1. Author Response:

    Reviewer #1 (Public Review):

    ...The limitations of this study, although minor for the conclusion drawn by this study, are (1) CTD deletion generally confers modest cellular phenotypes compare to DBD deletion and is fully resistant to MMC and cisplatin.It remains unknown why CTD deletion elicits less impact despite its strong impairments in ligand-induced conformational changes...

    This is an intriguing aspect of our results. We have added discussion of this aspect (Page 22, line 397). We agree that the relationship between our various assays is not simple. This points to complex functions of BRCA2 that are only beginning to be revealed and understood. Because the assays are very different, involving cells with all interacting components available vs. individual isolated proteins, we cannot at this point directly relate the protein structural changes to precise biological functions. However, we do note that the ΔDBDΔCTD and ΔDBD cells lines behave similarly and are both sensitive to MMC and Cis Pt. Purified ΔCTD is deficient in structural response, while the similar variant in cells is not sensitive the DNA crosslinking agent. All deletion variant proteins are defective in response to ssDNA while again the ΔCTD in cells is not overly sensitive to DNA crosslinking agents. Thus, we observe structural transition defects in all c-terminal deletion mutants while only those variants missing the DBD are sensitive in cell assays probing the function of BRCA2 in DNA cross link repair. We and others (Le et al., 2020) observe complex interactions between different parts of BRCA2 with itself (inter= multimerization and intra= conformation molecularly), that can be modulated by binding partners, including DSS1. Although important and interesting, including DSS1 interaction is outside of the scope of our current study. We continue to investigate the structural response of BRCA2, these and other variants, to additional binding partners and hope that these studies will eventually contribute to a clearer connection between protein conformational changes and biological functions.

    ...and (2) the molecular behaviours of BRCA2 in mouse ES cells might not be directly translated to these in human somatic cells.

    It is of course possible that some aspects of BRCA2 behavior in human somatic cells and mouse ES cells differ. At least for diffusive behavior we have shown in our previous work (Reuter et. al., J. Cell Biol. 2014, in manuscript reference list) that BRCA2 behaves the same in HeLa cells as in mouse ES cells.

    Specific comments:

    (1) Thank you, does not need response.

    (2) …Surprisingly, they found that the deletion of DBD or CTD did not drastically affect foci formation, albeit slightly less efficient compared to full-length BRCA2. While the results and trends look promising, the number of samples analysed is somewhat limited (i.e., two or three technical replicates, rather than biological replicates) and the statistic tests have not been conducted.

    Statistical tests are in the source data files as indicated in the figure legend.

    For all cellular assays independent experiments have been performed at different days with cells at different passage numbers. Within all independent experiments we have included technical replicates (cell survivals: 2 or 3 wells; HR assays: 2 wells; microscopy experiments: at least 3 field of views per condition). To further support our observations, we have generated the single ΔDBD and ΔCTD cell lines and the cell line lacking both domains (ΔDBDΔCTD). Although in the original version of the manuscript we have included the results of statistical tests in the Source Data files, we have included additional information in the text and figure legend where appropriate).

    (3) Thank you, does not need response.

    (4) A silver stained gel of the proteins used has been added to supplementary figures (Figure 4 – supplement 4) and this issue is addressed in essential revision number 3.

    Reviewer #2 (Public Review):

    We apologize for not having included sufficient data replicates in our original submission. We have performed additional replicates of the cell survival experiments and foci counting experiments in figures 1D-F and 2B-C. The figures and legends have been revised to include the additional data. Statistical test results are included in the figure legends, main text and Source Data Files accompanied with Figures 1-3. The overall results are the same and the conclusions from them do not change. We agree that this strengthens our work and was a necessary improvement.

    Concerning identifying specific functions for the BRCA2 c-terminal domains, we agree this is a fascinating and important area to investigate. Our work addressed the role of these domains in protein behavior that we have previously described placing the suggested (though unspecified) assays out of the current scope. Indeed, the effect of additional BRCA2 interactors, including DSS1, is also of interest and part of our ongoing/future work. We trust the reviewer and others will be interested to follow this as it develops. As a general approach to effective scientific communication, we find the most value to the scientific community is achieved in timely reporting clearly understandable experiments and results so that others can evaluate and build on them. Concerning multimerization vs phase separation, this is also an interesting topic. Our current experimental work does not provide any data to distinguish these phenomena. We also believe that “phase separation” is a bit of a hype and is often misused or ill defined (see for example; “Evaluating phase separation in live cells: diagnosis, caveats, and functional consequences.” McSwiggen DT, Mir M, Darzacq X, Tjian R. Genes Dev. 2019 Dec 1). Because we do not provide any quantitative biophysical data on this topic we prefer not to contribute to the qualitative discussion. We trust this important distinction will be addressed by ongoing appropriate biophysical and theoretical work.

    Reviewer #3 (Public Review):

    1. Concerning comparison of cell sensitivity of our BRCA2 deletion variants and “completely non-functional BRCA2 allele”; This is indeed a good idea and would be interesting to pursue. However, we note that this would require making specific mutations from the human protein in mouse ES cell lines and thus require possibly substantial work determining if they mutations behave the same of differently. Although cell lines expressing (patient derived and other) BRCA2 truncations and deletion variants are described as “completely non-functional” this description does not entirely make sense to us. Cells lacking an essential protein (BRCA2) are, we assume by definition, dead or dying. That some tumor derived cell lines survive with apparently severe BRCA2 defects may attest to their other genetic alterations. A “clean” comparison in mouse ES cells does not exist. For our survivals in mouse ES cells we used a RAD54 deletion cell line as a well characterized comparison as HR defective in response to ionizing radiation. Though not perfect this at least provides a means of comparing sensitivity (Figure 1C) where the two BRCA2 deletion variants are even more sensitive.

    2. Concerning mechanistic importance (insight) from SPT analysis. The function of BRCA2 and other DNA repair proteins logically require them to become localized/temporary immobile at sites of damage where they need to exercise biochemical activities. This is seen as a high local concentration in “foci”. In order to accumulate in this way or simply become localized to do its work a protein has to change its diffusive behavior, either more of the protein moves to / through a place or more of it stay immobile for a longer time. This is what we can quantify by SPT. Here we show that, perhaps contrary to expectations, the in vitro defined DNA binding domain is not required for this immobilization or change in diffusive behavior. This lack of effect could be described as a negative result, however just as important to communicate and valid as if we had detected an effect. We discussed the mechanistic implication and motivation for SPT study of BRCA2 in a previous publication (Reuter et al, JCB, 2014 in the reference list of our current manuscript). There we also explain how the number of proteins that change mobility and the magnitude of their change in mobility is consistent with the expected amount of damage inflicted.

    General comments:

    Our statement that BRCA2 c-terminal domains have a role beyond (meaning after) delivering RAD51 is based on the observation that RAD51 still forms high local concentration at sites of damage (foci). We agree that the cell biology observations do not directly test DNA binding or RAD51 filament formation. Addressing these specific biochemical activities in vivo is challenging. On the other hand we have to admit that in vitro biochemistry (which we find essential to understanding) can show what is possible and not necessarily what is actually happening in cells. Our cell experiments are at one level aimed to define what we can quantitatively in the authentic molecular environment where all binding partners, specific and non-specific, are present. We hope that continued advances in observing molecular dynamics in cells and more complex yet defined in vitro conditions will converge in the future. We hope that our work here contributes to this progress. Concerning the observation that mouse ES cells survive “tolerate loss of CTD, DBD and both”; we agree this is an intriguing result especially given the highly conserved nature of this part of BRCA2. We do cover this topic specifically in the second and third paragraphs of the discussion section.

    Questions that should be addressed include the following: Are proliferation rates compromised compared to WT cells?

    We did not observe compromised growth rates compared to WT cells. We have included this observation in the results (page 7, line 110).

    Are they experiencing replication stress in the absence of any exogenous damage?

    The difference in number of spontaneous RAD51 foci we observe in untreated cells lacking the DBD could be an indication for increased replication-associated DNA damage. This interesting topic is ongoing work of a departmental collaborator and hence is here. We have however highlighted this observation in the discussion (page 20, line 368).

    Are ES cells special in relevant aspects?

    Mouse ES cells are highly proficient in homologous recombination and gene targeting, which makes them useful subjects for HR studies. Mouse ES cells have a relative high number of spontaneous BRCA2 and RAD51 foci, most likely caused by their rapid cell division and DNA replication. As mouse ES cells are non-transformed cells we use these cells in our experiments to avoid cancer cells which often include mutations influencing processes such as DNA repair.

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  2. Evaluation Summary:

    This work is of interest to readers in the field of genome stability, DNA repair and associated human diseases. The manuscript describes systematic analyses of the crucial DNA repair mediator BRCA2 and its variants lacking the DNA binding domain or RAD51 interacting C-terminal domain, and the conclusions present a conceptual advance as to how BRCA2 promotes DNA repair. The work is a technical tour de force that includes evaluation of the DNA damage response, gene targeting and single particle tracking in mouse embryonic stem cells, as well as biophysical analyses of the human counterparts. The key claims of the manuscript are largely supported by the data.

    (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. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    The breast cancer protein 2, BRCA2, is best known for its roles in DNA repair by homologous recombination (HR) and in protecting stalled replication forks. In these processes, BRCA2 is thought to play a primary role in delivering RAD51 to affected ssDNA-containing sites. However, BRCA2 is a large protein of ~400 kDa, mostly composed of disordered structure, and how it acts during HR repair remains not fully understood.

    This work tackles this fundamental question and aims to uncover essential functions of the C-terminal region of BRCA2, composed of the ssDNA binding domain (DBD) and RAD51 binding C-terminal domain (CTD). Using mouse embryonic stem (mES) cells in which BRCA2 DBD and/or CTD deletion variants are endogenously expressed as HeloTag fusions, the authors systematically analysed (1) cellular survival upon genotoxic treatments and HR competency, (2) nuclear localisation and (3) diffusion dynamics. The work was further extended to the structural analyses of purified human BRCA2 with analogous deletions, assessing the impact of RAD51 or ssDNA for their conformational changes.

    The authors show that, while DBD and CTD are both important for normal cellular survival upon DSB-inducing IR and for HR activity, these deletion variants are capable of forming RAD51 or BRCA2 foci and mobility changes following IR, comparably to full-length BRCA2. Conversely, they found the clear impact of deletion of DBD or CTD in their oligomeric states and structural plasticity.

    Together, the authors conclude that the cellular survivals upon IR and HR competency are best reflected by the BRCA2 structure plasticity, rather than RAD51/BRCA2 foci formation or mobility. Accordingly, the authors propose that BRCA2's role in promoting HR is not simply delivering RAD51 to DNA damage sites, but requires its conformational changes. This also raises a caution to the widely used readouts, such as RAD51 foci formation, to infer the functionality of BRCA2. Overall, I feel that their conclusion is justified by the results presented in this manuscript.

    The major strength of this work lies in their comprehensive analyses of BRCA2 variants using a wide range of state-of-the-art in vivo and in vitro techniques, allowing straightforward comparison of their impact on cellular function, molecular behaviour and structural changes. The limitations of this study, although minor for the conclusion drawn by this study, are (1) CTD deletion generally confers modest cellular phenotypes compare to DBD deletion and is fully resistant to MMC and cisplatin. It remains unknown why CTD deletion elicits less impact despite its strong impairments in ligand-induced conformational changes; and (2) the molecular behaviours of BRCA2 in mouse ES cells might not be directly translated to these in human somatic cells.

    My specific comments on each experimental data are outlined below:

    (1) Survival assays of respective mES cell lines show that CTD is important for normal resistance to IR and olaparib, but not for MMC or cisplatin, while DBD is important for all aforementioned treatments. Their analysis of HR competency, inferred by Cas9-induced gene targeting efficiency, revealed that the deletion of DBD, and of CTD to a lesser extent, impact on efficient integration of the reporter, concluding that these domains are important for HR repair of two-ended DSB. These results are robust and convincing.

    (2) They then moved onto the analyses of the IR-induced RAD51 and BRCA2 foci formation. Surprisingly, they found that the deletion of DBD or CTD did not drastically affect foci formation, albeit slightly less efficient compared to full-length BRCA2. While the results and trends look promising, the number of samples analysed is somewhat limited (i.e., two or three technical replicates, rather than biological replicates) and the statistic tests have not been conducted.

    (3) HeloTag also allowed them to assess the mobility of these BRCA2 variants in mES cells, using single-particle tracking (SPT). Focusing on S-phase cells, they show that the increase of the immobile fraction of BRCA2, detectable at 2-4 hours upon ionising radiation, is not severely affected by the deletion of DBD or CTD. The conclusion was drawn from the datasets from two independent experiments of at least 15 cells and ~10,000 tracks per condition, which, in my opinion, is respectful.

    (4) Equivalent human BRCA2 deletion variants were purified from human HEK293 cells and subjected to scanning force microscopy (SFM) imaging. This analysis revealed that, while full-length BRCA2 commonly forms large oligomers of more than four molecules (70%), all the truncation variants showed somewhat reduced capacity to form tetramers or larger oligomers (i.e. ~44-54%). Upon RAD51 incubation, the majority of full-length BRCA2 (74%) became monomeric, while the C-terminal deletion appeared to respond less, with 40-55% becoming monomers and 30% remaining as dimers. The addition of ssDNA made full-length BRCA2 structure extended but elicited no structural impact on the truncated variants. These conclusions were drawn from the analysis of ~260-500 particles per sample, and look to me, credible. It would nevertheless be good to see the quality of purified BRCA2 variants by silver staining or mass-spectrometry to eliminate potential complications associated with other co-purified factors.

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  4. Reviewer #2 (Public Review):

    In the manuscript by Paul W. Maarten and Sidhu A.. et al., the authors surveyed the importance of the DNA binding domain (DBD) and C-terminal domain (CTD) of BRCA2 in response to DNA damaging agents in cells, and the conformations adopted by recombinant constructs. The characterization of these domains are paramount in understanding basic BRCA2 function for novel future exploitation in cancer therapeutics. While the DBD and CTD domain have notable functions in DNA binding, nuclear localization upon DSS1 binding, RPA exchange, and replication fork protection, their role in response to damage and conformational modulation had been unexamined. Studying BRCA2 domain deletion in human cell lines is difficult as human BRCA2 contains a NLS in the C-terminus of the protein. The authors exploit the fact that the murine BRCA2 that has an additional N-terminal nuclear localization sequence to overcome lethality and the study of deletion mutants in human cell lines. Cell survival assays show the DNA binding domain of BRCA2 is most important for cell survival when treated with DNA damaging agents IR, Olaparib, MMC, and Cisplatin. The authors also show this system is functional as they observe the DBD domain is the most important for gene targeting assays that are repaired by homologous recombination. By assessing various DNA damaging agents, the authors highlight the multiple roles of BRCA2 in varying DNA repair processes from DSB repair, BIR, crosslink repair, etc. Interestingly, the C-terminus of BRCA2 does not appear to play a role in to cells when treated with MMC or Cisplatin but plays an important role in mediating self-organization. The authors describe that both the DBD and CTD domains of BRCA2 are important for RAD51 foci formation following IR. Assessing BRCA2 single-particle tracking in live cells, the authors show that the deletion of the DBD and the CTD domain leads to an increased immobile fraction following IR treatment. Using biophysical single molecule analysis, the authors analyzed recombinant BRCA2 DBD , CTD, and double mutants in the presence of ssDNA and interacting protein RAD51. The authors determined these domains are important for BRCA2 self-interactions and BRCA2 conformational rearrangements in the presence of ssDNA supporting in vivo analysis. Biophysical analysis show that the DBD and CTD are important for BRCA2 conformational dynamics that are observed with binding protein RAD51 or DNA substrates.

    Strengths:
    • These studies exploit a murine cellular system to overcome cellular lethality observed in BRCA2 depletion in human cell lines, which allows them to study the mouse BRCA2 protein and associated domain deletions.
    • The authors also utilize bright photostable fluorophore's called JF646 Halo Tag ligand to study BRCA2, the deletion mutants, and RAD51 using live cell imaging. This is a great technical advancement in observing BRCA2 function in vivo.
    • The in vivo and in vitro studies both support important roles of the DBD and CTD domain in BRCA2 dynamics.

    Weaknesses:
    • The importance of the in vivo work with these domains and the findings presented is confounded by a lack of biological replicates and clear presentation of statistical analysis within figures in the manuscript.
    • As both domains are important for response to DNA damaging agents (IR, Olaparib, MMC, and Cisplatin) if a function specification could be made to the deletion mutations this would be most valuable to the field. Assaying molecules with varying substrates (Ex-forked substrates, crosslinked substrates, ssDNA substrates containing DNA lesions) or other protein players (DSS1) may aid in teasing out these roles.
    • The discussion focuses on DSS1 and the DBD domain, yet the paper lacks any experimental analysis of BRCA2-DSS1. A biophysical analysis with recombinant protein DSS1 may greatly enhance the impact of this work on the field.
    • It is unclear if the larger BRCA2 assemblies or the deletion mutants in the manuscript form via an oligomerization mechanisms or a phase separated mechanism. Speculation from authors would be valuable.

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  5. Reviewer #3 (Public Review):

    The biochemical and genetic characterization of BRCA2 has been an ongoing challenge in the DNA repair field as the protein is large, prone to degradation, and expressed at low levels in most cell types. While certain features of BRCA2 have been described previously including its ability to bind and load RAD51 onto resected DNA substrates, much remains to be discovered. In this study, the authors combine genetic studies in mouse ES cells with biochemical analysis to examine the spatial dynamics and molecular architecture of BRCA2. Notably, they utilize an innovative approach coupling endogenous tagging of mouse BRCA2 with a HALO tag to monitor BRCA2 movement within live cells by single particle tracking.

    I applaud the authors for achieving a highly technical approach to epitope tagging both endogenous BRCA2 alleles in mouse ES cells and combining this strategy with a HALO tag providing additional utility for a variety of cell biological experiments. By analyzing the endogenous alleles, the authors' system provides physiological levels of protein expression as transcription will be driven by the endogenous promoter thus preserving stoichiometric protein interactions within the cell and avoiding artifacts caused by overexpression.

    The authors determine the influence of the DNA binding domain (DBD) and c-terminal binding (CTD) on the dynamic activities of BRCA2. They begin by exposing cells containing 3 different deletion mutants ΔDBD, ΔCTD, and the double mutant ΔDBDΔCTD to four different types of DNA damage (IR, PARPi, MMC, and cisplatin). Notably, ΔDBD displays significant impairment in survival in response to all 4 types of DNA damage. The ΔCTD, in contrast, demonstrates less sensitivity to IR and Olaparib, however, complements as well as WT BRCA2 in response to crosslinking agents MMC and cisplatin. My only criticism in this aspect of the work is that it would have been informative to include a truncated BRCA2 (mimic of a patient pathogenic mutation) or null allele to compare to the survival of the ΔDBD and ΔCTD mutants. I realize that these alleles may be inviable but the authors should clearly state if that was indeed the case.

    The authors then go on to demonstrate that the ΔDBD and ΔCTD mutants are recruited to sites of IR damage in a similar manner to WT BRCA2 based on number and intensity of foci. I think it would be informative if the authors provided statistical significance for the graphs depicting the quantitation of foci number and intensity as there do appear to be differences between the mutants and the WT protein. There appears to be a delay in the kinetics of recruitment, especially at the 2 hr timepoint, for the mutants compared to WT BRCA2, which could indicate a defect in the recognition of the DNA damage. Only at the 2 hr timepoint following IR are there less RAD51 foci, and of a lesser intensity, in the three deletion mutants compared to WT BRCA2. Another possibility is the results could be interpreted as a defect in RAD51 loading and/or stabilization of the nucleoprotein filament. While immunofluorescence imaging of DNA repair foci have become common practice to measure protein recruitment to damage, it is impossible to know exactly what is happening in these foci with any granularity.

    Next, the authors measure BRCA2 movement in the mouse ES cells taking advantage of the HALO tag to track single particles. While technically and visually alluring, it is difficult to extract mechanistic insight from the results. DNA damage induces changes in diffusion leading to BRCA2 molecules with restricted mobility; the authors demonstrated this phenomenon in a prior publication. The deletion mutants appear to have little effect upon BRCA2 mobility.

    Finally, the authors utilize scanning force microscopy to analyze binding of the purified human BRCA2 proteins to RAD51 and ssDNA. In the absence of RAD51/ssDNA binding, there is a notable shift in the deletion mutants from oligomeric forms to monomeric compared to full length WT BRCA2. Upon binding to RAD51, there is a dramatic change from multimeric to monomeric forms for the WT BRCA2 (~7% to 74%) with a slight suppression of these changes shown for the deletion mutants. While WT BRCA2 forms extended molecular assemblies upon binding ssDNA, not surprisingly, deletion of the DBD or CTD fail to demonstrate any significant changes in physical architecture. In both situations, the mutant proteins respond to RAD51 and ssDNA in a dampened manner likely due to altered or loss of binding. While the architectural effects of RAD51 and ssDNA binding to BRCA2 are measurable by SFM, it is difficult to reconcile these changes in shape and oligomerization to defects in response to DNA damage and at which specific steps in homologous recombination these physical forms would impact.

    Strengths:

    1. Generation of mouse ES cells with both endogenous alleles of BRCA2 containing the deletion mutations in addition to a HALO tag is an incredible technical breakthrough and will be a highly valuable reagent for genetic and cell biological studies of mouse BRCA2.
    2. The deletion mutants ablating either the DBD or the CTD, or both, is a great genetic approach to understanding the role of these key domains in BRCA2. The response of these mutants (versus WT BRCA2 as a benchmark) to various DNA damage (IR, PARPI, MMC, cisplatin) provides interesting information delineating the roles of these two important domains in BRCA2. For example, the ΔCTD mutant is significantly sensitive to IR and Olaparib, yet complements as well as WT BRCA2 in response to the crosslinking agents MMC and cisplatin.
    3. The BRCA2 protein is notoriously difficult to purify and yet the authors succeeded in purifying 4 different forms of the protein for biophysical analysis. While it is difficult to interpret the various forms of BRCA2 by SFM, there are clear differences in the architecture between WT and the three c-terminal mutants. These differences are highlighted upon binding to RAD51 or ssDNA.

    Weaknesses:

    1. While the separation-of-function result for the CTD deletion in response to crosslinking agents MMC and cisplatin is a novel and compelling result, it would have been informative to compare the survival results and gene targeting assay using a BRCA2 null or mimic of patient mutation (truncating mutation) to see how these 3 mutants stack up against a completely non-functioning BRCA2 allele. Likely, the BRCA2 null alleles are inviable but perhaps a conditional system or truncating allele similar to a patient germline mutation would give a window into response compared to the DBD and CTD deletion mutants.
    2. It's not clear in the manuscript what new information we are learning about the mechanisms of BRCA2 in the single particle tracking (SPT) data. The differences in mobility between the mutants and WT BRCA2 seem minimal, but more importantly, it is not immediately clear how these data help us understand the normal cellular functions of BRCA2. No doubt, the technology and innovation to track single particle proteins in the nuclei of cells is impressive, but the authors should clearly explain how we can gain mechanistic insight from the SPT data that is presented in this manuscript.

    General Comments:

    It is unclear how missing the c-terminal domain (CTD) or the DNA binding domain (DBD) of BRCA2 can be interpreted as having "roles beyond delivering strand exchange protein RAD51" unless a complete biochemical workup of the deletion mutants was performed to detect any alterations in DNA binding, stimulation of RAD51 dependent strand exchange, etc... While interesting and certainly an impressive technical feat, foci imaging and single particle tracking do not provide much information on mechanism (i.e. whether BRCA2 is binding DNA and loading/nucleating RAD51).

    The interpretations in the discussion are not overstated, however, I somewhat disagree with the notion that the data, as presented, clarifies the role of BRCA2 beyond its canonical functions of RAD51 loading and nucleation on resected DNA substrates. I would have liked if the authors discussed the idea that it is surprising that mouse ES cells can tolerate complete loss of the DBD, CTD, and loss of both together. Questions that should be addressed in include some of the following: Are proliferation rates compromised compared to WT cells? Are they experiencing replication stress in the absence of any exogenous damage? Further, is there something unique about mouse ES cells that may differentiate BRCA2 behavior that would be expected in somatic human cells?

    It is interesting to note that many years ago Ashworth and Taniguchi published back-to-back papers in Nature (2008) describing BRCA2 reversion alleles from in vitro screens of BRCA2 mutant cells selected in cisplatin or PARPi such that some of these reversions resulted in huge deletions of the entire DBD of BRCA2, and yet, they promoted resistance to PARPi. In this context, I would much appreciate if the authors commented on their findings that their constructed DBD deletion is not resistant to PARPi and if they offered some speculation as to why the reversions in those previous studies were.

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