Characterization of the REC114‐MEI4‐IHO1 complex regulating meiotic DNA double‐strand break formation

  1. Hamida Laroussi
  2. Ariadna B Juarez‐Martinez
  3. Aline Le Roy
  4. Elisabetta Boeri Erba
  5. Frank Gabel
  6. Bernard de Massy
  7. Jan Kadlec

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Abstract

Meiotic recombination is initiated by the formation of DNA double‐strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4, and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled‐coil‐based tetramers. Using AlphaFold2 modeling combined with biochemical characterization, we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1‐REC114‐MEI4 complex and suggest that REC114 could act as a potential regulatory platform mediating mutually exclusive interactions with several partners.

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  1. Version published to 10.15252/embj.2023113866
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    Reply to the reviewers

    Response to reviewers' comments

    We thank the reviewers for their constructive evaluation of our manuscript. In the following point-by-point response, we explain how we will implement the suggested modifications.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary:

    The formation of meiotic double-stranded DNA breaks is the starting point of meiotic recombination. DNA breaks are made by the topoisomerase-like SPO11, which interacts with a number of regulatory factors including REC114, MEI4 and IHO1. Despite the key role this process has in the continuation, and genetic variation, or eukaryotic life, there is very little known about how this process is regulated. Laroussi et al make use of biochemical, biophysical and structural biological approaches to extensively characterise the REC114-MEI4-IHO1 complex.

    This is an outstanding biochemical paper. The experiments are well planned and beautifully executed. The protein purifications used are very clean, and the figures well presented. Importantly Laroussi et. al describe, and carefully characterise through point mutational analysis, the direct physical interaction between IHO1 and REC114-MEI4. This is an interaction that has, at least in yeast, previously been suggested to be driven by liquid-liquid separation. The careful and convincing work presented here represents an important paradigm-shift for the field.

    I am fully supportive of publication of this excellent and important study.

    We thank the reviewer for his/her positive comments, appreciation of the importance of our study and suggested modifications.

    Major comments:

    Point 1:

    My only major concern is regarding Figure 4, and specifically the AF2 model of the coiled-coil tetramer of IHO1. Given the ease with which MSAs of coiled-coils can become "contaminated" with non-orthologous sequences, I would urge some caution with this model. This is especially since the yeast ortholog of IHO1, Mer2, has been previously reported to be an anti-parallel tetramer (albeit, not very well supported by the data). The authors have several choices here. 1) They could simply reduce the visibility of the IHO1 tetramer model, and indicate caution in the parallel tetramer model. 2) They could consider using a structure prediction algorithm that doesn't use an MSA (e.g. ESMFold). 3) They could try to obtain experimental evidence for a parallel coiled-coil tetramer, e.g. through EM, SAXS or FRET approaches. I would like to make it crystal clear, however, that I would be *very* supportive of approach 1) or 2). An experimental approach is *not* necessary.

    Assuming the authors don't take a wet-lab approach, this shouldn't take more than a couple of weeks.

    This is a very good suggestion. We are aware of the previously reported anti-parallel architecture of the yeast IHO1 ortholog Mer2 (Claeys Bouuaert et al., Nature 2021). It should be noted, that in the recent preprint, posted by the Claeys Bouuaert lab (BioRxiv, https://doi.org/10.1101/2022.12.16.520760), a high confidence model of yeast Mer2 (and for human) parallel tetrameric coliled-coil is presented, apparently consistent with their previous XL-MS results (Claeys Bouuaert et al., Nature 2021).

    To clarify this issue we will follow the suggestions of Reviewer 1 and 2.

    1. As suggested also by Reviewer 2, we will produce a tethered dimer of IHO1125-260, connected by a short linker and determine its MW by SEC-MALLS (and SAXS).
    2. In the meantime we followed the suggestion of Reviewer 1 and modelled the IHO1130-281 by the ESMfold, which is another recent powerful AI-based program that does not use multiple sequence alignments. Remarkably, the predicted structure is very similar to the one predicted by AlphaFold, also predicting the parallel arrangement of IHO1. This model will be included as a supplementary figure.
    3. We will also point out in the text that these models, despite being very convincing, remain models.

    Minor comments:

    Point 2:

    The observation that REC114 and MEI4 can also form a 4:2 complex is very interesting and potentially important. Did the authors also try to model this higher order complex in AF2?

    Yes, we did this with the hope that we could identify residues whose mutation could limit the fast exchange between the 2:1 and 4:2 states. Unfortunately, no convincing additional contacts are modelled by AlphaFold. This PAE plot will be included as a supplementary figure.

    Point 3:

    Similarly to above, what does the prediction of the full-length REC114:MEI4 2:1 complex look like? Presumably the predicted interaction regions align well with experimental data, but it would be interesting to see (and easy to run).

    The AlphaFold modelling of the FL REC114:MEI4 (2:1) complex will be included as supplementary figure. It is consistent with the model comprising only the interacting regions. No additional convincing contacts are predicted.

    Point 4:

    Did the authors carry out SEC-MALS experiments on any IHO1 fragment lacking the coiled-coil domain? It was previously reported for Mer2 that the C-terminal region can form dimers, for example (OPTIONAL).

    We can easily do that. We have the N- and C- terminal regions lacking the coiled-coil expressed as MBP fusions and they will be analysed by SEC-MALLS.

    Point 5:

    Given that full-length REC114 is used for the IHO1 interaction studies, do the authors have any data as to the stoichiometry of the REC114FL-MEI41-127 complex? (OPTIONAL)

    We have repeatedly analysed the REC114-MEI4-IHO1 complex sample by SEC-MALLS and native mass spectrometry, but in both cases the sample is too complex to be interpreted. This is like due to the fast exchange between REC114-MEI4 2:1 and 4:2 complexes and low binding affinity of IHO1 for REC114.

    Point 6:

    Did the authors try AF2 modelling of the REC114-IHO1 interaction using orthologs from other species?

    Yes, but not extensively. We will repeat this modelling again.

    **Referees cross commenting**

    I will add cross-comments to the comments of Reviewer #2

    Firstly, the comments made by Reviewer #2 are technically correct. Firstly, reviewer #2 points out that the oligomerization states that the authors report could, in part, be artifactual the based on the his-tag purification method. This is indeed correct. However, given that none of the oligomerization states reported are per se unusual, given what is already known (including pre-prints from the Keeney and Claeys Bouuaert laboratories), I think the authors could forego this step.

    Secondly, the use of an experimental structural method, such as SAXS, would certainly add value to the paper. Also Reviewer #2 is correct in pointing out the availability of the ESRF beamlines to the authors. However, while SAXS is a useful method, I personally consider the use of mutants to validate the interactions, an even stronger piece of evidence that the AlphaFold2 interactions are correct. I must disagree somewhat with Reviewer #2 with their argument that SAXS would validate the fold. Certainly if one of the AF2 predicted structures is radically wrong, then SAXS would produce scattering data, and a subsequent distance distribution plot that is incompatible with the AF2 model. However, a partly correct AF2 model, of roughly the right shape, might still fit into a SAXS envelope.

    Reviewer #2 shares my concern on the parallel coiled-coil of IHO1, and their suggested solution is very elegant.

    In my view, due to the time constraints imposed by the partially competing work from the Keeney and Claeys Bouuaert laboratories (recently on biorxiv). I would support the authors if they chose the quickest route to publication.

    Reviewer #1 (Significance (Required)):

    General assessment: The strengths of the paper are as follows:

    1. Quality of experiments - The proteins used have been properly purified (SEC) and properly handled. The experiments are carefully carried out and controlled.
    1. Detail - The authors carry out the considerable effort of characterising protein interactions. through separation-of-function mutants. This adds to the quality of the paper, and renders the AF2 models as convincing as experimentally determined structures
    1. Conceptual advances - The most important conceptual advance is the direct binding of the N-term of IHO1 to REC114. That this is the same region as used by both TOPOVIBL and ANKRD31 points to a complex regulation.
    1. Integrity - the authors have taken great care not to "oversell" the results. The data are presented, and analysed, without hyperbole.

    Limitations - The only limitation of the paper is the lack of in vivo experiments to test their findings. However given the time and effort required, and the pressing need to publish this exciting study, this is not a problem.

    Advance: The paper provides advances from a number of directions, both conceptual and mechanistic. Mechanistically the description of the REC114-MEI14 complex is important, and in particular the observation that it can also form a higher order 4:2 structure. Likewise, while IHO1 was inferred to be a tetramer (based on work on Mer2) this was never proven formally. Most importantly, is the physical linkage between IHO1 and REC114, and that this is an interaction that is incompatible with TOPOVIBL and ANKRD31.

    Audience:

    Given the central role of meiotic recombination in eukaryotic life, any studies that shed additional light on the regulation of meiosis are suitable for a broad audience. However, this subject paper will be more specifically of interest to the meiosis community. The elegant methodology will also be of interest to structural biologists and protein biochemists.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This manuscript addresses the structure of the REC114-MEI4-IHO1 complex, which controls the essential process of programmed DSB induction by SPO11/TOPOVIBL in meiosis.

    The manuscript carefully combines biochemistry, biophysics and modelling in an integrative manner to report the architecture of the full REC114-MEI4-IHO1 complex that is not itself amenable to direct structure elucidation such as by X-ray crystallography. These are important results that will be of interest to the recombination and meiosis fields. The data are generally convincing and interpretations appear correct, so the manuscript is certainly suitable for publication. I have included some suggestions below that I believe would strengthen the manuscript and enhance our confidence in the findings. Whilst the manuscript is publishable in its current format, I believe the suggestions given below would make it into a much stronger paper.

    We thank the reviewer for his/her positive comments on our study and the suggestions below.

    I have two general suggestions:

    Point 1:

    Analyses have been performed on fusion proteins (His, His-MBP etc). we have previously observed that bulky tags such as MBP can interfere with oligomeric state through steric hindrance, and that His-tags can mediated formation of larger oligomers, seemingly through coordination of metals leached from IMAC purification. This latter point has also been observed by others

    https://www.sciencedirect.com/science/article/pii/S1047847722000946.

    Where possible, I would repeat SEC-MALS experiments using untagged proteins, or at least following incubation with EDTA to mitigate the potential for His-mediated oligomerization.

    We agree with this reviewer’s comment that expression tags can have unexpected impact of the protein behaviour.

    1. For REC114-MEI4 complex the stoichiometry is assessed by several techniques. Figure 1f,g shows analytical ultracentrifugation, which was performed on the minimal REC114226-254-MEI41-43 complex that contains no fusion tag showing that this stoichiometry is independent of fusion tags. We will nevertheless repeat the SEC-MALLS on REC114-MEI41-127 after removing the His-tag of MEI4 as suggested.
    2. For the REC114 dimer, we cannot remove the His-MBP tag since this short fragment of REC114226-254 is no stable without MBP. The dimerization of Rec114 was already reported in (Claeys Bouuaert et al., Nature 2021). The dimerization is sensitive to specific point mutations within REC114. We will however, repeat the SEC-MALLS experiment following incubation with EDTA to mitigate the potential for His-mediated oligomerization.
    3. The presented SEC-MALLS on IHO1 fragments (Figure 4b) was done on proteins without fusion tags. Reviewer 1 and 2 also agreed that additional repeats of the experiments without fusion tags are not necessary.

    The authors have relied upon mutagenesis to validate Alphafold2 models. Their results are convincing but only confirm that contacts involved in structures rather than the specific fold per se. Their finding would be greatly strengthen by collecting SEC-SAXS data and fitting models directly to the scattering data. This is extremely sensitive, so a close fit provides the best possible evidence of accuracy of the model. SAXS is affected by unstructured regions and tags, so would have to be performed using structural cores of untagged proteins rather than full-length constructs. Given the local availability of world-class SAXS beamlines at the ESRF, which is next door to the leading author's institute, it seems that the collection of SAXS data would be practical for them.

    The usage of SAXS is discussed in the specific points below. We will attempt to do SEC-SAXS on the REC114-MEI4 complex. Due to instability of REC114226-254 without MBP, SAXS cannot be done. We will also do SAXS on the IHO1 tetramer.

    My specific comments are below:

    Point 2:

    Figure 1d

    The SEC-MALS shows multiple species, with 2:1 and 4:2 representing a minority of total species present. What are the larger oligomers? Could these be an artefactual consequence of the His-tags (as described above)?

    This SEC-MALLS will be repeated without the His-tag on MEI4.

    Point 3:

    Figure 1f,g

    The AUC changes over concentration and pH are intriguing - have they tried MALS in these conditions? This would be much more informative as it would reveal the range of species present. Low concentrations could be analysed by peak position even if scattering is insufficient to provide interpretable MW fits. I would advise doing this without his tag or adding EDTA (as described above).

    We will perform this experiment as suggested.

    Point 4:

    Figure 2

    I would like to see the models validated by SAXS using minimum core untagged constructs. This could be sued to test the validity of the 2:1 model directly, and to model the 4:2 complex by multiphase analysis and/or docking together of 2:1 complexes.

    The hydrophobic LALALAII region of MEI4 is interesting and the mutagenesis data do agree with the model. However, it is important to point out that any decent model would place this hydrophobic helix in the core of the complex, and so would be disrupted by mutagenesis. Hence, the mutagenesis results confirm that the hydrophobic helix is critical for the interaction, but does not confirm that the specific alphafold model is more valid than any other model in which the helix is similarly in a core position.

    We will attempt to perform the SEC-SAXS measurements. The challenge here will be obtaining a sample that is monodisperse in solution being required for SAXS. We showed the fast exchange between the 2:1 and 4:2 oligomeric state. The AUC data indicates that the sample has a predominantly 2:1 stoichiometry at 0.2 mg/ml, pH 4.5 and 500mM NaCl. Given the small size of the complex, the signal at 0.2 mg/ml is likely to be noisy.

    Point 5:

    Figure 3

    This would also benefit from SAXS validation of the structural core. The mutagenesis here provides convincing evidence in favour of the structure as specific hydrophobics ether disrupt or have no effect, exactly as predicted. Hence, their data strongly support the dimer model. As this provides the framework for the 2:1 complex, these data make me far more confident of the previous 2:1 model in figure 2. I am wondering whether it would be better to present these data first such that the reader can see the 2:1 model being built upon these strong foundations?

    We agree with this suggestion and will present the REC114 dimerization data before the REC114-MEI4 complex. However, REC114226-254 is not stable without the MBP tag so is not suitable for SAXS analysis.

    Point 6:

    Figure 4

    The MALS data convincingly show formation of a tetramer. How do we know that it is parallel? The truncation supports this but coiled-coils do sometimes form alternative structures when truncated (e.g. anti-parallel can become parallel when sequence is removed), and alphafold seems to have a tendency of predicting parallel coiled-coils even when the true structure of anti-parallel (informal observation by us and others). A simple test would be to make a tethered dimer of 110-240, with a short flexible linker between two copies of the same sequence - if parallel it should form a tetramer of double the length, if anti-parallel it should form a dimer of the same length - determinable by MALS (and SAXS).

    To address this point we will perform this experiment as suggested by Reviewer 2. We will produce a tethered dimer of IHO1 110-240, connected by a short linker and determine its MW by MALS (and possibly SAXS). We also performed ESMfold modelling (Reviewer 1, Point 1), resulting in the same model. As the IHO1 tetramer is likely suitable for SAXS analysis, we will also perform SAXS on it.

    Point 7:

    Figures 5/6

    The interaction is clear albeit low affinity (but within the biologically interesting range). It would be nice to obtain MALS (using superose 6) data showing the stoichiometry of the complex - are the data too noisy to be interpretable owing to dissociation? The alpahfold model and mutagenesis data strongly support the conclusion that the IHO1 N-term binds to the PH domain, as presented.

    We have repeatedly analysed the REC114-MEI4-IHO1 complex sample by SEC-MALLS (on Superose 6) and native mass spectrometry, but in both cases the sample is too complex to be interpreted. This is likely due to the fast exchange between REC114-MEI4 2:1 and 4:2 complexes and low binding affinity of IHO1 for REC114.

    **Referees cross commenting**

    Just to clarify a couple of points regarding consultation comments from reviewer 1:

    The suggestion regarding tags was mostly directed to the cases in which MALS data are noisy, with multiple oligomeric species (such as figure 1d). In these cases, i wondered whether the large MW species may be artefactual and could be resolved by removal of the tags. In cases where oligomers agree with those reported by other labs, I agree that there's no need to explore these further.

    In terms of SAXS, I agree that fitting models into envelopes will not distinguish between similar folds. However, fitting models directly to raw scattering data is extremely sensitive and I have never seen good fits with low chi2 values for incorrect models (even when very similar in overall shape to the correct structure).

    Reviewer #2 (Significance (Required)):

    The manuscript carefully combines biochemistry, biophysics and modelling in an integrative manner to report the architecture of the full REC114-MEI4-IHO1 complex that is not itself amenable to direct structure elucidation such as by X-ray crystallography. These are important results that will be of interest to the recombination and meiosis fields. The data are generally convincing and interpretations appear correct, so the manuscript is certainly suitable for publication. I have included some suggestions below that I believe would strengthen the manuscript and enhance our confidence in the findings. Whilst the manuscript is publishable in its current format, I believe the suggestions given below would make it into a much stronger paper.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Laroussi et al used Alphafold models to predict the assembly of REC114-MEI4-IHO1 complex, and verified the structure using different biochemical experiments. Both Alphafold predictions and experiment data are convincing for the overall protein complex assembly. Importantly, they identified a motif on IHO1 that share the same binding site on REC114 with TOPOVIBL and ANKRD31, suggesting that REC114 acts as a regulatory base coordinating different binding partners during meiosis progression. Overall, I believe this is a nice biochemistry paper, but lacks insights into the biology (I believe those in vivo data is beyond the scope of this paper), at least more discussions are needed in terms of these findings.

    We thank the reviewer for the supportive comments on our manuscript and its evaluation. We agree with the reviewer, that including in vivo data, that might provide further biological insights, would be useful. However, there is currently no good cellular model for meiotic recombination in mouse and thus our structure-based mutations will need to be tested in transgenic mice. Such data will take a long time to obtain and would delay the publication these in-vitro results that already provide novel insight into the REC114-MEI4-IHO1 complex architecture. We will, nevertheless, as suggested, strengthen the discussion of the biological implications of our findings.

    Some minor points:

    Point 1:

    Any data showing MEI4 forms a dimer on its own?

    As mentioned in the manuscript, full-length MEI4 is difficult to produce in bacteria or insect cells. Thus, we worked with the N-terminal fragment which in absence of REC114 is nor very stable. We will perform SEC-MALLS to assess its oligomeric state. Alphafold suggests dimeric arrangement of MEI4, but only with low confidence.

    Point 2:

    In Fig2 and Sup Fig4, HisMBP-MEI4, you see more MBP than the fusion protein, especially more obvious in the mutants. What's your explanation?

    The N-terminus of MEI4 is well produced when co-expressed with REC114. For the pull-down experiments in Figure 2 we expressed it as His-MBP fusion in absence of REC114. In this situation, there is a degradation between MBP and MEI4. We find this very often for proteins that not very stable, which is the case of MEI4 without REC114. This is the best way we could produce at least some MEI4 in absence of REC114. The MBP protein could probably be removed by other chromatography techniques, but we think that for the purpose of the pull-down its presence is not interfering with the REC114-MEI4 binding.

    Point 3:

    TOPOVIBL and ANKRD31, I am curious if you have looked at the conserved residues on these motifs.

    We show a strong conservation of the IHO1 among vertebrates (Fig. 6c). We will further analyse the sequence conservation in more distant species.

    Point 4:

    Reference needed when stating that IHO1 was not interacting with REC114 in previous biochemical assay in the discussion part.

    This will be corrected

    Point 5:

    Also, have you run AlphaFold that gives multiple models? Sometimes, with short motifs, 1 or 2 models of several models give good confidence for the interaction.

    Using in-house Alphafold installation producing 25 models did not reveal better models.

    Reviewer #3 (Significance (Required)):

    While most of the interactions between REC114 and MEI4 or IHO1 were established with Y2H or other biochemical assays before. This paper used the AlphaFold, and finally verified the findings with biochemical experiments, which helps to establish the exact motifs/residues involved in the interaction. For example, the MEI4-REC114 interfaces are novel, more interestingly, the IHO1 shares the same interface with ANKRD31 and TOPOVIBL. Thus, this finding of REC114-MEI4-IHO1 complex assembly would be interesting to people with different working areas. I would like to see more studies on the coordination IHO1 with ANKRD31 or TOPOVIBL in the future.

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    Referee #3

    Evidence, reproducibility and clarity

    Laroussi et al used Alphafold models to predict the assembly of REC114-MEI4-IHO1 complex, and verified the structure using different biochemical experiments. Both Alphafold predictions and experiment data are convincing for the overall protein complex assembly. Importantly, they identified a motif on IHO1 that share the same binding site on REC114 with TOPOVIBL and ANKRD31, suggesting that REC114 acts as a regulatory base coordinating different binding partners during meiosis progression. Overall, I believe this is a nice biochemistry paper, but lacks insights into the biology (I believe those in vivo data is beyond the scope of this paper), at least more discussions are needed in terms of these findings.

    Some minor points:

    Any data showing MEI4 forms a dimer on its own? In Fig2 and Sup Fig4, HisMBP-MEI4, you see more MBP than the fusion protein, especially more obvious in the mutants. What's your explanation? Nice finding on the IHO1 N terminus, which shares the same binding sites on REC114 with TOPOVIBL and ANKRD31, I am curious if you have looked at the conserved residues on these motifs. Reference needed when stating that IHO1 was not interacting with REC114 in previous biochemical assay in the discussion part. Also, have you run AlphaFold that gives multiple models? Sometimes, with short motifs, 1 or 2 models of several models give good confidence for the interaction.

    Significance

    While most of the interactions between REC114 and MEI4 or IHO1 were established with Y2H or other biochemical assays before. This paper used the AlphaFold, and finally verified the findings with biochemical experiments, which helps to establish the exact motifs/residues involved in the interaction. For example, the MEI4-REC114 interfaces are novel, more interestingly, the IHO1 shares the same interface with ANKRD31 and TOPOVIBL. Thus, this finding of REC114-MEI4-IHO1 complex assembly would be interesting to people with different working areas. I would like to see more studies on the coordination IHO1 with ANKRD31 or TOPOVIBL in the future.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript addresses the structure of the REC114-MEI4-IHO1 complex, which controls the essential process of programmed DSB induction by SPO11/TOPOVIBL in meiosis.

    The manuscript carefully combines biochemistry, biophysics and modelling in an integrative manner to report the architecture of the full REC114-MEI4-IHO1 complex that is not itself amenable to direct structure elucidation such as by X-ray crystallography. These are important results that will be of interest to the recombination and meiosis fields. The data are generally convincing and interpretations appear correct, so the manuscript is certainly suitable for publication. I have included some suggestions below that I believe would strengthen the manuscript and enhance our confidence in the findings. Whilst the manuscript is publishable in its current format, I believe the suggestions given below would make it into a much stronger paper.

    I have two general suggestions:

    1. Analyses have been performed on fusion proteins (His, His-MBP etc). we have previously observed that bulky tags such as MBP can interfere with oligomeric state through steric hindrance, and that His-tags can mediated formation of larger oligomers, seemingly through coordination of metals leached from IMAC purification. This latter point has also been observed by others https://www.sciencedirect.com/science/article/pii/S1047847722000946. Where possible, I would repeat SEC-MALS experiments using untagged proteins, or at least following incubation with EDTA to mitigate the potential for His-mediated oligomerisation.
    2. The authors have relied upon mutagenesis to validate Alphafold2 models. Their results are convincing but only confirm that contacts involved in structures rather than the specific fold per se. Their finding would be greatly strengthen by collecting SEC-SAXS data and fitting models directly to the scattering data. This is extremely sensitive, so a close fit provides the best possible evidence of accuracy of the model. SAXS is affected by unstructured regions and tags, so would have to be performed using structural cores of untagged proteins rather than full-length constructs. Given the local availability of world-class SAXS beamlines at the ESRF, which is next door to the leading author's institute, it seems that the collection of SAXS data would be practical for them.

    My specific comments are below:

    Figure 1d The SEC-MALS shows multiple species, with 2:1 and 4:2 representing a minority of total species present. What are the larger oligomers? Could these be an artefactual consequence of the His-tags (as described above)?

    Figure 1f,g The AUC changes over concentration and pH are intriguing - have they tried MALS in these conditions? This would be much more informative as it would reveal the range of species present. Low concentrations could be analysed by peak position even if scattering is insufficient to provide interpretable MW fits. I would advise doing this without his tag or adding EDTA (as described above).

    Figure 2 I would like to see the models validated by SAXS using minimum core untagged constructs. This could be sued to test the validity of the 2:1 model directly, and to model the 4:2 complex by multiphase analysis and/or docking together of 2:1 complexes. The hydrophobic LALALAII region of MEI4 is interesting and the mutagenesis data do agree with the model. However, it is important to point out that any decent model would place this hydrophobic helix in the core of the complex, and so would be disrupted by mutagenesis. Hence, the mutagenesis results confirm that the hydrophobic helix is critical for the interaction, but does not confirm that the specific alphafold model is more valid than any other model in which the helix is similarly in a core position.

    Figure 3 This would also benefit from SAXS validation of the structural core. The mutagenesis here provides convincing evidence in favour of the structure as specific hydrophobics ether disrupt or have no effect, exactly as predicted. Hence, their data strongly support the dimer model. As this provides the framework for the 2:1 complex, these data make me far more confident of the previous 2:1 model in figure 2. I am wondering whether it would be better to present these data first such that the reader can see the 2:1 model being built upon these strong foundations?

    Figure 4 The MALS data convincingly show formation of a tetramer. How do we know that it is parallel? The truncation supports this but coiled-coils do sometimes form alternative structures when truncated (e.g. anti-parallel can become parallel when sequence is removed), and alphafold seems to have a tendency of predicting parallel coiled-coils even when the true structure of anti-parallel (informal observation by us and others). A simple test would be to make a tethered dimer of 110-240, with a short flexible linker between two copies of the same sequence - if parallel it should form a tetramer of double the length, if anti-parallel it should form a dimer of the same length - determinable by MALS (and SAXS).

    Figures 5/6 The interaction is clear albeit low affinity (but within the biologically interesting range). It would be nice to obtain MALS (using superose 6) data showing the stoichiometry of the complex - are the data too noisy to be interpretable owing to dissociation? The alpahfold model and mutagenesis data strongly support the conclusion that the IHO1 N-term binds to the PH domain, as presented.

    Referees cross commenting

    Just to clarify a couple of points regarding consultation comments from reviewer 1:

    The suggestion regarding tags was mostly directed to the cases in which MALS data are noisy, with multiple oligomeric species (such as figure 1d). In these cases, i wondered whether the large MW species may be artefactual and could be resolved by removal of the tags. In cases where oligomers agree with those reported by other labs, I agree that there's no need to explore these further.

    In terms of SAXS, I agree that fitting models into envelopes will not distinguish between similar folds. However, fitting models directly to raw scattering data is extremely sensitive and I have never seen good fits with low chi2 values for incorrect models (even when very similar in overall shape to the correct structure).

    Significance

    The manuscript carefully combines biochemistry, biophysics and modelling in an integrative manner to report the architecture of the full REC114-MEI4-IHO1 complex that is not itself amenable to direct structure elucidation such as by X-ray crystallography. These are important results that will be of interest to the recombination and meiosis fields. The data are generally convincing and interpretations appear correct, so the manuscript is certainly suitable for publication. I have included some suggestions below that I believe would strengthen the manuscript and enhance our confidence in the findings. Whilst the manuscript is publishable in its current format, I believe the suggestions given below would make it into a much stronger paper.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The formation of meiotic double-stranded DNA breaks is the starting point of meiotic recombination. DNA breaks are made by the topoisomerase-like SPO11, which interacts with a number of regulatory factors including REC114, MEI4 and IHO1. Despite the key role this process has in the continuation, and genetic variation, or eukaryotic life, there is very little known about how this process is regulated. Laroussi et al make use of biochemical, biophysical and structural biological approaches to extensively characterise the REC114-MEI4-IHO1 complex.

    This is an outstanding biochemical paper. The experiments are well planned and beautifully executed. The protein purifications used are very clean, and the figures well presented. Importantly Laroussi et. al describe, and carefully characterise through point mutational analysis, the direct physical interaction between IHO1 and REC114-MEI4. This is an interaction that has, at least in yeast, previously been suggested to be driven by liquid-liquid separation. The careful and convincing work presented here represents an important paradigm-shift for the field.

    I am fully supportive of publication of this excellent and important study.

    Major comments:

    My only major concern is regarding Figure 4, and specifically the AF2 model of the coiled-coil tetramer of IHO1. Given the ease with which MSAs of coiled-coils can become "contaminated" with non-orthologous sequences, I would urge some caution with this model. This is especially since the yeast ortholog of IHO1, Mer2, has been previously reported to be an anti-parallel tetramer (albeit, not very well supported by the data). The authors have several choices here. 1) They could simply reduce the visibility of the IHO1 tetramer model, and indicate caution in the parallel tetramer model. 2) They could consider using a structure prediction algorithm that doesn't use an MSA (e.g. ESMFold). 3) They could try to obtain experimental evidence for a parallel coiled-coil tetramer, e.g. through EM, SAXS or FRET approaches. I would like to make it crystal clear, however, that I would be very supportive of approach 1) or 2). An experimental approach is not necessary.

    Assuming the authors don't take a wet-lab approach, this shouldn't take more than a couple of weeks.

    Minor comments:

    1. The observation that REC114 and MEI4 can also form a 4:2 complex is very interesting and potentially important. Did the authors also try to model this higher order complex in AF2?
    2. Similarly to above, what does the prediction of the full-length REC114:MEI4 2:1 complex look like? Presumably the predicted interaction regions align well with experimental data, but it would be interesting to see (and easy to run).
    3. Did the authors carry out SEC-MALS experiments on any IHO1 fragment lacking the coiled-coil domain? It was previously reported for Mer2 that the C-terminal region can form dimers, for example (OPTIONAL).
    4. Given that full-length REC114 is used for the IHO1 interaction studies, do the authors have any data as to the stoichiometry of the REC114FL-MEI41-127 complex? (OPTIONAL)
    5. Did the authors try AF2 modelling of the REC114-IHO1 interaction using orthologs from other species?

    Referees cross commenting

    I will add cross-comments to the comments of Reviewer #2

    Firstly, the comments made by Reviewer #2 are technically correct. Firstly, reviewer #2 points out that the oligomerization states that the authors report could, in part, be artifactual the based on the his-tag purification method. This is indeed correct. However, given that none of the oligomerization states reported are per se unusual, given what is already known (including pre-prints from the Keeney and Claeys Bouuaert laboratories), I think the authors could forego this step.

    Secondly, the use of an experimental structural method, such as SAXS, would certainly add value to the paper. Also Reviewer #2 is correct in pointing out the availability of the ESRF beamlines to the authors. However, while SAXS is a useful method, I personally consider the use of mutants to validate the interactions, an even stronger piece of evidence that the AlphaFold2 interactions are correct. I must disagree somewhat with Reviewer #2 with their argument that SAXS would validate the fold. Certainly if one of the AF2 predicted structures is radically wrong, then SAXS would produce scattering data, and a subsequent distance distribution plot that is incompatible with the AF2 model. However, a partly correct AF2 model, of roughly the right shape, might still fit into a SAXS envelope.

    Reviewer #2 shares my concern on the parallel coiled-coil of IHO1, and their suggested solution is very elegant.

    In my view, due to the time constraints imposed by the partially competing work from the the Keeney and Claeys Bouuaert laboratories (recently on biorxiv). I would support the authors if they chose the quickest route to publication.

    Significance

    General assessment: The strengths of the paper are as follows:

    1. Quality of experiments - The proteins used have been properly purified (SEC) and properly handled. The experiments are carefully carried out and controlled.
    2. Detail - The authors carry out the considerable effort of characterising protein interactions. through separation-of-function mutants. This adds to the quality of the paper, and renders the AF2 models as convincing as experimentally determined structures
    3. Conceptual advances - The most important conceptual advance is the direct binding of the N-term of IHO1 to REC114. That this is the same region as used by both TOPOVIBL and ANKRD31 points to a complex regulation.
    4. Integrity - the authors have taken great care not to "oversell" the results. The data are presented, and analysed, without hyperbole.

    Limitations - The only limitation of the paper is the lack of in vivo experiments to test their findings. However given the time and effort required, and the pressing need to publish this exciting study, this is not a problem.

    Advance: The paper provides advances from a number of directions, both conceptual and mechanistic. Mechanistically the description of the REC114-MEI14 complex is important, and in particular the observation that it can also form a higher order 4:2 structure. Likewise, while IHO1 was inferred to be a tetramer (based on work on Mer2) this was never proven formally. Most importantly, is the physical linkage between IHO1 and REC114, and that this is an interaction that is incompatible with TOPOVIBL and ANKRD31.

    Audience: Given the central role of meiotic recombination in eukaryotic life, any studies that shed additional light on the regulation of meiosis are suitable for a broad audience. However, this subject paper will be more specifically of interest to the meiosis community. The elegant methodology will also be of interest to structural biologists and protein biochemists.

  6. Version published to 10.1101/2023.01.11.523614v1 on bioRxiv