Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments

Nature volume 640, pages 1103–1111 (2025)Cite this article

Subjects

Abstract

The tumour-suppressor protein BRCA2 has a central role in homology-directed DNA repair by enhancing the formation of RAD51 filaments on resected single-stranded DNA generated at double-stranded DNA breaks and stimulating RAD51 activity1,2. Individuals with BRCA2 mutations are predisposed to cancer; however, BRCA2-deficient tumours are often responsive to targeted therapy with PARP inhibitors (PARPi)3,4,5,6. The mechanism by which BRCA2 deficiency renders cells sensitive to PARPi but with minimal toxicity in cells heterozygous for BRCA2 mutations remains unclear. Here we identify a previously unknown role of BRCA2 that is directly linked to the effect of PARP1 inhibition. Using biochemical and single-molecule approaches, we demonstrate that PARPi-mediated PARP1 retention on a resected DNA substrate interferes with RAD51 filament stability and impairs RAD51-mediated DNA strand exchange. Full-length BRCA2 protects RAD51 filaments and counteracts the instability conferred by PARPi-mediated retention by preventing the binding of PARP1 to DNA. Extending these findings to a cellular context, we use quantitative single-molecule localization microscopy to show that BRCA2 prevents PARPi-induced PARP1 retention at homologous-recombination repair sites. By contrast, BRCA2-deficient cells exhibit increased PARP1 retention at these lesions in response to PARPi. These results provide mechanistic insights into the role of BRCA2 in maintaining RAD51 stability and protecting homologous-recombination repair sites by mitigating PARPi-mediated PARP1 retention.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: BRCA2 modulates distinct states of RAD51 filaments.
Fig. 2: PARP1 inhibition destabilizes RAD51 filaments, which is counteracted by BRCA2.
Fig. 3: BRCA2 prevents PARPi-induced PARP1 retention on DNA.
Fig. 4: PARP1–DNA retention interrupts RAD51 strand-exchange activity.
Fig. 5: BRCA2 prevents PARPi-induced PARP1 retention at HR repair sites.

Similar content being viewed by others

Data availability

All data needed to evaluate the conclusions in the paper are incorporated in the paper and/or Supplementary Information. Datasets of smFRET traces and FACS measurements have been deposited into Zenodo (https://doi.org/10.5281/zenodo.14713371)85. Raw imaging and STORM data constitute a sizable dataset that will be made available by the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

Matlab codes for SMLM and pair correlation function (PCF) analyses and Python codes used in the smFRET assays are available from GitHub (https://github.com/d-in-crtl/SMLM and https://github.com/HamiltonGeorge/RothenbergLab_ALEX_pipeline).

References

  1. Jensen, R. B., Carreira, A. & Kowalczykowski, S. C. Purified human BRCA2 stimulates RAD51-mediated recombination. Nature 467, 678–683 (2010).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bell, J. C., Dombrowski, C. C., Plank, J. L., Jensen, R. B. & Kowalczykowski, S. C. BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA. Proc. Natl Acad. Sci. USA 120, e2221971120 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. McCabe, N. et al. BRCA2-deficient CAPAN-1 cells are extremely sensitive to the inhibition of poly(ADP-ribose) polymerase: an issue of potency. Cancer Biol. Ther. 4, 934–936 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921 (2005).

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Fong, P. C. et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N. Engl. J. Med. 361, 123–134 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Lord, C. J. & Ashworth, A. PARP inhibitors: synthetic lethality in the clinic. Science 355, 1152–1158 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wooster, R. et al. Identification of the breast-cancer susceptibility gene Brca2. Nature 378, 789–792 (1995).

    Article  ADS  CAS  PubMed  Google Scholar 

  8. Phelan, C. M. et al. Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families. Nat. Genet. 13, 120–122 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Tutt, A. & Ashworth, A. The relationship between the roles of BRCA genes in DNA repair and cancer predisposition. Trends Mol. Med. 8, 571–576 (2002).

    Article  CAS  PubMed  Google Scholar 

  10. Nyberg, T. et al. Prostate cancer risks for male BRCA1 and BRCA2 mutation carriers: a prospective cohort study. Eur. Urol. 77, 24–35 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wong, A. K., Pero, R., Ormonde, P. A., Tavtigian, S. V. & Bartel, P. L. RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene Brca2. J. Biol. Chem. 272, 31941–31944 (1997).

    Article  CAS  PubMed  Google Scholar 

  12. Sharan, S. K. et al. Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature 386, 804–810 (1997).

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Whelan, D. R. et al. Spatiotemporal dynamics of homologous recombination repair at single collapsed replication forks. Nat. Commun. 9, 3882 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  14. Davies, A. A. et al. Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol. Cell 7, 273–282 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Prakash, R., Zhang, Y., Feng, W. R. & Jasin, M. Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins. Cold Spring Harb. Perspect. Biol. 7, a016600 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Anand, R., Ranjha, L., Cannavo, E. & Cejka, P. Phosphorylated CtIP functions as a co-factor of the MRE11–RAD50–NBS1 endonuclease in DNA end resection. Mol. Cell 64, 940–950 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 447, 346–346 (2007).

    Article  ADS  CAS  Google Scholar 

  18. Sung, P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241–1243 (1994).

    Article  ADS  CAS  PubMed  Google Scholar 

  19. Belan, O. et al. Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein. Mol. Cell 83, 2925–2940 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sung, P. & Robberson, D. L. DNA strand exchange mediated by a Rad51–Ssdna nucleoprotein filament with polarity opposite to that of Reca. Cell 82, 453–461 (1995).

    Article  CAS  PubMed  Google Scholar 

  21. Baumann, P., Benson, F. E. & West, S. C. Human RAD51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Ogawa, T., Yu, X., Shinohara, A. & Egelman, E. H. Similarity of the yeast Rad51 filament to the bacterial Reca filament. Science 259, 1896–1899 (1993).

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Yu, X., Jacobs, S. A., West, S. C., Ogawa, T. & Egelman, E. H. Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA. Proc. Natl Acad. Sci. USA 98, 8419–8424 (2001).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pellegrini, L. et al. Insights into DNA recombination from the structure of a RAD51–BRCA2 complex. Nature 420, 287–293 (2002).

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Subramanyam, S., Ismail, M., Bhattacharya, I. & Spies, M. Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics. Proc. Natl Acad. Sci. USA 113, E6045–E6054 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Joo, C. et al. Real-time observation of RecA filament dynamics with single monomer resolution. Cell 126, 515–527 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Jimenez-Sainz, J. et al. BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair. eLife 11, e79183 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Marsden, C. G. et al. The tumor-associated variant RAD51 G151D induces a hyper-recombination phenotype. PLoS Genet. 12, e1006208 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Yin, Y. et al. A basal-level activity of ATR links replication fork surveillance and stress response. Mol. Cell 81, 4243–4257 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Laspata, N., Muoio, D. & Fouquerel, E. Multifaceted role of PARP1 in maintaining genome stability through its binding to alternative DNA structures. J. Mol. Biol. 436, 168207 (2024).

    Article  CAS  PubMed  Google Scholar 

  31. Zandarashvili, L. et al. Structural basis for allosteric PARP-1 retention on DNA breaks. Science 368, eaax6367 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Adolph, M. B. et al. RADX controls RAD51 filament dynamics to regulate replication fork stability. Mol. Cell 81, 1074–1083 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Simandlova, J. et al. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells. J. Biol. Chem. 288, 34168–34180 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Xue, H. et al. A two-step mechanism governing PARP1–DNA retention by PARP inhibitors. Sci. Adv. 8, eabq0414 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Adamowicz, M. et al. XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair. Nat. Cell Biol. 23, 1287–1298 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Hucl, T. et al. A syngeneic variance library for functional annotation of human variation: application to BRCA2. Cancer Res. 68, 5023–5030 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Whelan, D. R. et al. Super-resolution visualization of distinct stalled and broken replication fork structures. PLoS Genet. 16, e1009256 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Whelan, D. R. & Rothenberg, E. Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination. Proc. Natl Acad. Sci. USA 118, e2021963118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Nakamura, K. et al. Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination. Mol. Cell 81, 1084–1099 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li, M. & Yu, X. C. Function of BRCA1 in the DNA damage response is mediated by ADP-ribosylation. Cancer Cell 23, 693–704 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Scott, D. E. et al. A small-molecule inhibitor of the BRCA2–RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death. Cell Chem. Biol. 28, 835–847 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Noordermeer, S. M. & van Attikum, H. PARP inhibitor resistance: a tug-of-war in BRCA-mutated cells. Trends Cell Biol. 29, 820–834 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Dai, M. F. et al. Safety and hematological toxicities of PARP inhibitors in patients with cancer: a systematic review of randomized controlled trials and a pharmacovigilance analysis. Expert Rev. Anticancer Ther. 24, 613–622 (2024).

    Article  CAS  PubMed  Google Scholar 

  44. Frenel, J. S. et al. Efficacy of subsequent chemotherapy for patients with BRCA1/2-mutated recurrent epithelial ovarian cancer progressing on olaparib versus placebo maintenance: post-hoc analyses of the SOLO2/ENGOT Ov-21 trial. Ann. Oncol. 33, 1021–1028 (2022).

    Article  CAS  PubMed  Google Scholar 

  45. Schlacher, K. et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 993–993 (2011).

    Article  CAS  Google Scholar 

  46. Cong, K. et al. Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency. Mol. Cell 81, 3128–3144 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Vugic, D. et al. Replication gap suppression depends on the double-strand DNA binding activity of BRCA2. Nat. Commun. 14, 446 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Fanale, D. et al. BRCA1/2 variants of unknown significance in hereditary breast and ovarian cancer (HBOC) syndrome: looking for the hidden meaning. Crit. Rev. Oncol. Hematol. 172, 103626 (2022).

    Article  PubMed  Google Scholar 

  49. Jimenez-Sainz, J. & Jensen, R. B. Imprecise medicine: BRCA2 variants of uncertain significance (VUS), the challenges and benefits to integrate a functional assay workflow with clinical decision rules. Genes 12, 780 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rouleau-Turcotte, E. & Pascal, J. M. ADP-ribose contributions to genome stability and PARP enzyme trapping on sites of DNA damage; paradigm shifts for a coming-of-age modification. J. Biol. Chem. 299, 105397 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Beneyton, A. et al. The dynamic process of covalent and non-covalent PARylation in the maintenance of genome integrity: a focus on PARP inhibitors. NAR Cancer 5, zcad043 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Goehring, L. et al. Dormant origin firing promotes head-on transcription–replication conflicts at transcription termination sites in response to BRCA2 deficiency. Nat. Commun. 15, 4716 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Petropoulos, M. et al. Transcription–replication conflicts underlie sensitivity to PARP inhibitors. Nature 628, 433–441 (2024).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  54. Dias, M. P., Moser, S. C., Ganesan, S. & Jonkers, J. Understanding and overcoming resistance to PARP inhibitors in cancer therapy. Nat. Rev. Clin. Oncol. 18, 773–791 (2021).

    Article  PubMed  Google Scholar 

  55. Appleby, R., Joudeh, L., Cobbett, K. & Pellegrini, L. Structural basis for stabilisation of the RAD51 nucleoprotein filament by BRCA2. Nat. Commun. 14, 7003 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. Park, P. H. et al. Amplification of the mutation-carrying BRCA2 allele promotes RAD51 loading and PARP inhibitor resistance in the absence of reversion mutations. Mol. Cancer Ther. 19, 602–613 (2020).

    Article  CAS  PubMed  Google Scholar 

  57. Walmsley, C. S. et al. Convergent evolution of BRCA2 reversion mutations under therapeutic pressure by PARP inhibition and platinum chemotherapy. NPJ Precis. Oncol. 8, 34 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Jensen, R. Purification of recombinant 2XMBP tagged human proteins from human cells. Methods Mol. Biol. 1176, 209–217 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Carreira, A. et al. The BRC repeats of BRCA2 modulate the DNA-binding selectivity of RAD51. Cell 136, 1032–1043 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Pommier, Y. et al. Differential trapping of PARP1 and PARP2 by clinical PARP inhibitors. Eur. J. Cancer 48, 87–87 (2012).

    Article  Google Scholar 

  61. Murai, J. et al. Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib. Mol. Cancer Ther. 13, 433–443 (2014).

    Article  CAS  PubMed  Google Scholar 

  62. Matta, E., Kiribayeva, A., Khassenov, B., Matkarimov, B. T. & Ishchenko, A. A. Insight into DNA substrate specificity of PARP1-catalysed DNA poly(ADP-ribosyl)ation. Sci. Rep. 10, 3699 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  63. Bryant, H. E. et al. PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination. EMBO J. 28, 2601–2615 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Eustermann, S. et al. The DNA-binding domain of human PARP-1 interacts with DNA single-strand breaks as a monomer through its second zinc finger. J. Mol. Biol. 407, 149–170 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Rudolph, J., Mahadevan, J., Dyer, P. & Luger, K. Poly(ADP-ribose) polymerase 1 searches DNA via a ‘monkey bar’ mechanism. eLife 7, e37818 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Shao, Z. et al. Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo. Nucleic Acids Res. 48, 9694–9709 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Lahiri, S. & Jensen, R. B. DNA strand exchange to monitor human RAD51-mediated strand invasion and pairing. Methods Mol. Biol. 2153, 101–113 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study. Nat. Methods 15, 669–676 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Bronson, J. E., Fei, J. Y., Hofman, J. M., Gonzalez, R. L. & Wiggins, C. H. Learning rates and states from biophysical time series: a Bayesian approach to model selection and single-molecule FRET data. Biophys. J. 97, 3196–3205 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  70. van de Meent, J. W., Bronson, J. E., Wiggins, C. H. & Gonzalez, R. L. Empirical Bayes methods enable advanced population-level analyses of single-molecule FRET experiments. Biophys. J. 106, 1327–1337 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  71. van de Meent, J. W., Bronson, J. E., Wood, F., Gonzalez, R. L. Jr. & Wiggins, C. H. Hierarchically-coupled hidden Markov models for learning kinetic rates from single-molecule data. JMLR Workshop Conf. Proc. 28, 361–369 (2013).

    PubMed  PubMed Central  Google Scholar 

  72. Cui, T. J. et al. Argonaute bypasses cellular obstacles without hindrance during target search. Nat. Commun. 10, 4390 (2019).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  73. Gotz, M. et al. A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories. Nat. Commun. 13, 5402 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  74. Jensen, R. B., Ozes, A., Kim, T., Estep, A. & Kowalczykowski, S. C. BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage. DNA Repair 12, 306–311 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Coleman, K. E. et al. USP1-trapping lesions as a source of DNA replication stress and genomic instability. Nat. Commun. 13, 1740 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  76. Yin, Y. & Rothenberg, E. Probing the spatial organization of molecular complexes using triple-pair-correlation. Sci. Rep. 6, 30819 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  77. Lee, W. T. C. et al. Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling. Nat. Commun. 12, 2525 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  78. Pinkard, H. et al. Pycro-Manager: open-source software for customized and reproducible microscope control. Nat. Methods 18, 226–228 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Yin, Y. D., Lee, W. T. C. & Rothenberg, E. Ultrafast data mining of molecular assemblies in multiplexed high-density super-resolution images. Nat. Commun. 10, 119 (2019).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  80. Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat. Methods 10, 653–658 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Simultaneous multiple-emitter fitting for single molecule super-resolution imaging. Biomed. Opt. Express 2, 1377–1393 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  82. Sengupta, P. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 8, 969–975 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Holden, S. J., Uphoff, S. & Kapanidis, A. N. DAOSTORM: an algorithm for high-density super-resolution microscopy. Nat. Methods 8, 279–280 (2011).

    Article  CAS  PubMed  Google Scholar 

  84. Ovesny, M., Krizek, P., Borkovec, J., Svindrych, Z. K. & Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30, 2389–2390 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Rothenberg, E. BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments. Zenodo https://doi.org/10.5281/zenodo.14713371 (2025)

Download references

Acknowledgements

We thank members of the Rothenberg, Jensen and Huang laboratories for technical assistance, reagents and critical discussions; C. Joo and T. Cui for sharing the Matlab script for smFRET analysis; M. Cammer from the NYU Langone Shared Resource Microscopy Core for help with PLAs and analysis; M. B. Adolph and D. Cortez for purified RADX; H. Xue for purified CF647–HALO–PARP1; K. Zahn for precision protease; E. Nudler and V. Ephstein for help with radioactivity assays; and R. Ezhilarasan for help with the IR treatments. This research was supported in part by the V Foundation BRCA–Convergence Team Award to T.T.H., R.B.J. and E.R. and by the Laura Chang and Arnold Chavkin Charitable donation (to E.R. and T.T.H.), by the Goldberg Family Foundation (to T.T.H.) and the Gray Foundation (to R.B.J.). Research in the Rothenberg, Jensen and Huang laboratories was supported by grants from the NIH—GM134947, AI153040, CA247773 and CA288368 (to E.R.); CA270788 and CA215990 (to R.B.J.); and GM139610, ES031658 and CA288368 (to T.T.H.). Funding for research resources at the Laura and Isaac Perlmutter Cancer Center was provided by a Cancer Center Support Grant (NCRR S10 RR023704-01A1). Research in the Rothenberg laboratory was also supported by funds from Pfizer and Ionis Pharma.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University Grossman School of Medicine, New York, NY, USA

    Sudipta Lahiri, George Hamilton, Liana Goehring, Tony T. Huang & Eli Rothenberg

  2. Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT, USA

    Sudipta Lahiri, Gemma Moore & Ryan B. Jensen

Authors
  1. Sudipta Lahiri
    View author publications

    Search author on:PubMed Google Scholar

  2. George Hamilton
    View author publications

    Search author on:PubMed Google Scholar

  3. Gemma Moore
    View author publications

    Search author on:PubMed Google Scholar

  4. Liana Goehring
    View author publications

    Search author on:PubMed Google Scholar

  5. Tony T. Huang
    View author publications

    Search author on:PubMed Google Scholar

  6. Ryan B. Jensen
    View author publications

    Search author on:PubMed Google Scholar

  7. Eli Rothenberg
    View author publications

    Search author on:PubMed Google Scholar

Contributions

S.L. and E.R. conceptualized the project, designed the experiments and interpreted the data. S.L. performed all the experiments and analysed the data, and E.R. provided guidance and supervision. Further input was received from R.B.J. and T.T.H. S.L., R.B.J. and E.R. wrote the manuscript with input from all the authors. G.H. automated the acquisition of STORM data, FRET ALEX acquisition modes and related calibrations. G.M. cloned PARP1 in the phCMV1 2XMBP vector to create the full-length 2XMBP–PARP1 construct, which was used to purify PARP1 from HEK293T cells. L.G. provide guidance and assistance with PLAs. L.G. and S.L. analysed the immunofluorescence and PLA data. S.L. and G.H. analysed the smFRET data.

Corresponding authors

Correspondence to Ryan B. Jensen or Eli Rothenberg.

Ethics declarations

Competing interests

R.B.J. is named on patent US9150897B2, which references the phCMV1-2XMBP vector used to purify full-length BRCA2 and PARP1 used in this study. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Kinetic analysis of BRCA2 modulating distinct states of RAD51 filament.

(a) Autoradiogram of DNA strand exchange reaction with 3′ tailed DNA and increasing concentrations of RAD51 in the presence of ATP. (b) A pseudo-3D plot, Transition Density plot (TDP), generated from HMM analysis of the smFRET trajectories for RAD51 only bound to 3′ tailed DNA highlighting the transition densities corresponding to each state (N, IE, E). (c) Dwell time histograms (bin size = 90 msec) for each of the FRET states as obtained from HMM analysis (Supplementary Table 1) and FRET population histograms for unprocessed and unnormalized raw data from ebFRET output (n = 891; r = 3). (d-e) TDP generated from HMM analysis of the smFRET trajectories for BRCA2-RAD51 bound to 3′ tailed DNA highlighting the transition densities corresponding to each state (N′, IE′, E′) along with dwell time histograms (bin size = 90 msec) for each of the FRET states as obtained from HMM analysis (Supplementary Table 1) (n = 670; r = 3). In both cases, individual states were selected from the TDP, and the corresponding dwell-time histograms were fitted using a single exponential function to obtain the apparent rates of transitions out of the states The number of analyzed molecules and replicates are denoted by n and r, respectively. [BRCA2] = 0.04 µM | [RAD51] = 0.4 µM | [ATP] = 2 mM.

Source Data

Extended Data Fig. 2 Purification of PARP1 From HEK293T cells and characterization of biochemical activity.

(a) Coomassie blue stain and western blot of purified full-length PARP1. Lane 1,4: Marker, lane 2: 2XMBP-PARP1, lane 3: purified PARP1, lane 5: western blot for PARP1. (b) In vitro PARylation assay using recombinant PARP1 in the presence of NAD+ and an activator oligonucleotide (0.2 µM) mimicking DNA strand break. Stain free gel shows PARP1 loading in all lanes. (c) In vitro PARylation inhibition assay using recombinant PARP1 in the presence of NAD+, activator oligonucleotide and PARPi. (d) PARylated PARP1 dissociation assay using PARP1 and NAD+ and 40 bp ds-DNA (0.4 nM). (e) EMSA analyses of PARP1 binding to 0.4 nM of dsDNA and 3′ tailed DNA. (f) EMSA analyses of PARP1 binding to 0.4 nM of ss-DNA. (g) Quantification of EMSA results from e and f. Data shown is mean ± s.d (r = 3).

Source Data

Extended Data Fig. 3 Kinetic analysis of BRCA2-RAD51 binding on resected DNA in presence of PARP1 + PARPi.

(a-b) A TDP generated from HMM analysis of the smFRET trajectories for RAD51 and PARP1 + PARPi (a) bound to 3′ tailed DNA highlighting the transition densities corresponding to each state (PR + N, IE, E). From the TDP, individual states were selected, and the corresponding dwell-time histograms (bin size = 90 msec) were fitted using a single exponential function to obtain the apparent rates of transitions out of the states along with the FRET population histograms for unprocessed and unnormalized raw data from ebFRET output (b) (n = 850; r = 3). (c-d) A TDP generated from HMM analysis of the smFRET trajectories for BRCA2-RAD51 and PARP1 + PARPi (c) bound to 3′ tailed DNA with transition states (N′, IE′, E′) and corresponding dwell-time histograms (bin size = 90 msec) along with the FRET population histograms for unprocessed and unnormalized raw data from ebFRET output (d) (n = 652; r = 4). Brighter colors represent more frequent transitions, and the frequency scale is shown to the right of the graphs. The number of analyzed molecules and replicates are denoted by n and r, respectively. [BRCA2] = 0.04 µM | [RAD51] = 0.4 µM | [ATP] = 2 mM. [PARP1] = 0.4 µM | NAD+ = 1 mM | [PARPi] = 0.1 µM talazoparib (BMN673).

Source Data

Extended Data Fig. 4 Disruption of RAD51 filament by PARPi-induced PARP1 retention potentiated by PARPi potency.

(a-b) Representative smFRET trajectory and normalized FRET histogram of RAD51 only (first panel), RAD51 with PARP1 + talazoparib (second panel), RAD51 with PARP1 + saruparib (third panel), RAD51 with PARP1 + olaparib (fourth panel) and finally RAD51 with PARP1 + veliparib (fifth panel). All four panels with PARPi show three distinct states (PR + N, IE, E), however, the shift in the population distribution is worth noting where talazoparib and saruparib causes maximum disruption to the RAD51 filament followed by olaparib and then little to no disruption by veliparib. (c) Quantification of the effect of different PARPi to the RAD51 filament stability. Lanes: 1: RAD51 only, 2: talazoparib, 3: saruparib (n = 433), 4: olaparib (n = 460) and 5: veliparib (n = 450). Data shown is mean ± s.d (r = 2). (d) Schematic and western blot showing bead-capture experiment to measure binding of RAD51 to 167 nucleotide 3′ tailed DNA pre-incubated with PARP1 ± NAD+ ± saruparib/olaparib/veliparib. The number of analyzed molecules and replicates are denoted by n and r, respectively. Cartoons were created in BioRender. Lahiri, S. (2025) https://BioRender.com/g65f931. [RAD51] = 0.4 µM | [ATP] = 2 mM [PARP1]smFRET = 0.4 µM | NAD+ = 1 mM | [PARPi] = talazoparib (0.1 µM), saruparib (0.1 µM), olaparib (10 µM) and veliparib (100 µM).

Source Data

Extended Data Fig. 5 BRCA2 protects RAD51 filaments from dismantling by FBH1 and RADX.

Normalized FRET histograms of (a) DNA only (first panel), RAD51 only (second panel), RAD51 with FBH1 (third panel) (n = 320), BRCA2 with RAD51(fourth panel) and finally BRCA2-RAD51 with FBH1 (fifth panel) (n = 276) (b) DNA only (first panel), RAD51 only (second panel), RAD51 with RADX (third panel) (n = 446), BRCA2 with RAD51(fourth panel) and finally BRCA2-RAD51 with RADX (fifth panel) (n = 391) (c) DNA only (first panel), RAD51 only (second panel), RAD51 with PARP1 + PARPi (third panel), RAD51 with PARP1 + PARPi + FBH1 (n = 240) (fourth panel),RAD51 with PARP1 + PARPi + RADX (n = 220) (fifth panel) and BRCA2-RAD51 with PARP1 + PARPi (sixth panel). (d) Effect of different replication fork proteins that governs fork stability such as RADX and FBH1 on RAD51 filament stability, cartoon depiction on the right, defining the specificity that PARPi have on PARP1 mediated changes to RAD51 filaments. Data shown is mean ± s.d (r = 2). The number of analyzed molecules and replicates are denoted by n and r, respectively. Data for DNA only, DNA with RAD51 and DNA with BRCA2-RAD51 was replotted here for comparison purposes. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/l07t264. [BRCA2] = 0.04 µM | [RAD51] = 0.4 µM | [ATP] = 2 mM. [RADX] = 0.04 µM | [FBH1] = 0.04 µM. [PARP1] = 0.4 µM | NAD+ = 1 mM | [PARPi] = 0.1 µM talazoparib (BMN673).

Source Data

Extended Data Fig. 6 Characterization of PARP1 interaction with BRCA2 and RAD51.

(a) Control western blots showing bead-capture experiment to measure binding of RAD51 to ss/dsDNA co-incubated with PARP1 ± NAD+ ± PARPi. (b) Coomassie stain and western blot for human XRCC1 protein highlighting the quality of the protein used for the protein pull-down assays. (c) First and last washes from the pulldown assays performed in Fig. 3d. (d) Protocol for Protein-A Magnetic Beads pull-down assays for untagged PARP1 with XRCC1, BRCA2 and RAD51 in absence and presence of PARPi. (e) Protein A Magnetic Beads pull-down assay without PARPi. Lane 1: Marker, lane 2: “Control” no IgG1 against PARP1, lane 3: IgG1 against PARP1 + recombinant PARP1 (0.1 µM), lane 4-5: IgG1 for PARP1 + recombinant PARP1 (0.1 µM) ± NAD+ (0.25 µM) with XRCC1/2X MBP-BRCA2/RAD51 (0.1 µM each). (f) Protein A Magnetic Beads pull-down assay with PARPi. Lane 1: Marker, lane 2: “Control” no IgG1 against PARP1, lane 3–5: IgG1 against PARP1 + NAD+ + PARPi with XRCC1/2XMBP-BRCA2/RAD51. (g) Control experiment demonstrating addition of NAD+ or PARPi does not affect BRCA2-RAD51 mediated strand exchange reaction (Lane5,6). Lanes 1–4 are also shown in Fig. 1c. (h) TDP generated from HMM analysis of the smFRET trajectories for PARP1 + NAD+ + PARPi bound to 3′ tailed DNA followed by addition of RAD51 highlighting the transition densities corresponding to each state (Nucleation, N) for RAD51 and (PARP1 Retention, PR) for PARP1 respectively. Brighter colors represent more frequent transitions, and the frequency scale is shown to the right of the graphs. (i) Dwell time histograms (bin size = 90 msec) for each of the FRET states as obtained from HMM analysis along with the FRET population histograms for unprocessed and unnormalized raw data from ebFRET output (Supplementary Table 1) (n = 632; r = 2). The number of analyzed molecules and replicates are denoted by n and r, respectively. [BRCA2] = 0.04 µM | [RAD51] = 0.4 µM | [ATP] = 2 mM. [PARP1] = 0.4 µM | NAD+ = 1 mM | [PARPi] = 0.1 µM talazoparib (BMN673).

Source Data

Extended Data Fig. 7 Characterization of BRCA2-RAD51 and PARP1-RAD51 interaction levels following DSB induction.

(a) Experimental schematic depicting PLA design to quantify levels of BRCA2-RAD51 5 h post DNA damage. (b) Representative immunofluorescence images of BRCA2-RAD51 (magenta) PLA foci and DAPI staining to visualize nuclei (blue) for DLDpar cells at 5 hr post CPT (0.1 μM) or IR (12 Gy) treatment (positive control). Quantification for total number of foci and the average foci per nuclease is shown alongside. Individual data points represent single PLA foci per nucleus (n = 180). (c) Single Antibody controls for BRCA2, RAD51 and PARP1 antibodies. (d) Experimental schematic depicting PLA design to quantify levels of RAD51-PARP1 5 hrs post DNA damage. Quantification for total number of foci per nucleus is shown alongside as a violin plot (r = 2). Individual data points represent single PLA foci per nucleus. Values on graph indicate P-values of unpaired two-sample t-tests between each sample set. DLDpar (n = 349) and DLD−/− (n = 350). The number of cells analyzed, and replicates are denoted by n and r, respectively. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/d94j248. *P < 0.05, **P < 0.01, and ***P < 0.001 and ****P < 0.0001.

Source Data

Extended Data Fig. 8 Characterization of seDSB damage, DDR and initial resection.

(a) U2OS NT and 0.1 µM CPT treated cells were pulse-labeled with 10 µM EdU for 30 min and analyzed by flow cytometry for DNA synthesis (EdU) and DAPI staining for DNA content (10,000 events per sample) (r = 2). (b) Experimental schematic of inducing seDSB repair foci and EdU labeling strategy to visualize nascent DNA (naDNA). (Left) Levels of γH2AX (n = 95) and 53BP1 (n = 82) localizing at nascent DNA 1 h after CPT treatment. Individual data points represent result from single cell (r = 2). (Right) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow) and 53BP1 (magenta). Scale bar = 2 μm. (c) Western blot analysis for DNA Damage Response (DDR) pathway of U2OS cells treated with 0.1 µM CPT for 1 h. (d) Experimental schematic depicting spatiotemporal mapping of the arrivals, accumulations, and departures of MRE11(n1Hr = 248; n3Hrs = 234) and RPA (n1Hr = 363; n3Hrs = 238) at individual seDSBs. (e-f) Kinetics of MRE11 and RPA localizing at nascent DNA throughout repair over 3 h recovery (r = 2). (g) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow), MRE11 (green) and RPA (red). Scale bar = 2 μm. Values on graph indicate P-values of unpaired two-sample t-tests between NT and CPT-treated cells. The number of analyzed regions of interest (ROI) and replicates are denoted by n and r, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001 and ****P < 0.0001. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/e34f095.

Source Data

Extended Data Fig. 9 Time-Course Analysis of BRCA2, RAD51 and PARP1 recruitment in HR repair.

(a) Representative epifluorescence (inset) and super-resolution SMLM-STORM images of a single nucleus, post damage, and stained for naDNA (yellow), RAD51 (cyan) and BRCA2 (magenta). Scale bar = 2 μm. (b) Spatial and temporal mapping of BRCA2, RAD51 and PARP1 proteins respectively, over 16 h of recovery post damage. Values on the graph represents the percent change in localization of BRCA2, RAD51 and PARP1 to naDNA compared to (nontreated) NT cells. Data shown is mean ± s.d (r = 2). (c-e) Kinetics of BRCA2, RAD51 and PARP1 localizing at nascent DNA throughout repair (over 16 hr of recovery for BRCA2-RAD51 and 5 h recovery for PARP1). For all quantification graphs individual data points represent result from single cell. Values on graph indicate P-values of unpaired two-sample t-tests between NT, CPT-treated and CPT followed by PARPi treated cells. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/a58b868. *P < 0.05, **P < 0.01, and ***P < 0.001 and ****P < 0.0001. [CPT] = 0.1 µM.

Source Data

Extended Data Fig. 10 Effects of BRCA2 depletion and BRCA2-RAD51 interruption on PARPi-induced PARP1 retention.

(a) Experimental schematic depicting U2OS cells NT and 0.1 µM CPT treated cells were pulse-labeled with 10 µM EdU for 30 min followed by PARPi treatment at the mature phase of recombinase assembly. (b-d) Quantification of levels of RAD51, PARP1 and BRCA2 at naDNA over 5 h of recovery in U2OS cells WT, compared to (e,f) U2OS cells lacking BRCA2 expression (siBRAC2), in presence and absence of PARPi. (g) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow), PARP1 (red) and RAD51 (cyan). Scale bar = 2 μm. Western blots showing siRNA-mediated knockdown of BRCA2 in U2OS cells. Stain free gel used as a loading control. (h) Experimental schematic depicting U2OS cells in media containing CAM833 followed by PARPi treatment at the mature phase of recombinase assembly. Cartoon showing CAM833 disrupting the protein-protein interaction between RAD51 and BRCA2. High-magnification SMLM images of damage induced RAD51 filament (cyan) in U2OS cells (left) and their suppression by CAM833 (right) at naDNA (yellow), under the same experimental conditions. Scale bar = 0.2 μm. (i,j) Levels of RAD51 and PARP1 at naDNA over 5 h of recovery in U2OS cells with CAM833 in media. CAM833 potentiates toxicity and synergizes with PARPi leading to PARP1 retention. We note that for both RAD51 and PARP1 the CPT only and CPT with PARPi data plotted in this panel is also used in panels e,f for comparison. Quantification is from two independent experiments and values on graph indicate P-values of unpaired two-sample t-tests between each sample set. (k,l) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow), PARP1 (magenta) and RAD51 (cyan) in presence and absence of PARPi. Scale bar = 2 μm. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/q21l624. *P < 0.05, **P < 0.01, and ***P < 0.001 and ****P < 0.0001. CPT = 0.1 µM | [CAM833] = 25 µM.

Source Data

Supplementary information

Supplementary Information

Supplementary Figs. 1–17 and Supplementary Tables 1–4.

Reporting Summary

Peer Review File

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lahiri, S., Hamilton, G., Moore, G. et al. BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments. Nature 640, 1103–1111 (2025). https://doi.org/10.1038/s41586-025-08749-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41586-025-08749-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing