A novel role for the peptidyl-prolyl cis-trans isomerase Cyclophilin A in DNA-repair following replication fork stalling via the MRE11-RAD50-NBS1 complex

  1. Marisa Bedir
  2. Emily Outwin
  3. Rita Colnaghi
  4. Lydia Bassett
  5. Iga Abramowicz
  6. Mark O’Driscoll

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Abstract

We previously reported that non-homologous end-joining (NHEJ)-defective human LIG4 -/- pre-B lymphocytes were unexpectedly sensitive to killing by the cyclic peptide Cyclosporin A (CsA), a common component of bone marrow transplantation conditioning and maintenance regimes. We also found that CsA induced DNA double strand breaks (DSBs) in LIG4 syndrome patient fibroblasts, specifically upon transit through S-phase. The molecular basis underlying these CsA impacts has not been described hitherto. We postulated that CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair, as CYPA is the primary physiological target interactor of CsA.

CYPA is the founding member of the Cyclophilin family of peptidyl-prolyl cis-trans isomerases (PPIs). CsA inhibits the PPI activity of CYPA through occupation of the latter’s enzymatic active site. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression-interaction analysis, we describe novel impacts of CYPA loss and inhibition of its PPI activity on DNA repair. Prompted by findings from our CYPA-BioID proximity interactome, we validate CYPA interactions with different components of the DNA end resection machinery. Moreover, we characterise a novel and direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 (MRN) complex, providing evidence that the PPI function of CYPA actively influences DNA repair via direct protein-protein interaction at the level of DNA end resection. Consequently, we demonstrate that CYPA loss or inhibition impairs Homologous Recombination Repair (HRR) following DNA replication fork stalling.

Additionally, we define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability herein. Leveraging the novel insights into CYPA biology we have uncovered; we explore examples of how CYPA PPI inhibition may be exploited to selectively kill cells from a variety of different cancers with a shared characteristic genomic instability profile. These findings propose a potential new disease application or repurposing strategy for the non-immunosuppressive CsA analogue class of Cyclophilin inhibitors.

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    Reply to the reviewers

    Revision summary.

    Additional new data.

    • CYPA expression levels in Scrm Vs KO Vs R55A isogenic cell lines as new Fig 1C.
    • ATR signaling: western blot analysis of HU-induced p-CHK1 (S345) in Scrm, KO and R55A isogenic cell lines as new Suppl Fig 1B.
    • MRN expression: western blot analysis of expression of NBS1, MRE11, RAD50 and MCM2 is Scrm, KO and R55A isogenic cell lines as new Suppl Fig 7A.
    • NBS1 subcellular fractionation: western blot analysis of NBS1 from whole cell extract Vs cytoplasmic extract Vs nuclear extract comparing expression/distribution in Scrm, KO and R55A isogenic cell lines, as new Suppl Fig 7B.
    • CYPA immunofluorescence (IF) staining on untreated and HU treated U2OS, as new Suppl Fig 7C.
    • CYPA immunofluorescence (IF) staining on untreated and HU treated U2OS following pre-extraction, as new Suppl Fig 7D.
    • DepMap Project Score Cancer Gene Dependency cell survival (“fitness”) following PPIA/CYPA-KO in breast carcinoma cell lines mapped against BRCA2 status, as a new Suppl Table 5.
    • DepMap Project Score Cancer Gene Dependency cell fitness following PPIA/CYPA-KO in Neuroblastoma cell lines, as a new Suppl Spreadsheet 4.
    • DepMap Project Score Cancer Gene Dependency cell fitness following PPIA/CYPA-KO in Multiple Myeloma cell lines, as a new Suppl Spreadsheet 4.
    • DepMap Project Score Cancer Gene Dependency cell fitness following PPIA/CYPA-KO in Chronic Myelogenous Leukaemia cell lines, as a new Suppl Spreadsheet 4.

    Revised and/or additional text.

    The Abstract, Introduction, Materials & Methods, Results and Discussion have been amended as necessary, to facilitate the issues raised by the Reviewers.

    Reviewer #1: We thank this reviewer for their understanding and appreciation of our CYPA study as espoused by their comprehensive summary of the content, importance, and potential implications of our work; “The manuscript presents clear and comprehensive data, demonstrating the profound impact of CYPA on DNA repair.” Furthermore, we very much appreciate their robust and complementary words regarding the significance of our work and its wide appeal; “The significance of this study is twofold: it adds a new layer to our understanding of DNA repair mechanisms and, importantly, it could point the way to novel therapeutic strategies for cancer. It will spark interest from molecular biologists to clinicians and pharmaceutical researchers.”

    Query:

    It's surprising to find that the loss of CYPA abolished HU-induced NBS1 foci, as the MRE11 interactive domain of NBS1 should remain intact in CYPA deficient conditions and the N-terminus of NBS1 is dispensable for ATM activation (Kim et al., 2017; Stracker and Petrini, 2011). A more detailed mechanistic explanation of this phenotype would be appreciated. The authors should check the subcellular localization of NBS1 and the stability of MRN in wildtype and CYPA KO cells. Additionally, including the kinetics of NBS1 foci formation using multiple timepoints in wildtype and CYPA KO cells after damage will further support the observation.

    RESPONSE:

    Regarding NBS1 foci formation, we note that rather than abolish HU-induced NBS1 foci formation, CYPA loss (through KO) and/or inhibition (through p.R55A) in fact results in a “…spontaneously elevated yet unresponsive amount of NBS1 foci/cells when compared to scrambled” (see original Fig 9A legend and associated Results section text). We have reinforced this observation in the revised Results section entitled ‘CYPA influences NBS1 and MDC1 foci formation’ and in the Discussion section. We do describe a kinetic impairment of RAD51 foci formation in the CYPA-engineered lines up to 16hrs post HU-treatment (Fig 6D). Our mechanistic working model is that CYPA interacts directly with NBS1 via a Pro residue within the short linking peptide between the FHA and BRCT1, and that this likely influences the relative dynamic positioning of the FHA with BRCA1-BRCT2, at least following acute HU treatment; replication fork stalling, likely biased towards ATR-dependent signaling initially, rather than that of ATM. The relative positioning of these functional domains can impact MRN function, and we discuss this possible mechanism in the section entitled ‘CYPA and the MRN complex’, with reference to the detailed structure-function analyses and complementary DDR activation models described by

    • Williams, R.S., et al., Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand break processing and repair. Cell, 2009. 139(1): p. 87-99.
      and
    • Lloyd, J., et al., A supramodular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA damage. Cell, 2009. 139(1): p. 100-11.
      and
    • Rotheneder, M., et al., Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions. Mol Cell, 2023. 83(2): p. 167-185.e9.

    The N-terminal FHA-BRCT region of NBS1 does indeed influence MRN recruitment and HRR execution, a point we highlight in the section entitled ‘CYPA influences NBS1 and MDC1 foci formation’, with reference to the seminal original observations of

    • Sakamoto, S., et al., Homologous recombination repair is regulated by domains at the N-
      and C-terminus of NBS1 and is dissociated with ATM functions. Oncogene, 2007. 26(41): p.6002-6009
      and
    • Tauchi, H., et al., The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50-hMRE11-NBS1 complex
      DNA repair activity. J Biol Chem, 2001. 276(1): p. 12-15.
      and
    • Zhao, S., W. Renthal, and E.Y. Lee, Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses. Nucleic Acids Res, 2002. 30(22): p. 4815-22.
      and
    • Cerosaletti, K.M. and P. Concannon, Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation. J Biol Chem, 2003.
      278(24): p. 21944-21951.

    HU-unresponsive NBS foci (indicative of MRN dysfunction) and MDC1 foci formation are consistent with the DNA-R (i.e., DR-GFP reporter systems: Fig 3A-C and impaired RAD51 foci formation: Fig 6D) and resection-related phenotypes (Fig 6A-B) we report here and are also consistent with the relative resistance to HU-induced killing we report for CYPA-KO and CYPA-R55A cells (Fig 11A and as reported by Manthey, K.C., et al., NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse. J Cell Sci, 2007. 120(Pt 23): p. 4221-9).

    At the reviewer’s request we include additional novel experimental data showing that MRN expression is stable and equivalent in control, CYPA-KO and CYPA-R55A cells (Suppl Fig 7A). We also provide evidence that NBS1 subcellular distribution (via extract fractionation) is not altered upon CYPA loss and/or inhibition (Suppl Fig 7B).

    Query:

    The authors showed that the interaction between CYPA and MRN didn't change after HU treatment. The authors should also include co-localization analysis of CYPA and NBS1 after HU.

    RESPONSE:

    At the reviewer’s suggestion we undertook a series of IF analyses concerning endogenous CYPA (i.e., +/- HU, +/- pre-extraction). We found that endogenous CYPA failed to form foci following HU thereby precluding CYPA-NBS1 foci co-localization analysis (Suppl Fig 7C-D).

    Query:

    The paper demonstrated that BRCA2 knockdown cells were sensitive to CsA. The authors should also examine CsA sensitivity in BRCA2 deficient cancer cells. In addition, the authors could elaborate more on their criteria for selecting cancers for CYPA inhibition, whether it is based on high genomic instability or an addiction to HRR for survival.

    RESPONSE:

    Despite repeated attempts we have been unable to successfully routinely culture the TNBC suspension line HCC1599 (BRCA2 c.4154_5572del1419 and p.K1517fs*23), consistent with its reported ~5 days population doubling time. Although not a tumour line per se, we also failed to effectively culture the FANC-D1 patient FB line HSC62 (BRCA2 c.8488-1 G>A (IVS19-1G>A)) to enable survival analysis. We provide new quantification analysis of the CsA survival on the H1299 conditional shBRCA2 line (Fig 11E). Additionally, we include a comprehensive new analysis of cell survival (“fitness”) of a range of breast carcinoma cell lines following PPIA/CYPA-KO, extracted from DepMap Project Score Cancer Gene Dependency portal (https://score.depmap.sanger.ac.uk/), and also specify the BRCA2 status of each line. Interestingly, we find that reduced BRCA2 copy number is more commonly associated with loss of fitness following PPIA/CYPA loss (Suppl Table 5). We also include similar cell line fitness datasets for each of the cancers for whom we demonstrate elevated sensitivity to CYPAi (i.e., Neuroblastoma, Multiple Myeloma and CML) (Suppl Spreadsheet 4). Fascinatingly, PPIA/CYPA loss clearly results in loss of fitness in most of these cancer cell lines. Collectively, these new independent comprehensive datasets support our argument that targeting CYPA in select cancer scenarios shows impact in the preclinical setting and may represent an effective new strategy.

    The unifying features of the cancers showing elevated sensitivity to CYPAi are indeed high genomic instability, denoted by elevated RS and hence a dependency upon replication fork protection machinery. This would be consistent with the observed lethality of our CYPA-panel to shBRCA2, siXRCC3 and siRAD51C. The cancers are additionally characterised by aberrantly elevated HRR (i.e. an addiction to/dependency on HRR). This would be consistent with the observed lethality of our CYPA-panel to siCtIP, siRAD52, siXRCC3, and siRAD51C. At the Reviewer’s request we have reinforced and better clarified this point in the section Potential rational applications of CYPA inhibition in select cancers and in the Discussion.

    Reviewer #2:

    We thank this reviewer for their positive and supportive comments concerning our work; “Authors have quite conclusively explored the interaction between NBS1 and cyclophilinA as well as the putative proline residue important for this interaction.” We appreciate the constructive feedback concerning the range of consequences/impacts of CYPA impairment and we concur with their contention that “This manuscript will have broad interest from groups working on genomic stability, immunology as well as cancer therapy.”; a general view also voiced by Reviewer #1.

    We do stress that whilst other prolyl isomerases have previously been linked to DNA repair (e.g., most notably the Parvulin family member PIN1), this is the first time that CYPA has been directly implicated in DNA repair, and the first time CYPA has been shown to directly interact with a known DNA-R protein (i.e. NBS1).

    We believe that the comprehensive CYPA-BioID we describe is worthy of report and should serve as a very useful starting point for additional studies concerning CYPA biology, which is undoubtedly complex. The interactome will also function as a useful tool in helping dissect the clinically significant wider biological consequences of CYPA inhibition. Our interactome findings demonstrate that CYPA may influence DNA-R via multiple, and not necessarily mutually exclusive, routes. We do not argue that CYPA’s role in DNA-R is exclusively via NBS1/MRN. This is clearly demonstrated by our validation of CYPA interactions via co-IP with endogenous CYPA with proteins including PCNA, 53BP1, CHAMP1 and ILF2-3 complex (Fig 5). These are completely novel observations that furthermore reinforce the validity and efficacy of our experimental approach in leveraging the CYPA-BioID to provide new biological insight into this druggable prolyl cis-trans isomerase.

    Query:

    Authors show delayed S-phase transit along with reduced replication speed indicating replication stall. However, authors have not discussed how cyclophilinA might regulate replication (other than hypothesizing regarding altered dynamism of FHA-BRCT). It is conceivable that it could be an indirect effect on cellular metabolism or if authors believe it could be due to direct disruption to core replication machinery or signaling. In this regard, it will be helpful to see if there is shortening of (premature entry) G1 phase and comment on the status of the associated G1/S checkpoint.

    RESPONSE:

    The reviewer makes a very interesting and astute observation concerning the DNA replication phenotypes we report following CYPA loss and/or inhibition. The bases of these phenotypes are likely multifactorial, and we have revised the associated Discussion text to reflect this. Specifically, we highlight the elevated and unresponsive NBS1 and MDC1 foci seen in the CYPA-KO lines (Fig 9. i.e., persistent protein-DNA complexes) and dependence upon fork protection factors (XRCC3, RAD51C, BRCA2: Fig 11). We also report that a range of DNA replication factors are found in the CYPA-BioID (Fig 5A). Untangling the functional significance of these putative interactions would involve further study. Are they direct/indirect interactors? If direct, are they prolyl isomerase substrates or chaperone clients or regulated by liquid-liquid phase separation (LLPS)? Similarly, the CYPA-BioID throws-up an extensive set of RNA binding factors (Suppl Table 2), many of whom may conceivably contribute to the replication–transcription fork conflicts/collisions under conditions of CYPA-dysfunction. As this is the first comprehensive report of the cellular impacts of CYPA loss and inhibition, we thought it worth reporting the DNA replication associated phenotypes specifically to demonstrate the pleiotropic impact of loss and inhibition of this particular prolyl isomerase, to underscore its significance/importance. Although we have indeed found cell cycle phase transition impairments in our CYPA-KO and CYPA-R55A cells (for both G1-S and G2-M), these constitute additional studies requiring more thorough molecular-mechanistic characterization. We chose to focus on DNA repair for this first manuscript, as the CYPA-NBS1 interaction was the physical relationship for which we have assembled the most detailed and interconnected datasets, to-date. We do intend to pursue the cell cycle work as it too is derived from our CYPA-BioID (Suppl Spreadsheet 1), and we have already validated some of those relevant interactions by CYPA co-IP, but this is very much a work-in-progress. With this manuscript we’re endeavoring to tread a fine line by showcasing a wide range of cellular phenotypes resultant from CYPA loss and inhibition, but then also showing a deeper level of characterisation with at least one relevant interactor known to function in a range of DNA-R pathways wherein we’ve found impairments and dependencies.

    Query:

    In connection to this, it will also be interesting to see if the ATR/Chk1 signaling axis is intact in CYPA KO cells with or without additional DNA damage compared to WT.

    RESPONSE:

    At the reviewer’s request we include new data showing that HU-induced ATR-dependent CHK1 phosphorylation is normal in CYPA-KO and CYPA-R55A cells, and that ATR does not appear to be spontaneously activated in the absence of replication stress in these cells (Suppl Fig 1B).

    Query:

    Authors show that the P112 residue of NBS1 is important for the binding of cyclophilinA. What is the status of interaction among components of the MRN complex in CYPAKO cells and P112G NBS1? Further, what are the authors' thoughts on rescue experiments and whether P112G containing NBS1 to perform resection function.

    RESPONSE:

    We include new data showing normal expression of MRN components and normal subcellular localisation of NBS1 in the CYPA-KO and CYPA-R55A cells (Suppl Fig 7A-B). Regarding the interaction status of P112G, we show that this fails to co-IP endogenous CYPA when transiently expressed in HEK293 cells, in marked contrast to WT-NBS1 (Fig 8A). Furthermore, we show that ablation of another FHA Pro residue (P64) does not impair co-IP with endogenous CYPA under similar conditions, suggesting P112G is unique in this regard. Our recombinant protein interaction work demonstrates that CYPA-Step directly interacts with a HIS-(FHA-BRCT1) peptide and that P112G abolishes this interaction (Fig 8B). Regarding rescue experiments, we’ve found that stable overexpression of NBS1 can be neomorphic, resulting in resistance to certain DNA damaging agents, thereby complicating cell-based rescue analyses. We stress that along with our engineered KO and R55A (isomerase-dead) lines we have employed the well-known CYPAi Cyclosporin A (CsA) to reproduce several of the DNA-R related phenotypes (e.g., Fig 1, Fig 3, Fig 6, Fig 10, Fig 11). To further examine impacts upon resection specifically, a logical approach would be to engineer P112G into a full-length recombinant (baculoviral produced) human MRN complex for in vitro kinetic assessment using various labelled DNA substrates. But we think that this specialist and not insignificant undertaking is outside the scope of our report of the extensive cellular consequences of CYPA loss and dysfunction and it’s potential (pre)clinical significance with regards CYPAi repurposing.

    Query:

    What are the protein levels of MRN, RAD51 etc. in CYPAKO cells? It will be important control to delineate the effects of CYPA on global transcription and translation vs specific and direct effect on end-resection. Can overexpression of NBS1 rescue the observed resection and focus phenotypes?

    RESPONSE:

    Basal levels of RAD51 foci/cell are comparable between Scrm and both CYPA-KO and R55A cells (Fig 6D). We also find comparable levels of MRN components between these lines (Suppl Fig 7A). Importantly, we observe the pRPA/resection defect following an acute (up to 3hrs) treatment with CsA; conditions unlikely to grossly impair translation to an extent that would result in reduced expression of the relevant DNA-R proteins. Furthermore, microarray based transcriptomic analyses of these isogenic lines did not show evidence of a global impact upon transcription following CYPA-KO or R55A, nor was there evidence of reduced expression of any genome stability/DNA-R genes. We did not include this negative data so as to maintain the focus on the functional link with DNA repair.

    Reviewer #3: This critically negative review is myopic, unbalanced, self-contradictory and frustratingly mis-represents some of our key findings. The dismissive tone of the text unnecessarily and unprofessionally crosses into the pejorative (“Either evidence is lacking or experiments were not performed in a convincing way”). The stark contrast between this review and the summations of Reviewer #1 and Reviewer #2 serve to highlight this hyper-negative approach.

    It is very frustrating that this reviewer describes our findings as “…an interesting story…”, that “…the identification of NBS1 as a novel substrate of CYPA is significant” , that the “..manuscript may provide new insight…”, and that “…the role of CYPA in DNA repair is fairly well described using its inhibitor or KO cells”, and yet then concludes by stating “… the current manuscript suffers lack of evidence to support the main conclusion”. This is self-contradictory and unbalanced. Again, the contrast with Reviewer #1 and Reviewer #2 in this regard is stark.

    Major critical theme no. 1.

    Expression of CYPA-R55A: “…vastly different…”

    RESPONSE.

    This reviewer dismisses the entirety of the R55A model cell line work based upon the apparent “…vastly different…” expression levels of the reconstituted lines. This is an overstatement of the situation and notably not an issue for either Reviewer #1 or Reviewer #2. Nonetheless, we have replaced the original CYPA blot in Fig 1C with a clearer and more representative depiction of expression levels between the engineered lines and control. Importantly, the pRPA/resection work, siRAD52 and siXRCC3 dependency work were all corroborated/reproduced using the CYPA PPI inhibitor Cyclopsorine A (CsA). The plurality of our complementary approaches showing the influence of CYPA upon DNA-R is minimised and/or ignored by this Reviewer. Although not shown in this study, we find that the R55A cells are selectively sensitive to DNA cross-linker melphalan, in contrast to the CYPA-KO cells. Although we don’t yet understand the basis of this observation, this clearly indicates that R55A expression is a valid model in our hands and is not a like-for-like mimic of CYPA-KO simply because of reduced expression. We appreciate the reviewer could not know this.

    Major critical theme no. 2.

    CYPA-NBS1 work: “Another major concern is that the evidence to support that NBS1 is the major substrate of CYPA is lacking since all the experiments were performed with the CYPA mutant or CsA treatment.”

    RESPONSE:

    We do not claim that NBS1 is ”… the major substrate of CYPA.” . We do not claim that all the DNA-R deficits we have identified are specifically a consequence of impaired NBS1 function. These are misrepresentations of our findings and how we’ve presented and discussed them. This Reviewer ignores our comprehensive CYPA-BioID, and specifically our discussion pertaining to the DNA-R and Replication factors found therein (section entitled ‘CYPA Interacting protein partners’ and Fig 5A). We explicitly discuss the fact that “A recurring theme amongst these CYPA interactors is that all are involved in end-resection” whilst also demonstrating CYPA co-IP with 53BP1, CHAMP1 and ILF2-3 (Fig 5C-E). In the ‘Discussion’ section we describe a “homesostatic role for CYPA in genome stability”, including possible contributions to controlling LLPS of well-known DNA-R factors and the fact that several mitotic, kinetochore, centrosomal and spindle proteins are found in the CYPA-BioID.

    Major critical theme no. 3.

    A major repeated criticism levelled by this reviewer as a basis for dismissing the entirety our findings is that we have failed to demonstrate that the catalytic activity of CYPA is required for DSB repair.

    • Their conclusion should be supported by additional key experiments to prove that the catalytic activity of CYPA is indeed required for DSB repair…

    • Another major concern is that the evidence to support that NBS1 is the major substrate of CYPA is lacking since all the experiments were performed with the CYPA mutant or CsA treatment.

    • One major weakness of this study is that it focuses on characterizing the interaction between CYPA and NBS1, then jumps into a conclusion that the catalytic activity of CYPA is required for DSB repair based on its direct interaction with NBS1

    RESPONSE:

    As this criticism is repeated, the impression created, and no doubt intended, is that the manuscript is irreparably flawed (“…major weakness…”). This is an over-simplification and a misdirection. It’s notable that this critique isn’t raised in such a manner by either Reviewer #1 or Reviewer #2. This is likely because any modest inferences we made concerning the possible role of CYPA catalytic isomerase activity were based on a combination of differing but complementary approaches. Firstly, we routinely used the p.R55A engineered CYPA variant, although this Reviewer regards our use of this as invalid. This longstanding peptidyl prolyl isomerase (PPI)-dead mutant model has frequently been employed to invoke the catalytic function of CYPA. The mutant was originally proposed and characterized as catalytically-dead using the in vitro chymotrypsin-coupled prolyl isomerase assay using N-succinyl-AAPF-p-nitroanilide as a substrate as far back as 1992 (Zydowsky, L.D., et al., Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition. Protein Science, 1992. 1(9): p.1092-1099). In addition, we routinely use Cyclopsorin A (CsA), the longstanding clinically relevant CYPA PPI inhibitor, and we also use a different and more potent CYPA PPI inhibitor, namely NIM811 (N-methyl-4-isoleucine-cyclosporine) for the DR-GFP reporter assays of individual DNA-R pathway function (i.e.’ NHEJ, HRR and SSA).

    With regards to our findings concerning CYPA-NBS1 interaction, in the Discussion section we clearly state that mechanistic analyses of prolyl isomerase on the dynamism of NBS1 FHA-BRCT would require specialist approaches outside the scope of this manuscript, as the manuscript is firmly within the realm of cellular biology. This is ignored by this Reviewer. Specifically, we state that “A regulated cis-trans isomerisation of the E111-P112 peptide bond could conceivably dynamically alter the relative positioning of the FHA domain with the tandem BRCTs of NBS1 (Fig 7C-D). This may then impact on these domains’ abilities to dynamically interact with their respective phospho-threonine (for FHA) and phospho-serine (BRCT) containing targets, consequently likely shaping/impacting NBS1 recruitment dynamics and/or plasticity of its interactome [120-122]. Demonstrating this hypothesis would require additional structural analysis using techniques such as 2D-NMR which is outside the scope of this manuscript.”

    Minor comments: 1.

    Fig. 1E; is the survival between KO and R55A statistically significant? If so, do the authors have an explanation? Why is the reconstitution of R55A more toxic than KO alone?

    RESPONSE:

    Yes, R55A is slightly more sensitive compared to KO for this endpoint. The irony that this observation runs contrary to the Reviewer’s dismissal of the R55A model line is not lost on us (Major critical theme no. 1). As is well-known for PARP1, inhibition is not equivalent to absence. A possible speculative explanation is that the R55A isomerase-dead could have additional dominant impacts compared to the KO situation. Nonetheless, we suspect this Reviewer would object to such speculation in the absence of ever more data.

    Minor comments: 2.

    In Fig. 3D, the NHEJ activity of CsA- or NIM811-treated cells is significantly downregulated in comparison to control, which raises the possibility of the pleiotropic effect of CYPA inhibition. The authors should discuss this issue.

    RESPONSE:

    Not necessarily indicative of a pleiotropic effect if one accepts that absence of a protein is not always biologically equivalent to the presence of an inhibited version the same protein. Of note, we do see somewhat reduced NHEJ following siCYPA (Fig 3A), something not mentioned by this Reviewer. Furthermore, we explicitly discuss and show interaction between CYPA and 53BP1, CHAMP1 and ILF2-3 complex, all players in NHEJ and in the intricate balance between NHEJ and resection-mediated recombination directed repair pathways.

    Minor comments: 3.

    In Figure 8A, since the expressions of Flag-NBS1 WT, P112G, and P64G are very different, the conclusion that the isomerization of CYPA is essential for NBS1 cannot be supported. Given the variation of input levels, it appears that the P64G mutation actually enhances the interaction with endogenous CYPA. Is this reproducible? This co-IP result may need to be quantified from independent sets for statistical analysis.

    RESPONSE:

    We do not claim that “…isomerization of CYPA is essential for NBS1…”. Fig 8A data is derived from a transient transfection. Whilst there is some variation in expression, we do not make any precise quantitative conclusions from these co-IPs. Nonetheless, FLAG-NBS1-P112G clearly interacts less with endogenous CYPA in this system. Importantly, and ignored by this Reviewer, the associated recombinant protein work shown in Fig 8B clearly confirms that NBS1-P112G is profoundly compromised in its ability to interact with CYPA.

    Minor comments: 4.

    A defect in DSB repair generally hypersensitizes cells to DNA replication stress, including HU. In this regard, resistance of the CYPA KO (or R55A cells) to HU is interesting, but it may be due to the nonspecific effect of the CYPA loss in multiple DNA damage signaling and repair processes. Alternatively, cell cycle may be affected nonspecifically, rendering cells resistant to replication-associated genotoxic stress. This needs to be addressed further. Analysis of overall cell cycle profile may be required.

    RESPONSE:

    Resistance to HU is likely multifactorial and cell cycle transition kinetics may be relevant here. That is why we linked the DNA replications phenotypes to this discussion in the section entitled “Impaired CYPA function reveals novel genetic dependencies/vulnerabilities”. A comprehensive analysis of cell cycle profile and phase transits is outside the scope of the current manuscript (see response to Reviewer #2).
    Impaired HU-induced pRPA has been linked to HU-resistance via NBS1 previously: Manthey, K.C., et al., NBS1 mediates ATR-dependent RPA hyperphosphorylation following replication-fork stall and collapse. J Cell Sci, 2007. 120(Pt 23): p. 4221-9.

    Minor comments: 5.

    Text not to mention Abstract is too dense. The manuscript will benefit a lot from extensive editing and rearrangement of figures to make the story more succinct for journal submission.

    RESPONSE:

    The Reviewer’s view concerning a lack of succinctness is not shared by Reviewer #1 and Reviewer #2. We have endeavored to draft a concise and accessible manuscript, the main body of which comes in at just over 23x sides of A4 (including Materials & Methods). Considering we guide the reader through 12x multipart figures, 5x supplementary tables and 8x supplementary figure, we believe we have achieved succinctness. Nonetheless, we will of course take direction from the appropriate journal editorial team regarding house style and format.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, O'Driscoll and colleagues identify the role of cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase, in promoting DNA repair. They propose that the catalytic activity of CYPA is required for the action of the MRE11-RAD50-NBS1 (MRN) complex and thus double-strand break (DSB) repair. This study originated from their previous finding that cyclosporin A (CsA) induces replication-associated DNA breakage and genome instability in LIG4 syndrome patient fibroblasts. As CsA is an inhibitor of the CYPA, the authors reasoned that the negative effect of CsA in DNA repair results from its inhibition of CYPA and presumably its essential downstream substates in DNA repair. Using CRISPR/Cas-based U2OS knockout cells, they showed that the catalytic activity of CYPA is necessary for homology-directed repair. Series of BioID-proximity interactome analysis, biochemical studies (e.g., co-immunoprecipitation), and AIphaFold-derived structural determination revealed that CYPA directly interacts with the MRN complex, specifically through the Pro112 of NBS1, and its catalytic activity is required for damage-induced NBS1 foci formation, which all together led to the conclusion that the MRN complex is a direct substrate of CYPA and that CYPA controls DNA end resection and DSB repair via isomerization of NBS1.

    This is an interesting story as it reveals a new role of prolyl isomerization, which is mediated by CYPA, in promoting DSB repair. The identification of NBS1 as a novel substrate of CYPA is significant, and the manuscript may provide new insight into how prolyl isomerization of NBS1 regulates the function of the MRN complex that is engaged in DNA end resection during DSB repair. However, the authors' major claim that CYPA controls DSB repair via the MRN complex is not substantiated by the data provided at its current form. Either evidence is lacking or experiments were not performed in a convincing way. Their conclusion should be supported by additional key experiments to prove that the catalytic activity of CYPA is indeed required for DSB repair and NBS1 is a major substrate of CYPA, through which CYPA regulates DNA end resection at the stalled DNA replication fork.

    Major comments

    1. The authors reconstituted the CYPA knockout (KO) cells with WT or a catalytic mutant (R55A) for the structure-function analysis. However, re-expression levels of CYPA are vastly different between WT vs. R55A, R55A being expressed at much lower levels (not near to endogenous CYPA) (Fig. 1C). Consequently, the loss-of-function phenotypes of R55A may be simply explained by its inadequate reconstitution, thus failing to complement KO phenotypes. For instance, the lack of pRPA2 S4/S8 induction in R55A cells may be just due to the insufficient expression of R55A, thus resulting in the same phenotype as KO. Additionally, the R55A cells were compared to parental cells, not to the WT-reconstituted cells for the majority of functional analysis, so it is not clear whether WT is able to complement the KO phenotype in their system (Figs. 1, 2, 6, 9, 10, and 11). Whether the catalytic activity of CYPA is indeed responsible for the phenotypes of DNA repair deficiency is not supported. The authors should compare the phenotypes between WT- vs. R55A-reconstitued cells side-by-side for the key experiments. Ideally, expression of WT and R55A should be similar in KO cells to exclude the possibility that the R55A phenotypes merely result from insufficient mutant expression rather than true loss of catalytic activity.
    2. Another major concern is that the evidence to support that NBS1 is the major substrate of CYPA is lacking since all the experiments were performed with the CYPA mutant or CsA treatment. Whether the NBS1 P112G isomerization-defective mutant indeed exhibits a defect in DNA repair similarly to the CYPA mutant is not shown. For instance, one key experiment would be to test whether the P112G mutant fails to form damage-inducible NBS1 foci formation.
    3. In Figure 7A, the authors showed that the interaction between CYPA and NBS1 is dependent on the isomerization activity of CYPA. It should be checked whether the CYPA R55A mutant fails to interact with NBS1 in contrast to WT to support the main conclusion that NBS1 is controlled by the isomerization activity of CYPA.
    4. OPTIONAL) One major weakness of this study is that it focuses on characterizing the interaction between CYPA and NBS1, then jumps into a conclusion that the catalytic activity of CYPA is required for DSB repair based on its direct interaction with NBS1. How the isomerization of NBS1 affects its localization, stability, and/or function is not addressed. At its current form, the functional link between NBS1 isomerization and stalled fork processing is weak. Elucidating how the catalytic activity of CYPA controls the action of the MRN complex via the isomerization of NBS1 will add significant impact on the manuscript. Otherwise, the story fails to fully support the description of its title.

    Minor comments

    1. Fig. 1E; is the survival between KO and R55A statistically significant? If so, do the authors have an explanation? Why is the reconstitution of R55A more toxic than KO alone?
    2. In Fig. 3D, the NHEJ activity of CsA- or NIM811-treated cells is significantly downregulated in comparison to control, which raises the possibility of the pleiotropic effect of CYPA inhibition. The authors should discuss this issue.
    3. In Figure 8A, since the expressions of Flag-NBS1 WT, P112G, and P64G are very different, the conclusion that the isomerization of CYPA is essential for NBS1 cannot be supported. Given the variation of input levels, it appears that the P64G mutation actually enhances the interaction with endogenous CYPA. Is this reproducible? This co-IP result may need to be quantified from independent sets for statistical analysis.
    4. A defect in DSB repair generally hypersensitizes cells to DNA replication stress, including HU. In this regard, resistance of the CYPA KO (or R55A cells) to HU is interesting, but it may be due to the nonspecific effect of the CYPA loss in multiple DNA damage signaling and repair processes. Alternatively, cell cycle may be affected nonspecifically, rendering cells resistant to replication-associated genotoxic stress. This needs to be addressed further. Analysis of overall cell cycle profile may be required.
    5. Text not to mention Abstract is too dense. The manuscript will benefit a lot from extensive editing and rearrangement of figures to make the story more succinct for journal submission.

    Referees cross-commenting

    I agree with concerns on the pleiotropic effect of CYPA KO, which exhibit many distinct phenotypes in DNA repair and replication fork stability.

    Significance

    While establishing a new link between CYPA-dependent prolyl isomerization and DSB repair is significant, the current manuscript suffers lack of evidence to support its main conclusion. Specifically, although the role of CYPA in DNA repair is fairly well described using its inhibitor or KO cells, whether its isomerization activity is indeed essential and whether NBS1 is the major target for its action in DSB repair is not clear. Existence of many other targets cannot be excluded. Whether the role of CYPA is specific to replication-associated DSB repair processes or can be generally applicable to homologous recombination in any DSB repair is not shown. The role of MRN in stalled fork processing and in response to DSBs could be different, but how CYPA would modulate these distinct processes is not addressed. As such, the manuscript is targeting more specialized audience, but if the link between CYPA and the MRN complex can be further elaborated, including how isomerization affects the function of NBS1 (e.g., using the isomerization-defective NBS1 mutant), it could reach out to broader readership.

    My field of expertise includes DNA replication stress and replication-associated repair processes including stalled fork processing and recovery. I am familiar with most of the genetic, cellular, and biochemical experiments presented in the manuscript. I do not have significant expertise on the structural analysis of protein-protein interactions.

  7. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The current manuscript by Bedir et al. explores the role of cyclophilin A in DNA repair, particularly homologous recombination. Authors show that the absence of cyclophilin A or loss of PP1 activity affects end-resection via direct interaction with NBS1. Authors have conducted a series of experiments to confirm their findings. While the findings are interesting, further discussion/ experiments mentioned below will perhaps assure readers with respect to pointed direct vs consequence facilitated indirectly through global cellular effects of CYPA.

    1. Authors show delayed S-phase transit along with reduced replication speed indicating replication stall. However, authors have not discussed how cyclophilinA might regulate replication (other than hypothesizing regarding altered dynamism of FHA-BRCT). It is conceivable that it could be an indirect effect on cellular metabolism or if authors believe it could be due to direct disruption to core replication machinery or signaling. In this regard, it will be helpful to see if there is shortening of (premature entry) G1 phase and comment on the status of the associated G1/S checkpoint.
    2. In connection to this, it will also be interesting to see if the ATR/Chk1 signaling axis is intact in CYPA KO cells with or without additional DNA damage compared to WT.
    3. Authors show that the P112 residue of NBS1 is important for the binding of cyclophilinA.
    4. What is the status of interaction among components of the MRN complex in CYPAKO cells and P112G NBS1? Further, what are the authors' thoughts on rescue experiments and whether P112G containing NBS1 to perform resection function.
    5. What are the protein levels of MRN, RAD51 etc. in CYPAKO cells? It will be important control to delineate the effects of CYPA on global transcription and translation vs specific and direct effect on end-resection. Can overexpression of NBS1 rescue the observed resection and focus phenotypes?

    Significance

    Current study highlights the role of cyclophilin A or in large peptidyl-prolyl cis- trans isomerases activity in DNA repair. Although this is not the first study showing the relevance of cyclophilin A in DNA repair, they do highlight its role in homologous recombination and DNA repair. Authors have quite conclusively explored the interaction between NBS1 and cyclophilinA as well as the putative proline residue important for this interaction.

    One of the drawbacks of the study is the pleotrophic effects of CyclophilinA. This needs to be at least discussed. Authors themselves observe induction of DSBs, replication stall, reduced NHEJ, SSA as well as HR efficiencies. Taken together, the effects of CyclophilinA even on resection could be a combination of both direct and indirect effects.

    This manuscript will have broad interest from groups working on genomic stability, immunology as well as cancer therapy. I have expertise in NHEJ, mammalian replication and replication-stress response.

  8. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, the authors reveal a previously unexplored role of CYPA in DNA repair, particularly in the context of cells sensitive to the CsA. The authors' multi-faceted approach involved using CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, and specific DNA repair investigations. They suggest that CYPA, through its PPI activity, plays an active role in DNA repair, specifically in DNA end resection. They also demonstrate that inhibition or loss of CYPA results in impaired HRR following DNA replication fork stalling. Furthermore, the authors associate the loss and inhibition of CYPA with certain genetic vulnerabilities, suggesting potential therapeutic applications by exploiting CYPA PPI inhibition to selectively target cancer cells with characteristic genomic instability.

    The manuscript presents clear and comprehensive data, demonstrating the profound impact of CYPA on DNA repair. It would be suitable for publication after addressing the following points:

    1. It's surprising to find that the loss of CYPA abolished HU-induced NBS1 foci, as the MRE11 interactive domain of NBS1 should remain intact in CYPA deficient conditions and the N-terminus of NBS1 is dispensable for ATM activation (Kim et al., 2017; Stracker and Petrini, 2011). A more detailed mechanistic explanation of this phenotype would be appreciated. The authors should check the subcellular localization of NBS1 and the stability of MRN in wildtype and CYPA KO cells. Additionally, including the kinetics of NBS1 foci formation using multiple timepoints in wildtype and CYPA KO cells after damage will further support the observation.
    2. The authors showed that the interaction between CYPA and MRN didn't change after HU treatment. The authors should also include co-localization analysis of CYPA and NBS1 after HU.
    3. The paper demonstrated that BRCA2 knockdown cells were sensitive to CsA. The authors should also examine CsA sensitivity in BRCA2 deficient cancer cells. In addition, the authors could elaborate more on their criteria for selecting cancers for CYPA inhibition, whether it is based on high genomic instability or an addiction to HRR for survival.

    Significance

    The significance of this study is twofold: it adds a new layer to our understanding of DNA repair mechanisms and, importantly, it could point the way to novel therapeutic strategies for cancer. It will spark interest from molecular biologists to clinicians and pharmaceutical researchers.

  9. Version published to 10.1101/2023.06.27.546694v1 on bioRxiv