The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers
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Evaluation Summary:
This paper will be of interest to the chromosome biology field and SMC researchers in particular. The study provides cell biological, biochemical, and structural modeling evidence that a new Nse5-like protein named SIMC1 is a paralog of SLF1, and that the two compete for SLF2-Smc5/6 binding. The authors also show that SIMC1 targets SMC5/6 to polyomavirus replication centers through its SUMO binding motifs (SIMs), supporting a role for SIMC1 in Smc5/6 recruitment for viral restriction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1’s Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1’s helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.
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Evaluation Summary:
This paper will be of interest to the chromosome biology field and SMC researchers in particular. The study provides cell biological, biochemical, and structural modeling evidence that a new Nse5-like protein named SIMC1 is a paralog of SLF1, and that the two compete for SLF2-Smc5/6 binding. The authors also show that SIMC1 targets SMC5/6 to polyomavirus replication centers through its SUMO binding motifs (SIMs), supporting a role for SIMC1 in Smc5/6 recruitment for viral restriction.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors used BioID to identify SMC5 proximal proteins in HEK293T cells. In one of the top hits, they recognized an NSE5-like domain and proposed it (SIMC1) to be the new NSE5-like subunit of the human SMC5/6 complex. Accordingly, they showed its colocalization and co-immunoprecipitation with the NSE6/SLF2 subunit. They confirmed SIMC1-SLF2 direct physical interaction by their copurification. Furthermore, using cryoEM and AlphaFold modelling, they were able to determine the structure of the SIMC1-SLF2 dimer at 3.9A resolution. They also showed that the SIMC1-SLF2 dimer is mutually exclusive to the previously described SLF1-SLF2 dimer. To make their structural data more solid, the authors should have mutated contact residues to support their structural data and their conclusion that the similar SIMC1 and …
Reviewer #1 (Public Review):
The authors used BioID to identify SMC5 proximal proteins in HEK293T cells. In one of the top hits, they recognized an NSE5-like domain and proposed it (SIMC1) to be the new NSE5-like subunit of the human SMC5/6 complex. Accordingly, they showed its colocalization and co-immunoprecipitation with the NSE6/SLF2 subunit. They confirmed SIMC1-SLF2 direct physical interaction by their copurification. Furthermore, using cryoEM and AlphaFold modelling, they were able to determine the structure of the SIMC1-SLF2 dimer at 3.9A resolution. They also showed that the SIMC1-SLF2 dimer is mutually exclusive to the previously described SLF1-SLF2 dimer. To make their structural data more solid, the authors should have mutated contact residues to support their structural data and their conclusion that the similar SIMC1 and SLF1 surfaces bind SLF2. Otherwise, the above data are very strong and support the author's conclusions about the new NSE5-like subunit of the human SMC5/6 complex.
Further, the authors showed that the SMC5/6 complex is localized to SV40 T-large (LT) antigen-induced foci and PML bodies. This localization is dependent on the SIMC1 and its SUMO-interacting motifs. In addition, several SUMO pathway factors (known components of PML bodies) were identified in the SIMC1 BioID search. These data suggest a strong connection of SMC5/6 to SUMO-rich sites. Based on this connection, the authors speculate that SIMC1-SLF2 specifically regulates responses to viral challenges while SLF1-SLF2 is specifically involved in DNA damage responses. Given the roles of PML bodies in homologous recombination (DNA damage repair, alternative telomere lengthening), their experiments do not directly prove this assumption. Actually, SMC5/6 was already shown to be localized to PML bodies and involved in alternative telomere lengthening (https://pubmed.ncbi.nlm.nih.gov/17589526/). In conclusion, further experiments are required to show the exclusive roles of distinct SLF2 complexes and the specific involvement of SIMC1-SLF2 in antiviral responses.
Major requests:
1. The biochemical data on the SIMC1-SLF2 dimer are solid. However, the authors did not verify the structural data by mutating surface residues mediating SIMC1-SLF2 interaction (although they prepared various constructs and generated "combo" mutations at the different surfaces - Fig. 7). They should mutate contact residues to support their structural data and their conclusion that the same SIMC1/SLF1 surfaces bind SLF2.
2. The claims about the exclusive roles of SIMC1-SLF2 and SLF1-SLF2 should be better substantiated. The control experiment showed (no)localization of SLF1 to LT-induced foci and PML bodies (and vice versa SIMC1 (no)localization in DNA-damaged cells like in Raschle, 2015 - https://pubmed.ncbi.nlm.nih.gov/25931565/) should be performed.
3. In addition, the sensitivity of the SIMC1-/- cells to replication stress and DNA damage agents should be compared to the other SMC5/6 mutants.
4. SIMC1 BioID (under native/denaturing conditions) experiment suggests that multiple SUMO pathway factors are proximal to SIMC1. However, there is no evidence for the interaction between SIMC1 and ZNF451 (p. 12/line 228 + p.28/line 508). The coIP experiment (Fig. 3D) was performed in a biotin-streptavidin manner suggesting the proximity of these proteins but not the interaction. The coIP experiment should be performed with anti-myc or anti-GFP antibodies. -
Reviewer #2 (Public Review):
In this manuscript, the authors provide cell biological, biochemical, and structural modeling data to suggest that SIMC1 is a paralog of SLF1 and that the two compete for SLF2 binding likely because their C-terminal regions form similar helix-enriched structures to bind at the same surface of SLF2. As previously shown for SLF1, the authors found that SIMC1 bound to other subunits of Smc5/6. Distinct from SLF1, SIMC1 contains N-terminal SIMs and binds to SUMO pathway proteins including PML and SUMO. They found that SIMs and the SLF2-binding regions of SIMC1 are both required for targeting SIMC1 and SMC5/6 to the PML body upon LT expression. The authors showed that structural models of the dimerized portions of SIMC1:SLF2 and SLF1:SLF2 are similar to that of the yeast Nse5-6 structures, suggesting that they …
Reviewer #2 (Public Review):
In this manuscript, the authors provide cell biological, biochemical, and structural modeling data to suggest that SIMC1 is a paralog of SLF1 and that the two compete for SLF2 binding likely because their C-terminal regions form similar helix-enriched structures to bind at the same surface of SLF2. As previously shown for SLF1, the authors found that SIMC1 bound to other subunits of Smc5/6. Distinct from SLF1, SIMC1 contains N-terminal SIMs and binds to SUMO pathway proteins including PML and SUMO. They found that SIMs and the SLF2-binding regions of SIMC1 are both required for targeting SIMC1 and SMC5/6 to the PML body upon LT expression. The authors showed that structural models of the dimerized portions of SIMC1:SLF2 and SLF1:SLF2 are similar to that of the yeast Nse5-6 structures, suggesting that they are Nse5-6 homologs but with different roles in targeting the SMC5/6 complex.
Overall, this is a very interesting study. The experiments were well done and data are presented clearly. The report will be highly interesting to the SMC field.
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Reviewer #3 (Public Review):
In yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the Smc5/6 complex is associated with regulatory subunits Nse5 and Nse6, which have been shown to be required for chromatin association and also inhibit ATP turnover. In addition, the recruitment to sites of DNA damage requires a multi-BRCT domain protein, Brc1/Rtt107. The identity of the Nse5 and Nse6 regulatory subunits of the human SMC5/6 complex has long been a mystery. The Nse6 homologue, SLF2, was identified in a proteomics screen for factors required for bypass of DNA crosslinks. SLF1, the other recruiter identified in the same screen, showed very limited homology to Nse6 and also contained BRCT domains and ANKRIN repeats suggesting that it provided an analogous mechanism of recruitment of SMC5/6 to sites of DNA damage to that of …
Reviewer #3 (Public Review):
In yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the Smc5/6 complex is associated with regulatory subunits Nse5 and Nse6, which have been shown to be required for chromatin association and also inhibit ATP turnover. In addition, the recruitment to sites of DNA damage requires a multi-BRCT domain protein, Brc1/Rtt107. The identity of the Nse5 and Nse6 regulatory subunits of the human SMC5/6 complex has long been a mystery. The Nse6 homologue, SLF2, was identified in a proteomics screen for factors required for bypass of DNA crosslinks. SLF1, the other recruiter identified in the same screen, showed very limited homology to Nse6 and also contained BRCT domains and ANKRIN repeats suggesting that it provided an analogous mechanism of recruitment of SMC5/6 to sites of DNA damage to that of Brc1/Rtt107. The questions of whether SLF1 was the functional homologue of Nse5 in humans and whether it was required for all chromatin association remained.
The authors address this in their analysis. They used proteomics to identify SMC5/6 interactors in PML nuclear bodies. They identified a novel regulator, SIMC1, that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. They showed that SIMC1 and SLF2 interact through the Nse5 domain and form an alternative complex to SLF1/SLF2. They then suggested that the SIMC1/SLF2 complex is specific for recruitment of SMC5/6 for restriction of viral replication/transcription as the SIM and Nse5 domains were found to be important to localise SMC5/6 to polyomavirus replication centres at PML bodies, which are enriched for SUMO, and for recruitment of SMC5/6 to SV40 replication centres. Since PML bodies are associated with alternative lengthening of telomeres (ALT) and SMC5/6 has roles in telomere maintenance it would be interesting to know whether the SIMC1/SLF2 complex was also required in these circumstances but this is a question for a future study.
Overall, the manuscript is well written and the data is of high quality. It makes a step advance in terms of our understanding of how the Smc5/6 complex functions.
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