Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

Acceptance summary:

The authors use the photoconvertible fluorophore mEos3 fused to Sm5/6 complex components to study the dynamics of these complexes in live fission yeast cells at single molecule resolution by single-particle tracking photo-activated localization microscopy (sptPALM). Specific point mutations affecting the dsDNA binding, ssDNA binding or ATPase activity of the SMC5/6 complex or the interacting factors Brc1 and Nse6 were analyzed by sptPALM and genetic assays to measure DNA damage survival, mutation rates, and genomic rearrangements at stalled replication forks. The authors propose a model of two modes of chromatin association of the Smc5/6 complex by direct binding to dsDNA and indirect recruitment through Brc1 bound to γ H2A. Moreover, the authors conclude that ssDNA binding is not required for Smc5/6 chromatin association but for a more downstream action in maintaining genomic stability by homologous recombination.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Single-molecule live cell imaging of the Smc5/6 DNA repair complex" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Wolf-Dietrich Heyer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

In its current form, the manuscript might be better suited for a more specialized methods paper, but the impact of the work could be significantly raised by addressing the points #1-3 discussed below. As the requested additions are too extensive for a revision, we would invite a new submission of a revised manuscript with substantial new experiments to address these points, if you elect this course of action. We would make every effort to have the same review team evaluate the new manuscript.

Summary:

The authors use the photoconvertible fluorophore mEos3 fused to cohesin, condensin, and Sm5/6 complex components to study the dynamics of these complexes in live fission yeast cells at single molecule resolution by single-particle tracking photo-activated localization microscopy (sptPALM). Specifically, the authors find that Brc1 and Nse6 both promote chromatin loading of Smc5/6 but with brc1∆ having a less dramatic defect than nse6∆. The role of Brc1 is consistent with a recent report by (Oravcova et al., 2019). Consistent with Brc1 being recruited to chromatin by binding γ-H2A (Williams et al., 2010), brc1∆ and hta-SA mutants exhibit comparable defects in chromatin association of Smc5/6. Next, the authors show that mutation of each of the two ATP binding sites cause DNA damage sensitivity and defective chromatin association. Importantly, the authors find that dsDNA but not ssDNA binding is required for loading Smc5/6 on chromatin but not for prevention of gross chromosomal rearrangements.

Overall, the manuscript is well written and the data of high quality, but it does not advance the field much in terms of our understanding of the role of Smc5/6 in DNA repair. Moreover, the sptPALM method is not compared sufficiently with existing methods (ChIP and cytological analysis of foci), which appear to have a better dynamic range that sptPALM, to allow readers to choose between these methods for their experiments. Moreover, there are a number of technical issues that need clarification and resolution.

The key points to be addressed in case the authors elect to re-submit the manuscript:

1. This manuscript represents the first time sptPAML is used to derive the dynamic behavior of the Smc5/6 complex in live cells. There are a number of technical concerns that should be addressed.

• Lines 119/120: The authors state that DNA-bound proteins have constrained diffusion, which is clearly correct. The question is whether all constrained diffusion is caused by DNA binding, or whether binding to other stationary nuclear structures, such as the nuclear pore complex, or confinement in phase-separated compartments, could also lead to constraints?

• Line 162 ff: The authors document significant differences between cohesin and Smc5/6 mobility and mention ChIP experiments that suggest co-localization. This discrepancy should be examined and discussed more than is done on lines 374/5?

• What does it mean to be 50% chromatin bound be the sptPALM method? Does FRAP (or chromatin/soluble fractionation) also show a similar immobile fraction?

• The higher displacement value observed for Nse6-mEos3 compared with Nse4 could be due to the tag differentially affecting different subunits, particularly given that Figure S1 showed that cells containing Nse6-mEos3, but not Nse4-mEos3, are sensitive to MMS.

• Could the different displacement values of Nse4-mEos3 between brc1∆ vs nse6∆ cells be due to the mutant cells accumulating at different cell cycle phases given the slow growth of the mutants?

• Could the different mutants tested affect Nse4-mEos3 protein levels thus may affect data?

• Could the nse3-R254E or hinge mutants tested affect the Smc5/6 complex stability, thus affecting the signals?

• Figure 1B, the result raised the question whether the Rad21-mEos3 expression level is greatly reduced compared to the endogenous Rad21. Does mEos3 fusion affect the protein levels of the examined Smc5/6 subunits? Clarification of protein levels of the fusion proteins is important for this work, as the sptPAML data can be affected by protein levels.

• Figure 1C, it is unclear how cells in mitosis vs. interphase were assigned. Also, what are criteria for determining nucleus vs cytoplasm in these images?

• Judging from Figure 2C and S1B, Nse4 and Rad21 appear to be present mostly in large bright clusters, though significant amounts of weaker signals are also visible, particularly for Nse4. This could suggest that two populations of proteins (within and outside the clusters) exist. Do data in Figure 2A and B represent only the signals within the cluster or exhibiting weaker signals, or both (averaged).

2. What is the functional significance of the observed asymmetry in use of the two ATP binding sites? Moreover, the conclusion that "DNA-dependent ATP hydrolysis by the two binding pockets is not equivalent" could be strengthened by a direct assessment of the ATPase activity of the Smc5 (smc5-R77A) or Smc6 (smc6-R150A).

3. Why does suppression of gross chromosomal rearrangements require ssDNA binding but apparently not chromatin binding? The authors propose a model for HRR by Smc5/6 where dsDNA binding happens first and independent of DNA damage and ssDNA binding happens during DNA repair. It would strengthen the impact of the manuscript to test this model. For example: can DNA damage sensitivity and the chromatin association defect of dsDNA binding nse3-R254E mutant be rescue by fusion of Nse4 or Nse6 to Brc1? What distinguishes resistance to replication stress and suppression of gross chromosomal rearrangements?

Additional points for the authors to consider:

4. Figure 3 C, D: The results beg the question of the phenotype of the brc1 nse6 double mutant. This could be very informative to start addressing the models developed in the discussion.

5. Figure 3E: The treatment with MMS is rather long (5 hours). How was this time chosen? Likely DNA lesions would have been processed and perhaps even repaired and some cells dead at 5 hours. I therefore suggest to do a time course for Smc5/6 chromatin association. The kinetics of chromatin association might simply be different in the brc1∆ and nse6∆ mutants.

6. Figure 3F: The model in A predicts epistasis between the brc1 and the γ H2AX mutants, which should be tested here or has this been tested before?

7. Figure 5, the Alt 2017 paper suggests that while smc5-R609, 615E abolishes ssDNA binding, smc5-Y612G or smc6-x, and to a large extent Smc6-F528A, exhibit close to wild-type level of ssDNA binding. Here, these mutants showed similar Nse4 sptPALM behavior. Thus, these observations would argue that features other than ssDNA binding affect Nse4-chromatin association.

8. Figure 6B: The fluctuation tests lack error bars. Since Figure S6 is labeled as a repeat of Figure 6B left, I assume these are single determinations. The data should be reported as mean of n=3. The rate difference between Figure 6B left and Figure S6 are unusually large for a rate determination, which is usually quite robust. All the more a reason to bolster these data with n=3.

Text changes or clarifications:

9. Column graphs should be converted to scatter "superplots" as recently proposed (Lord et al., 2020) to show individual cell data and variability between experiments.

10. Line 35: insert 'function of the' in front of Smc5/6.

11. Line 42: It might help the reader to include the statement here, which is found in the beginning of the discussion (lines 355 ff), to indicate that SMC must have functions beyond HR.

12. Line 129: What is the evidence that the single particle of condensin contains a single condensin complex?

13. Page 10: Please provide a reference for the epistatic relationship between smc6-X (R706C) and smc6-74 (A151T) and rad51-d in response to MMS.

14. Figure 3A: The evidence that Brc1 is binding to gammaH2AX should be discussed.

15. Figure 5A: Please indicate the gene/mutations used in the scheme.

References:

Alt, A, Dang HQ, Wells OS, Polo LM, Smith MA, McGregor GA, Welte T, Lehmann AR, Pearl LH, Murray JM, Oliver AW. (2017) Specialized interfaces of Smc5/6 control hinge stability and DNA association. Nature Comm 8, 14011

Lord, S.J., Velle, K.B., Mullins, R.D., and Fritz-Laylin, L.K. (2020). SuperPlots: Communicating reproducibility and variability in cell biology. J Cell Biol 219.

Oravcova, M., Gadaleta, M.C., Nie, M., Reubens, M.C., Limbo, O., Russell, P., and Boddy, M.N. (2019). Brc1 Promotes the Focal Accumulation and SUMO Ligase Activity of Smc5-Smc6 during Replication Stress. Mol Cell Biol 39.

Williams, J.S., Williams, R.S., Dovey, C.L., Guenther, G., Tainer, J.A., and Russell, P. (2010). gammaH2A binds Brc1 to maintain genome integrity during S-phase. EMBO J 29, 1136-1148.

Reviewer #1:

The authors use the photoconvertible fluorophore mEos3 fussed to cohesin, condensin, and Sm5/6 complex components to study the dynamics of these complexes in live fission yeast cells at single molecule resolution by single-particle tracking photo-activated localization microscopy (PALM). Using the 'spot-on' program to analyze single particle tracts, the authors define differences between the three complexes in their nuclear dynamics, determine the effect of DNA binding and ATPase mutants in the Smc5/6 complex on constrained diffusion, which is interpreted a reporting on chromatin association. In addition, they define two distinguishable recruitment pathways for Smc5/6 complex. Overall, this is a novel and fruitful approach to study the enigmatic Smc5/6 complex, which generated significant novel insights not previously accessible. The authors should consider the following points in a revision.

1. Lines 119/120: The authors state that DNA-bound proteins have constrained diffusion, which is clearly correct. The question is whether all constrained diffusion is caused by DNA binding, or whether binding to other stationary nuclear structures, such as the nuclear pore complex, or confinement in phase-separated compartments, could also lead to constraints?

2. Line 162 ff: The authors document significant differences between cohesin and Smc5/6 mobility and mention ChIP experiments that suggest co-localization. Maybe this discrepancy could be discussed more than is done on lines 374/5?

3. Figure 3 C, D: The results beg the question of the phenotype of the brc1 nse6 double mutant. I think this could be very informative to start addressing the models developed in the discussion.

4. Figure 3F: The model in A predicts epistasis between the brc1 and the γ H2AX mutants, which should be tested here.

5. Figure 6B: The fluctuation tests lack error bars. Since Figure S6 is labeled as a repeat of Figure 6B left, I assume these are single determinations. The data should be reported as mean of n=3. The rate difference between Figure 6B left and Figure S6 are unusually large for a rate determination, which is usually quite robust. All the more a reason to bolster these data with n=3.

Additional points:

6. Line 35: insert 'function of the' in front of Smc5/6.

7. Line 42: It might help the reader to include the statement here, which is found in the beginning of the discussion (lines 355 ff), to indicate that SMC must have functions beyond HR.

8. Line 129: What is the evidence that the single particle of condensin contains a single condensin complex?

9. Figure 3A: The evidence that Brc1 is binding to gammaH2AX should be discussed.

10. Figure 5A: Please indicate gene/mutations used in the scheme.

Reviewer #2:

The manuscript entitled "Single-molecule live cell imaging of the Smc5/6 DNA repair complex" by Etheridge and colleagues introduces single-particle tracking PALM (sptPALM) microscopy to study chromatin association of the Smc5/6 complex. The authors use this technique in combination with genetics to dissect the chromatin recruitment of the Smc5/6 in unchallenged cells and after genotoxic stress. Specifically, the authors find that Brc1 and Nse6 both promote chromatin loading of Smc5/6 but with brc1∆ having a less dramatic defect than nse6∆. The role of Brc1 is consistent with a recent report by (Oravcova et al., 2019). Consistent with Brc1 being recruited to chromatin by binding γ-H2A (Williams et al., 2010), brc1∆ and hta-SA mutants exhibit comparable defects in chromatin association of Smc5/6. Next, the authors show that mutation of each of the two ATP binding sites cause DNA damage sensitivity and defective chromatin association. Importantly, the authors find that dsDNA but not ssDNA binding is required for loading Smc5/6 on chromatin but not for prevention of gross chromosomal rearrangements. The manuscript is well written and the data of high quality, but it does not advance the field much in terms of our understanding of the role of Smc5/6 in DNA repair. Moreover, the sptPALM method is not compared sufficiently with existing methods (ChIP and cytological analysis of foci), which appear to have a better dynamic range that sptPALM, to allow readers to choose between these methods for their experiments. In its current form, the manuscript might be better suited for a more specialized methods paper, but the impact of the work could be significantly raised by addressing some of the key questions raised by the study such as:

What is the functional significance of the observed asymmetry in use of the two ATP binding sites?

Why does suppression of gross chromosomal rearrangements require ssDNA binding but apparently not chromatin binding?

Additional major issues to address:

1. Page 8: The conclusion that "DNA-dependent ATP hydrolysis by the two binding pockets is not equivalent" would be strengthen by a direct assessment of the ATPase activity of the Smc5 (smc5-R77A) or Smc6 (smc6-R150A).

2. The authors propose a model for HRR by Smc5/6 where dsDNA binding happens first and independent of DNA damage and ssDNA binding happens during DNA repair. It would strengthen the impact of the manuscript to test this model. For example: can DNA damage sensitivity and the chromatin association defect of dsDNA binding nse3-R254E mutant be rescue by fusion of Nse4 or Nse6 to Brc1? What distinguishes resistance to replication stress and suppression of gross chromosomal rearrangements?

3. What does it mean to be 50% chromatin bound be the sptPALM method? Does FRAP (or chromatin/soluble fractionation) also show a similar immobile fraction?

4. Figure 3E: The treatment with MMS is rather long (5 hours). How was this time chosen? Likely DNA lesions would have been processed and perhaps even repaired and some cells dead at 5 hours. I therefore suggest to do a time course for Smc5/6 chromatin association. The kinetics of chromatin association might simply be different in the brc1∆ and nse6∆ mutants.

References:

Lord, S.J., Velle, K.B., Mullins, R.D., and Fritz-Laylin, L.K. (2020). SuperPlots: Communicating reproducibility and variability in cell biology. J Cell Biol 219.

Oravcova, M., Gadaleta, M.C., Nie, M., Reubens, M.C., Limbo, O., Russell, P., and Boddy, M.N. (2019). Brc1 Promotes the Focal Accumulation and SUMO Ligase Activity of Smc5-Smc6 during Replication Stress. Mol Cell Biol 39.

Williams, J.S., Williams, R.S., Dovey, C.L., Guenther, G., Tainer, J.A., and Russell, P. (2010). gammaH2A binds Brc1 to maintain genome integrity during S-phase. EMBO J 29, 1136-1148.

Reviewer #3:

The authors used sptPAML to examine the Smc5/6 subunits for their chromatin association in live fission yeast cells. They found that Smc5/6 appears to be in the nucleus throughout the cell cycle but exhibited more dynamic association with chromatin compared with cohesin. In addition, Nse6 appears to be more dynamic than Nse2, Nse4, and Smc5 or 6. The authors further examined several mutants in this assay. They found several mutants show defects of Nse4 chromatin associated and these include: 1) brc1∆ (or H2A-SA) and nse6∆ with only nse6∆ exhibit a complete defect, regardless of MMS treatment. 2) Smc5 or 6 ATP hydrolysis mutants, with Smc6 mutant exhibiting stronger effects. 3) a Nse3 mutant reduce in vitro dsDNA binding, and 4) hinge mutant defective afffecting ssDNA binding exhibiting defect only after MMS treatmen. Lastly, the authors show that the smc6-x and -74 increased rates of DNA deletion due to fork stalling (HR based events). Overall, this is an interestingly work that for the first time uses sptPAML to derive the dynamic behavior the Smc5/6 complex in live cells and using this assay to assess how different mutants of the complex can influence such behavior. While several conclusions are expected from previous reports, a few potentially significant conclusions are drawn. The work could benefit from technique clarifications and adjustment in interpretations as detailed below.

Given data are mostly acquired using sptPALM, an expert of this technique and analyses (spot-on, model fitting, PDF graphs) should be asked to assess data quality and interpretations. For example, can displacement values be strictly interpreted as reflecting chromatin-bound protein fraction; could they reflect other aggregation behavior of the proteins, such as phase separation? In addition, this reviewer felt that the interpretation of sptPALM data can be somewhat biased and the authors should consider other possibilities. For example, whether the higher displacement value observed for Nse6-mEos3 compared with Nse4 could be due to the tag differentially affecting different subunit, particularly given that Figure S1 showed that cells containing Nse6-mEos3, but not Nse4-mEos3, are sensitive to MMS. Also, could different displacement values of Nse4-mEos3 between brc1∆ vs nse6∆ cells be due to mutant cells are accumulated at different cell cycle phases given the slow growth of the mutants. Could the different mutants tested affect Nse4-mEos3 protein levels thus may affect data? Could the nse3-R254E or hinge mutants tested affect the Smc5/6 complex stability, thus affecting the signals?

Figure 1B, the result raised the question whether the Rad21-mEos3 expression level is greatly reduced than the endogenous Rad21. Along the same line, does the mEos3 fusion affect the protein levels of the examined Smc5/6 subunits? Clarification of protein levels of the fusion proteins is important for this work, as sptPAML data can be affected by protein levels.

Figure 1C, it is unclear how cells in mitosis vs. interphase were assigned. Also, what are criteria for determining nucleus vs cytosplam in these images?

Judging from Figure 2C and S1B, Nse4 and Rad21 appear to be present mostly in large bright clusters, though significantly amounts of weaker signals are also visiable, particularly for Nse4. This could suggest that two populations of proteins (within and outside the clusters). Do data in Figure 2A and B represent only the signals within the cluster or exhibiting weaker signals, or both (averaged).

Figure 5, the Alt 2017 paper suggests that while smc5-R609, 615E abolishes ssDNA binding, smc5-Y612G or smc6-x, and to a large extent Smc6-F528A, exhibit close to wild-type level of ssDNA binding. Here these mutants showed similar Nse4 sptPALM behavior. Thus, these observations would argue that features other than ssDNA binding affect Nse4-chromatin association.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo" for consideration by eLife. Your article has been reviewed by the same 3 peer reviewers as the initial submission, including Wolf-Dietrich Heyer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Kevin Struhl as the Senior Editor. The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Summary:

This is the resubmission of a previously rejected manuscript that was reviewed by the same reviewers as the original submission. The authors focused the manuscript on the Smc5/6 complex and the manuscript benefitted from the streamlining. The revision and the response clarified many of the technical concerns.

New experimentation with the Nse5-mEos3 fusion corroborates the data with the NSe6 fusion. Additional controls using FACS analysis exclude cell cycle differences as the cause for the observed effects in brc1 and nse6 deficient cells. Previous published work provides controls for protein levels as well as the analysis of the brc1 nse6 double mutant. New experimentation conducting a time course and additional clarification confirms that the 5 hr time point for the MMS treatment is adequate. New experimentation solidified the mutation rate data.

Essential revisions:

The authors report in their rebuttal that they have performed SPT at hourly intervals in wild type cells and found that the fraction bound was highest at 4-5 hours. These data should be included in the manuscript, maybe as a supplemental figure.