motif promotes DNA repair functions of Smc5-Smc6. We tested the ability of both human and yeast Nse1 to mediate ubiquitin E3 ligase activity in vitro and found no detectable activity associated with full-length Nse1 or the isolated RING domains. Interestingly, however, the Nse1 RING-like domain is required for normal Nse1-Nse3-Nse4 trimer formation in vitro and for damage-induced recruitment of Nse4 and Smc5 to subnuclear foci in vivo. Thus, we propose that the Nse1 RING-like motif is a protein-protein interaction domain required for Smc5-Smc6 holocomplex integrity and recruitment to, or retention at, DNA lesions.

Figure 1.

Structural analysis of the Nse1 RING-like motif. (A) A multiple sequence alignment between fission yeast Nse1 (SPCC550.05), human NSMCE1 (NP_659547), mouse NSMCE1 (AAH49558), rat RGD1307760, bovine NSMCE1 (NP_001030483), Xenopus MGC68739 (AAH60339), Aspergillus Nse1 (XP_748562), and budding yeast YDR288W (NP_013107) is depicted. Conserved cysteines are shaded in red. Alternate potential Zn²⁺ ligands of fission yeast Nse1 NH-RING are shaded in pink. Other conserved residues of interest are shaded in yellow and blue. Numbered circles indicate the eight predicted Zn²⁺ ligand positions in the interspecies Nse1 NH-RING consensus. The C₄HC₃ RING consensus, with expected spacing, is shown below this alignment, as well as the two loop positions. A shaded box below indicates the positions of the ligands mutated in this study, as well as the region deleted in the ΔRING mutant. Φ, conserved hydrophobic residue. (B) The fission yeast Nse1 homology model. Comparative modeling studies based on human Nse1 structure (2CT0.pdb) suggest that part of the fission yeast Nse1 sequence forms a RING-like structure, comprised of a central β-sheet (two strands, in blue), a short α-helix (in green) and two binding sites for Zn²⁺ ions (pink spheres). The positions of the central β-sheets and α-helix are also represented on the alignment in A for better understanding. The Cysteine (Cys) residues and a Histidine (His) residue that chelate Zn²⁺ are highlighted with red line labels. Cys202, Cys208, and His210 (highlighted with black line labels) are present in the structure, but sequence analysis and modeling studies indicate that they are not situated at the Zn²⁺ coordination sites. This model includes Nse1 residues Leu181 to Ile229.

Figure 2.

The Nse1 NH-RING domain is required for DNA repair. (A) Spot assays showing temperature and genotoxic stress sensitivities of the indicated Nse1 NH-RING mutants. Serial dilutions of the indicated strains were spotted onto rich media with or without the indicated doses of DNA-damaging agents. Plates were grown at the indicated temperature for 3–5 d. (B) nse1ΔRING cells properly segregate their DNA. Live cell microscopy showing the RFP-tagged nucleolar marker Gar1 segregation in nse1-1 and nse1ΔRING cells. Phase constrast (DIC or Nomarski) shows these mutants cell length when grown at 32°C. (C) nse1ΔRING and mus81Δ are synthetically sick mutations. Serial dilutions of the indicated strains were spotted as in A. Combined nse1ΔRING and mus81Δ mutations show additive growth defects and hypersensitivity to genotoxic stress when compared with single mutants. (D) Genetic interactions for nse1ΔRING. Top, tetrad dissections showing synthetic lethality of combined nse1ΔRING and rqh1Δ or nse2-1 mutations. Genetic backgrounds of resulting strains are represented by the various shapes circling each colony. Bottom, summary of genetic interactions (synthetic sickness or lethality) between nse1ΔRING and the indicated alleles. (E) rhp51Δ and nse1ΔRING display similar sensitivities to UV and are not additive. Kill curves showing the percentage of cell survival for each indicated strain, which equals the number of colony forming after exposure to increasing UV doses versus the number of colonies forming on untreated plates.

Figure 3.

The Nse1 NH-RING domain is required for damage-induced Smc5-Smc6 localization. Left, live cell microscopy monitoring endogenously tagged Nse4-GFP localization in untreated (−) or MMS-treated (+MMS) at 25°C, in wild-type or nse1ΔRING backgrounds. Right, percentage of cells showing one or more than one Nse4-GFP focus on the left panels were scored.

Figure 4.

Human and fission yeast Nse1 proteins and their NH-RINGs do not mediate ubiquitin E3 ligase activity in vitro. (A) In vitro ubiquitination assay testing GST fusions of fission yeast or human (h) Nse1 full-length, ΔRING, or RING-only constructs, as putative ubiquitin E3 ligases. GST-Slx8 is used as a positive control for E3-mediated polyubiquitin chain formation. WB, Western blot; Ubi, ubiquitin. White arrows in the bottom panel show the position of each GST fusion used. (B) Human Nse1 GST fusion was tested as ubiquitin E3 ligase with a panel of E2-conjugating enzymes. A reaction containing GST-Slx8 and the E2 enzyme UbcH5a was used as a positive control.

Figure 5.

Analysis of Nse1-Nse3-Nse4 trimer formation in the presence of Nse1 NH-RING mutations. (A) Nse1 interaction with Nse4 is bridged by Nse3. Protein interaction assays were performed in Sf9 insect cells coinfected with the indicated protein-expressing baculoviruses, using Nickel (Ni²⁺) purification columns and Western blotting for copurifying proteins. Nse1 can interact with Nse3 but not with Nse4 in the absence of Nse3, whereas the full trimer can be pulled down when all three proteins are coexpressed. (B) Nse1 NH-RING is not required for interaction with Nse3. Yeast two-hybrid interaction assays were performed using the indicated strains and spotted on media without (D-WLH) or with (D-WLH + 3-AT) selection to test for direct interaction between the expressed proteins. Nse1 does not interact with Nse4 in the absence of Nse3, but interacts with Nse3 in an NH-RING–independent manner. −, empty vector control; AD, Gal4 activation domain; DBD, Gal4 DNA-binding domain. (C) The Nse1 NH-RING domain is required for robust Nse1-Nse3 trimerization with Nse4 in vitro. Sf9 insect cells were simultaneously infected with baculoviruses to coexpress the indicated proteins. Complexes were purified by GST pulldown, eluted by PreScission Protease cleavage, and detected by Western blotting against the HA and Flag tags. * Nonspecific band appearing in every purification, slightly larger than Nse1 full-length. (D) Distinct Nse3 domains are required to contact Nse1 and Nse4. The indicated yeast two-hybrid strains were performed as in B using the indicated strains. Nse3 protein domains are illustrated on the right. Regions of high sequence homology between species are represented by light gray boxes. (E) The Nse1 NH-RING domain optimizes Nse1-Nse3-Nse4 trimer formation. Schematics summarizing interaction analyses. Nse1 and Nse3 N-termini interact together independently of Nse1 NH-RING. Nse3 MAGE domain interacts with Nse4 independently of Nse1. Nse1 NH-RING is required for stable trimer formation, as an Nse1 ΔRING mutant (right panel) loses contact with Nse4, but not with Nse3.