LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex

  1. Marella D Canny
  2. Michael P Latham

  • Curated by eLife

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    Evaluation Summary:

    This study on the Mre11 and Rad50 proteins is of interest to biologists studying DNA repair. Advances in the understanding of Mre11-Rad50 mechanism, for instance how structural states are linked to DNA end detection and DNA processing as addressed in this study, are of central importance to research on genome stability and DNA repair, with implications in human disease such as cancer and immune disorders. Enzymatically, RAD50 is an ATPase and MRE11 is a nuclease with both exo- and endo-nuclease activities. How all these functions are catalyzed by the complex remains unresolved. This study identifies three conformations of ATP-bound P. furiosus Mre11-Rad50 complex: open, partially open, and closed. The work would benefit from further experiments to clarify the functional difference between the partially open and open conformations.

    (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 Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double-strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional ‘partially open’ conformation using luminescence resonance energy transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with small-angle X-ray scattering data, which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.

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  1. eLife

    Author Response:

    Reviewer #1 (Public Review):

    This manuscript is a follow-up of an earlier manuscript using the LRET technology, but extends the study by identifying a new "open" state and using experimental distance constraints to provide molecular models of the different states. All in all, the manuscript is well written, the experiments are described in sufficient details and experiments are done to high quality with the appropriate controls. The data corroborate the partially open state as published early, but extend the study to a second, open state. It is very good to see that the observed states are not only present in the catalytic head but the authors also use the full-length protein and find similar states. However, in the present manuscript, I find the conceptual advance with respect to the mechanism of MR somewhat limited. The authors curiously do not include any DNA in their structural studies, so the observed states are only relevant for the free MR complex, but not the complex "in action" bound to DNA where quite different conformations might occur. As one consequence, the structurally proposed states do not directly correlate with the functional nuclease states that are necessarily bound to DNA. Perhaps as a consequence, in the author's model, Rad50 is merely a gate-keeper for Mre11, but this is not the case as recent structural work shows that Rad50 forms a joint DNA binding surface with Mre11. Likewise, biochemical studies are done with physiologically unclear/less relevant 3' exonuclease activity only, but not with the physiological important 5' endonuclease activity. In my opinion, it is important for a publication in a journal with the scope of eLife and addressed to a broad audience to provide structural analysis in the presence of DNA and validate the structures using the endonuclease activity.

    We thank the reviewer for these comments.

    Specific recommendations:

    1. Instead of using the physiological unclear exo activity, I suggest to use the more relevant endonuclease activity to validate the mutants.

    We now include plate- and gel-based endonuclease activity assays, using a variety of DNA substrates, for all of the validation mutants. We have expanded Fig. 3 and included a new Supplemental Fig. S4 to show this data. We have expanded the Results section of the modified manuscript to present and discuss these findings.

    1. Since the authors mutated one side of newly identified/proposed salt-bridges, I also suggest to test whether a charge reversal on both sides of the salt bridge rescues the phenoptype. I find this important because MR has quite many conformations, and mutating a single residue might not unambiguously validate the proposed conformation, a rescue by a charge reversed salt bridge is much stronger.

    We thank the Reviewer for this suggested experiment, and we tried to do it. Although we were successful in generating each of the charge reversal mutations in full-length Rad50, all of the mutants unfortunately had issues with either expression or purification. For example, the 6x His-tag for several of the new Rad50 mutants was not accessible to the TEV protease for cleavage indicating that the mutated proteins were mis-folded (the His-tag of the WT full-length Rad50 is readily cleaved off by TEV). As such, we did not feel confident using these proteins in subsequent MR activity assays.

    1. Since all LRET experiments are done without DNA, the authors do not capture relevant DNA processing states and comparison of structural (w/o DNA) and biochemical data (w/ DNA) is not really justified, in my opinion. Also, they might miss critical conformations. Is there a technical reason for not including DNA in the LRET studies?

    We have collected LRET data on ATP-bound MRNBD in the presence of a hairpin DNA or a ssDNA as substrates. We still observe three states in the presence of both DNAs; however, the open conformation appears to be slightly more compact (i.e., closer distance between Rad50NBD protomers) in the presence of ssDNA. As described above, we have added to the Results section of the modified manuscript and included a new figure (Fig. 4) describing these data.

    1. If the authors want to claim processive movement coupled to partially open/open state interchanges, they should provide experimental evidence. Where would the energy come from for such a movement, this is not clear from the model?

    On the surface, ATP hydrolysis by Rad50 would seem to be the perfect source of energy for the conformational changes that drive the sequential and/or processive nuclease functions of the MR complex. However, the D313K mutant is not as good at ATP hydrolysis as the wild type enzyme (Fig. 3E), and the data in Fig. 3 and Supplemental Fig. S4 clearly demonstrate that D313K is by far the best nuclease. If the free energy for the movement does not come from ATP hydrolysis, where else could it come? Richardson and co-workers measured a release of -5.3 kcal mol-1 (-22.17 kJ mol-1) of free energy for the hydrolysis of a DNA phosphodiester bond (Dickson, K.S. et al. 2000 J. Biol. Chem. 275:15828–15831). Thus, the free energy released from the Mre11 nuclease activity could be the driving force for the conformational changes we propose. We have made this point in the Discussion of the revised manuscript.

    1. The SAXS data for the "open" state do not validate the model, in my opinion. Experimental data and model are not inconsistent, but the curve looks to me as if the open state is perhaps much more flexible (i.e. an ensemble) or extended? Please comment.

    We agree with the Reviewer on this point. We have updated Fig. 5A (original Fig. 4) to include the two-state fits to the experimental SAXS data. Although the multi-state fit to the apo MR SAXS data is better than any of the single model fits (2 = 1.05 vs. 1.26, respectively), the 2 is still larger than the multi-state fits to the ATP-bound MR SAXS data. Thus, an additional unobserved conformation (perhaps the so-called “extended”) might be present in solution for apo MRNBD. We have added a sentence to the revised manuscript with this point.

    To explore the possibility that the previously described “extended” structure might be contributing to the SAXS data, we built a model of the extended conformation of Pf MRNBD based on the Tm MRNBD structure (PDB: 3QG5) and used Rosetta to connect the coiled-coils and add the linker to the Mre11 HLH. When this model was used in the FoXS calculations for the apo SAXS data, the 2 was 4.77 (versus 2 of 1.26 for the “open” model). The MultiFoXS two-state fit gave 90% open + 10% closed (2 of 1.04), whereas the three-state fit gave 65% open + 20% extended + 15% part open (2 of 0.84). Thus, there is some improvement when using the extended model, but since that model is not measurable in our LRET experiments and we are unsure of its validity as we have modeled it for Pf MR, we have chosen to omit it from the analysis.

    1. Distance errors for the full complex are much smaller than those for the catalytic module only (Fig. 1d). Does that mean that the full complex is more rigid, please comment?

    From looking at the data presented in Fig. 1D, it is logical to suggest that the full-length complex may be more rigid or better defined by the LRET data. However, we note that there are nearly as many distance errors which are similar between MRNBD and MR as there are MR errors less than MRNBD. And although many are not identical, most are of a similar magnitude. Because of this, we do not think the variations in LRET errors are systematic (i.e., related to a more rigid full-length complex).

  2. Version published to 10.7554/elife.69579 on eLife
  3. eLife

    Evaluation Summary:

    This study on the Mre11 and Rad50 proteins is of interest to biologists studying DNA repair. Advances in the understanding of Mre11-Rad50 mechanism, for instance how structural states are linked to DNA end detection and DNA processing as addressed in this study, are of central importance to research on genome stability and DNA repair, with implications in human disease such as cancer and immune disorders. Enzymatically, RAD50 is an ATPase and MRE11 is a nuclease with both exo- and endo-nuclease activities. How all these functions are catalyzed by the complex remains unresolved. This study identifies three conformations of ATP-bound P. furiosus Mre11-Rad50 complex: open, partially open, and closed. The work would benefit from further experiments to clarify the functional difference between the partially open and open conformations.

    (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.)

  4. eLife

    Reviewer #1 (Public Review):

    This manuscript is a follow-up of an earlier manuscript using the LRET technology, but extends the study by identifying a new "open" state and using experimental distance constraints to provide molecular models of the different states. All in all, the manuscript is well written, the experiments are described in sufficient details and experiments are done to high quality with the appropriate controls. The data corroborate the partially open state as published early, but extend the study to a second, open state. It is very good to see that the observed states are not only present in the catalytic head but the authors also use the full-length protein and find similar states. However, in the present manuscript, I find the conceptual advance with respect to the mechanism of MR somewhat limited. The authors curiously do not include any DNA in their structural studies, so the observed states are only relevant for the free MR complex, but not the complex "in action" bound to DNA where quite different conformations might occur. As one consequence, the structurally proposed states do not directly correlate with the functional nuclease states that are necessarily bound to DNA. Perhaps as a consequence, in the author's model, Rad50 is merely a gate-keeper for Mre11, but this is not the case as recent structural work shows that Rad50 forms a joint DNA binding surface with Mre11. Likewise, biochemical studies are done with physiologically unclear/less relevant 3' exonuclease activity only, but not with the physiological important 5' endonuclease activity. In my opinion, it is important for a publication in a journal with the scope of eLife and addressed to a broad audience to provide structural analysis in the presence of DNA and validate the structures using the endonuclease activity.

    Specific recommendations:

    1. Instead of using the physiological unclear exo activity, I suggest to use the more relevant endonuclease activity to validate the mutants.

    2. Since the authors mutated one side of newly identified/proposed salt-bridges, I also suggest to test whether a charge reversal on both sides of the salt bridge rescues the phenoptype. I find this important because MR has quite many conformations, and mutating a single residue might not unambiguously validate the proposed conformation, a rescue by a charge reversed salt bridge is much stronger.

    3. Since all LRET experiments are done without DNA, the authors do not capture relevant DNA processing states and comparison of structural (w/o DNA) and biochemical data (w/ DNA) is not really justified, in my opinion. Also, they might miss critical conformations. Is there a technical reason for not including DNA in the LRET studies?

    4. If the authors want to claim processive movement coupled to partially open/open state interchanges, they should provide experimental evidence. Where would the energy come from for such a movement, this is not clear from the model?

    5. The SAXS data for the "open" state do not validate the model, in my opinion. Experimental data and model are not inconsistent, but the curve looks to me as if the open state is perhaps much more flexible (i.e. an ensemble) or extended? Please comment.

    6. Distance errors for the full complex are much smaller than those for the catalytic module only (Fig. 1d). Does that mean that the full complex is more rigid, please comment?

  5. eLife

    Reviewer #2 (Public Review):

    This study investigates the ATP-dependent global conformational changes of the P. furiosus Mre11-Rad50 (MR) complex using Luminescent Resonance Energy Transfer (LRET), the molecular docking program HADDOCK, and Small-angle X-ray scattering (SAXS). LRET use a luminescent lanthanide donor and a fluorophore acceptor pair, and provides a measurement of the distance between donor and acceptor molecules calculated from the donor and fluorophore lifetimes. Authors engineered a set of LRET probes throughout the NBD of Pf Rad50 via single-cysteine mutations. The network of distances provided three conformations of the ATP-bound Pf MR(NBD) complex in solution: open, partially open, and closed, which were modelled by HADDOCK. These models were tested using site-directed mutagenesis by disrupting interacting domains and evaluated using SAXS. The work also investigates how these conformational changes induced by ATP-binding affect the exonuclease activity of Mre11 dimer. Overall, this is a solid and very well presented study. However, this work also presents some concerns. It is unclear how SAXS modelled data obtained from HADDOCK models corroborate the three conformations proposed by the authors. Additionally, simulation of SAXS data from the open conformation - obtained under ATP-binding saturating conditions - matches well with SAXS experimental curves of the apo-MR complex. This is unclear and should also be addressed. Experiments including the non-hydrolysable ATP analogue AMP-PNP and in the absence of nucleotide should bring light on the question on the coupling between ATP binding and hydrolysis with MR complex conformational changes.

  6. eLife

    Reviewer #3 (Public Review):

    In this study, first, multiple LRET pairs were designed, purified and analyzed, which revealed three distinct groups. The measured distances between the respective pairs were then given as constraints for molecular modeling. The authors then modelled three distinct conformations: closed (nuclease-inactive), partially open and open (latter two being active). Based on the models, several mutants that disrupt the equilibrium between the conformations were designed and biochemically analyzed. This an interesting study that is focused on an important problem. Overall, the study appears to be well done, described and presented. The functional difference between the partially open and open conformations remains however unclear.

  7. Version published to 10.1101/2021.08.04.455035v1 on bioRxiv