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In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search

Nature Structural & Molecular Biology volume 30, pages 1582–1591 (2023)Cite this article

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An Author Correction to this article was published on 22 November 2023

A Publisher Correction to this article was published on 07 September 2023

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Abstract

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks (DSB). HR uses an undamaged homologous DNA sequence as a template for copying the missing information, which requires identifying a homologous sequence among megabases of DNA within the crowded nucleus. In eukaryotes, the conserved Rad51–single-stranded DNA nucleoprotein filament (NPF) performs this homology search. Although NPFs have been extensively studied in vitro by molecular and genetic approaches, their in vivo formation and dynamics could not thus far be assessed due to the lack of functional tagged versions of Rad51. Here we develop and characterize in budding yeast the first fully functional, tagged version of Rad51. Following induction of a unique DSB, we observe Rad51–ssDNA forming exceedingly long filaments, spanning the whole nucleus and eventually contacting the donor sequence. Emerging filaments adopt a variety of shapes not seen in vitro and are modulated by Rad54 and Srs2, shedding new light on the function of these factors. The filaments are also dynamic, undergoing rounds of compaction and extension. Our biophysical models demonstrate that formation of extended filaments, and particularly their compaction–extension dynamics, constitute a robust search strategy, allowing DSB to rapidly explore the nuclear volume and thus enable efficient HR.

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Fig. 1: Rad51 forms filaments on DSB to perform homology search in living cells.
Fig. 2: Rad51 forms micrometer-long filaments on ssDNA.
Fig. 3: Rad51 filament structures are regulated by Rad52, Srs2 and Rad54.
Fig. 4: In vivo dynamics of Rad51 filaments.
Fig. 5: Fast and robust search is facilitated by Rad51 filament dynamics.

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Data availability

All data are available in the main text or the supplementary materials. Raw images presented in the paper are openly available in Zenodo (https://doi.org/10.5281/zenodo.8104491). All primary data are available in Zenodo (https://doi.org/10.5281/zenodo.8362494). Strains and raw images quantified but not shown in the paper are available upon request. Source data are provided with this paper.

Code availability

Codes for polymer model are available at https://github.com/open2c/polychrom, https://openmm.org/ and https://github.com/mirnylab/. Other codes are available in the supplementary materials.

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Acknowledgements

We thank M. Lisby (University of Copenhagen, Denmark) and L. Symington (Columbia University, NY, USA) for sharing strains, M. Garnier for his help on image analysis, A. Dubos-Taddei for his help in exploring videos, the members of the Taddei and Mirny laboratories, A. Piazza and E. Coïc for helpful discussions, B. Futcher (State University of New York, Stoney Brook, NY, USA) and V. Borde (Institut Curie, Paris, France) for sharing reagents and G. Almouzni for her critical reading of the paper. A.T.’s team was financially supported by funding from the Labex DEEP (grant nos. ANR-11-LABEX-0044 DEEP and ANR-10-IDEX-0001-02 PSL), PIC3I IC-CEA 2020 and Centre National de la Recherche Scientifique (CNRS) grant no. 80prime PhONeS. S.L. received funding from the CNRS MITI. L.A.M. is supported by Chaire d’excellence internationale Blaise Pascal by the Île-de-France region. We acknowledge the PICT-IBiSA at the Pasteur Imaging Facility of the Institut Curie, member of the France Bioimaging National Infrastructure (grant no. ANR-10-INBS-04).

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Author notes

  1. These authors contributed equally: Judith Miné-Hattab, Marie Villemeur.

Authors and Affiliations

  1. Institut Curie, Université PSL, Sorbonne University, CNRS, Nuclear Dynamics, Paris, France

    Siyu Liu, Judith Miné-Hattab, Marie Villemeur, Leonid A. Mirny & Angela Taddei

  2. Institute for Integrative Biology of the Cell (I2BC), University of Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France

    Raphaël Guerois

  3. Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA

    Henrik Dahl Pinholt & Leonid A. Mirny

  4. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Leonid A. Mirny

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Contributions

S.L. and M.V. generated strains and performed experiments. S.L. and J.M.-H. quantified microscopy experiments. R.G. designed the Rad51 tagging strategy. L.A.M. performed the modeling and supervised H.D.P., who performed polymer simulations. A.T., S.L., M.V. and J.M.-H. contributed to the design of the experiments. All authors contributed to the interpretation of the data, the drafting of the figures and the writing and revision of the paper.

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Correspondence to Angela Taddei.

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An invention disclosure related to the results was submitted to Institut Curie Technology Transfer Office for filing a patent application.

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Extended data

Extended Data Fig. 1 Rad51-iGFP is expressed at wild-type level and form filaments upon DSB induction, in contrast to N and C-tagged Rad51.

a. Western blot on WT and Rad51-iGFP strains at different time after DSB induction, rad51Δ is shown as negative control. b. Representative fluorescent images of YFP-Rad51 (from27, N-terminal tag) and Rad51-iGFP after 2h Zeocin treatment, Z-projection is applied. c. Representative fluorescent images of Rad51-GFP (C-terminal tag) and Rad51-iGFP with I-SceI cutting site after 4h galactose induction, Z-projection is applied.

Source data

Extended Data Fig. 2 Quantification methods and Rad51-iGFP in haploid and diploid strain.

a. Quantification of Rad51 structures using the machine-learning-based image analysis tool, Ilastik28. Input: Z-projection fluorescent images. Output: left, number of nuclei; right, numbers of Rad51 foci, globular structures, and filaments. b. Representative images fluorescent images of a Rad51-iGFP in haploid and diploid strain at different time after DSB induction maximal Z-projections are shown. c. Percentages of Rad51 foci, globular structures, and filaments at different time after DSB induction in haploid and diploid strains quantified as in d (n > 100 cells examined per experiment, per condition with a total of n = 2416 over 2 experiments, data are presented as mean values +/− SEM).

Source data

Extended Data Fig. 3 Rad51-iGFP canform filaments in the presence of an intrachromosomal donor sequence.

a, b. Representative fluorescent images and percentages of Rad51-iGFP structures in the following strains after 2, 4 or 6h endonuclease induction: yAT4553 where HO creates a DSB at the MATalpha locus that can be repaired with the HM loci on the same chromosome (with a preference for HMR, located 90kb away from the cut site); yAT3880, where I-SceI creates a DSB at the lys2 locus in the absence of a donor sequence, yAT4552, where I-SceI creates a DSB at the lys2 locus in the presence of a donor sequence, 20kb upstream. maximal Z-projections are shown. (n>100 cells examined per experiment, per condition with a total of n = 6670 cells examined over 2 experiments; data are presented as mean values +/− SEM).

Source data

Extended Data Fig. 4 Rad51-iGFP upon replication stress and prolonged G1 synchronization.

a. Representative fluorescent images of Rad51-iGFP after 1, 2, 3 h release from 1 h 200mM hydroxyurea treatment, Z-projection is applied. b. Representative images (Transmitted-light image, fluorescent image, and merged image) of Rad51-iGFP with I-SceI cutting site after 2h galactose induction. Cells that are unambiguously in G1 phase, as attested by the absence of a bud, are circled. Z-projection is applied. c. Schemes for G1 synchronization and galactose induction, 10μM alpha factor is induced at the beginning of synchronization and every 30min after galactose induction. Representative images (Transmitted-light image and fluorescent image) and percentages of Rad51 structures of Rad51-iGFP with I-SceI cutting site after 3h galactose induction under alpha factor arrest or release, maximal Z-projections are shown. Scale bars: 2 µm.

Source data

Extended Data Fig. 5 Effects of Sgs1 and Exo1 on Rad51 filament intensities and distribution of filament length in diploid and haploid cells.

a. Comparison of total intensities of Rad51 filaments in WT, sgs1∆, exo1∆, sgs1∆ exo1∆ strains as indicated (n = 608 Rad51 filaments are analyzed, two-sided Wilcoxon rank sum test, **** represents p < 0.0001). On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the '+' marker symbol. b. Distributions of filament length in haploid (left) and diploid (right) strains, same data as in Fig. 2c. n = 1720 filaments are analyzed from 2 independent experiments.

Source data

Extended Data Fig. 6 Effects of Rad54 on Rad51 structures.

a. Representative images (Transmitted-light image and GFP channel fluorescent image) of Rad51-iGFP strains in WT and mutants 4 hours after DSB induction as indicated. b. Percentages of foci, globular structures, and filaments in (n>100 cells examined per experiment, per condition with a total of n = 3485 cells examined over 2 experiments) on the same strains as in A.

Source data

Extended Data Fig. 7 Variety of nuclear Rad51 filament shapes observed in haploid and diploid living cells expressing Rad51-iGFP and by immune-fluorescence in cells expressing the untagged Rad51 protein.

a. Representative fluorescence images a Rad51-iGFP Nup-mCherry strain at 2,4,6 hrs after DSB induction. GFP channel and RFP channel are combined, Z-projection is applied on fluorescent images. b. Representative filaments of WT haploid and diploid strains at different time after galactose induction. c. Untagged Rad51 localized by Immunofluorescence 6 hours after DSB induction show the same classes of filament shapes observed in cells expressing the untagged Rad51 protein.

Extended Data Fig. 8 Rad51 filaments in mutants.

a. Representative filaments of WT and mutants at 6h after galactose induction, Z-projection is applied. b. Distributions of class1 and class2 filament length (Rods and bent rods) in WT and srs2Δ 2,4 or 6 h after galactose induction, statistical test: two-side Wilcoxon rank sum test, 2 h, p = 0.1187;4h p = 0.0409;6h p = 0.0025. n = 540 Rad51 filaments analyzed from 2 experiments. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the '+' marker symbol. Scale bars: 2 µm.

Source data

Extended Data Fig. 9 Dynamics of Rad51 filaments in srs2∆ or rad54∆ haploid and wild-type diploid cells.

a. Examples of time-lapse images in WT and mutants upon DSB, images acquired every 2 minutes, Z-projection is applied. b. Number of frames from Rad51 foci to filaments (>6 pixel) in WT and mutants upon DSB (n = 41 movies from 4 experiments). On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the most extreme data points not considered outliers, and the outliers are plotted individually using the '+' marker symbol. c, d. Examples of time-lapse images in Rad51-iGFP diploid strain upon DSB, images acquired every 2 minutes, Z-projection is applied (Supplementary Movies 4, 5). Scale bar: 2µm.

Source data

Extended Data Fig. 10 The ATPase dead mutant Rad51-K191 does not form filament in living cells; Srs2 or Rad54 are not required for compaction/extension events.

a. Representative images (Transmitted-light image and GFP channel fluorescent image) and average percentages of foci, globular structures, and filaments in Rad51-iGFP and Rad51-K191R-iGFP strain 4 hrs after DSB induction. Z-projection is applied. Of note, gray levels are set between 100 and 4500 before 16 to 8-bit conversion to visualize wild-type Rad51 structures and to 100–1500 to visualize Rad51-K191R structures (n = 1186 cells in 2 experiments, data are presented as mean values +/− SEM). b–c. Examples of time-lapse images in Rad51-iGFP mutant strains upon DSB, images acquired every 2 minutes, Z-projection is applied (Supplementary Movies 6, 7). Red rectangle: Compacted Rad51 structures. Scale bars: 2 µm.

Source data

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Liu, S., Miné-Hattab, J., Villemeur, M. et al. In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search. Nat Struct Mol Biol 30, 1582–1591 (2023). https://doi.org/10.1038/s41594-023-01065-w

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