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Structure and function of the RAD51B–RAD51C–RAD51D–XRCC2 tumour suppressor

Nature volume 619, pages 650–657 (2023)Cite this article

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Abstract

Homologous recombination is a fundamental process of life. It is required for the protection and restart of broken replication forks, the repair of chromosome breaks and the exchange of genetic material during meiosis. Individuals with mutations in key recombination genes, such as BRCA2 (also known as FANCD1), or the RAD51 paralogues RAD51B, RAD51C (also known as FANCO), RAD51D, XRCC2 (also known as FANCU) and XRCC3, are predisposed to breast, ovarian and prostate cancers1,2,3,4,5,6,7,8,9,10 and the cancer-prone syndrome Fanconi anaemia11,12,13. The BRCA2 tumour suppressor protein—the product of BRCA2—is well characterized, but the cellular functions of the RAD51 paralogues remain unclear. Genetic knockouts display growth defects, reduced RAD51 focus formation, spontaneous chromosome abnormalities, sensitivity to PARP inhibitors and replication fork defects14,15, but the precise molecular roles of RAD51 paralogues in fork stability, DNA repair and cancer avoidance remain unknown. Here we used cryo-electron microscopy, AlphaFold2 modelling and structural proteomics to determine the structure of the RAD51B–RAD51C–RAD51D–XRCC2 complex (BCDX2), revealing that RAD51C–RAD51D–XRCC2 mimics three RAD51 protomers aligned within a nucleoprotein filament, whereas RAD51B is highly dynamic. Biochemical and single-molecule analyses showed that BCDX2 stimulates the nucleation and extension of RAD51 filaments—which are essential for recombinational DNA repair—in reactions that depend on the coupled ATPase activities of RAD51B and RAD51C. Our studies demonstrate that BCDX2 orchestrates RAD51 assembly on single stranded DNA for replication fork protection and double strand break repair, in reactions that are critical for tumour avoidance.

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Fig. 1: Cryo-EM structure of BCDX2.
Fig. 2: Structural comparison of BCDX2 and RAD51 filaments.
Fig. 3: Structural insights into RAD51B, RAD51C, RAD51D and XRCC2 VUS.
Fig. 4: Coupled ATPase activities of RAD51B and RAD51C.
Fig. 5: ATP hydrolysis by BCDX2 is required for RAD51 filament growth.
Fig. 6: Interaction of BCDX2 with ssDNA and RAD51.

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Data availability

Cryo-EM density maps and atomic models of BCDX2 have been deposited in the Electron Microscopy Data Bank (EMDB) and PDB under the following accession codes: BCDX2–ADP.AlFx (EMD-17206 and 8OUZ), BCDX2–ADP.AlFx–ssDNA (EMD-17207) and BCDX2–ADP.BeFx (EMD-17205 and 8OUY). All other data and materials reported here are available on request.

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Acknowledgements

The authors thank members of the West laboratory for support and advice; N. Cronin and A. Nans for cryo-EM data collection; H. N. Ho and G. Cameron for reagents; A. Kaczmarczyk and D. Rueda for help with single-molecule analyses; M. Tuppi for contributions to experimental design; R. Carzaniga, Q. Chen and D. Barry for assistance with electron microscopy and data analysis; A. Costa and D. Benton for critical reading of this manuscript; M. Spies for providing pCH1-RAD51opt; M. Modesti for providing phRPA-mStrawberry and M. Allen for pET-TEV; X. Zhang, P. Walker, the Structural Biology STP and the Peptide Chemistry STP. Work in the S.C.W. laboratory was funded by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2098), the UK Medical Research Council (CC2098) and the Wellcome Trust (CC2098), by the European Research Council (ERC-ADG-666400), by the BBSRC (BB/W01355X/1) and by the Louis-Jeantet Foundation. Work in the S.J.B. laboratory was supported by the Francis Crick Institute (CC2057), European Research Council Advanced Investigator grants (TelMetab 742437 and ChrEndProt 101053876), and a Wellcome Trust Senior Investigator grant (220808/Z/20/Z). For the purpose of Open Access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

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Author notes

  1. Ondrej Belan

    Present address: Department of Genetics, Harvard Medical School, Boston, MA, USA

  2. Ondrej Belan

    Present address: Division of Genetics, Brigham and Women’s Hospital, Boston, MA, USA

  3. Monica C. Rodrigo-Brenni

    Present address: AstraZeneca, Cambridge, UK

  4. These authors contributed equally: Luke A. Greenhough, Chih-Chao Liang, Ondrej Belan

Authors and Affiliations

  1. The Francis Crick Institute, London, UK

    Luke A. Greenhough, Chih-Chao Liang, Ondrej Belan, Simone Kunzelmann, Sarah Maslen, Monica C. Rodrigo-Brenni, Roopesh Anand, Mark Skehel, Simon J. Boulton & Stephen C. West

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Contributions

L.A.G., C.-C.L. and S.C.W. designed the study. L.A.G. purified proteins and carried out biochemical analyses, with contributions from M.C.R.-B., and performed cryo-EM and data analyses with C-C.L. O.B. carried out the single-molecule assays and was assisted by R.A. S.K., S.M. and M.S. performed biophysical assays, MS and HDX experiments and analysed the data. S.C.W. and S.J.B. supervised the experiments and data analyses. L.A.G. and S.C.W. prepared the manuscript with contributions from all authors.

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Correspondence to Stephen C. West.

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S.J.B. is a co-founder and VP Science Strategy at Artios Pharma. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Single particle analysis pipeline of BCDX2-ADP.AlFx and BCDX2-ADP.AlFx-ssDNA.

Summary of the data processing strategies, including intermediate 2D and 3D class averages, which yield the final reconstructions of BCDX2-ADP.AlFx (2.2 Å) and BCDX2-ADP.AlFx-ssDNA (2.9 Å).

Extended Data Fig. 2 Single particle analysis pipeline of BCDX2-ADP.BeFx.

Summary of the data processing strategy, including intermediate 2D and 3D class averages, which yields the final reconstruction of BCDX2-ADP.BeFx (3.4 Å).

Extended Data Fig. 3 Angular distribution, map and model resolution statistics and local resolution.

a, Angular distribution plots. b, Fourier Shell Correlation (FSC) plots. c, Map vs model FSCs. d, Local resolution estimates for BCDX2-ADP.AlFx (2.2 Å), BCDX2-ADP.AlFx (2.9 Å) and BCDX2-ADP.BeFx (3.4 Å).

Extended Data Fig. 4 Structural and biochemical analyses of BCDX2.

a, SDS-PAGE of BCDX2 and BNTDCDX2. b, Front (left) and back (right) views of BCDX2-ADP.BeFx cryo-EM map (3.4 Å) and atomic model. c, and d, NS-EM 2D class averages of BCDX2 showing movement of the mobile domain in the presence of ATP or ADP, respectively. e, Limited proteolysis of BCDX2. SDS-PAGE gel and immunoblots of Superdex 200 3.2/300 fractions for untreated and chymotrypsin treated BCDX2. For gel and immunoblot source data, see Supplementary Fig. 1.

Extended Data Fig. 5 Coupling of RAD51B and RAD51C ATPases.

a, b and c, Atomic models from the BCDX2-ADP.BeFx structure (3.4 Å) showing the binding of ATP by XRCC2, ATP by RAD51D and ADP by RAD51C, respectively. The Walker A lysine and threonine, Walker B aspartate, catalytic glutamate and the lysine finger from the adjacent subunit are indicated. Density of the nucleotide is shown as black mesh. Green spheres = Mg2+ ions. d, Sequence alignment of the RAD51, RAD51B, RAD51C, RAD51D and XRCC2 protein sequences. Highlighted in red are conserved catalytic glutamate residues (RAD51, RAD51B and RAD51C) and in cyan are lysine fingers (RAD51B, RAD51C, RAD51D and XRCC2). e, SDS-PAGE of the mutant proteins used in Fig. 4. For gel source data, see Supplementary Fig. 1. f, HPLC chromatograms (left) of standards (standardised to 2 µM ATP, ADP, ATPγS) and BCDX2 (standardised to 1 µM BCDX2). Bar chart (mean + s.d.) of bound nucleotides (right). All are n = 3 except wt and BK114ACDX2 which is n = 7 and n = 4 respectively. n values are independent experiments. Unpaired two-tailed t-test. g, Bar chart (mean + s.d.) of ATP hydrolysis rate. All are n = 3 except wt which is n = 6. n values are independent experiments. Unpaired two-tailed t-test. h and i, Difference plots between BCDX2-ADP.AlFx and BCDX2-ADP, and BCDX2-ADP.BeFx and BCDX2-ADP, respectively, showing the level of deuterium uptake after 3 (orange), 30 (red), 300 (blue) and 3,000 (black) seconds. Positive values = exposure; negative values = protection. RAD51B: 116 peptides, 91.1% coverage, 3.36 redundancy. RAD51C: 134 peptides, 88% coverage, 4.11 redundancy. RAD51D: 99 peptides, 93.0% coverage, 3.5 redundancy. XRCC2: 76 peptides, 93.6% coverage, 2.67 redundancy.

Extended Data Fig. 6 ATP hydrolysis by BCDX2 stimulates RAD51 filament assembly.

a, Representative NS-EM micrographs of RAD51 filaments in the absence and presence of BCDX2, showing filaments detected by curvilinear line analysis and crYOLO particle picking. Scale bar = 100 nm. b, Scatter plot (mean + s.d.) of number of crYOLO picked particles per micrograph (n = 631 micrographs). Unpaired two-tailed t-test. c, Force measured between the traps as a function of time in the absence (n = 6) and presence (n = 7) of BCDX2. Shaded area represents SEM. d, Representative kymographs and time-binned intensity histograms verses genomic position on RPAmStrawberry coated λ ssDNA for RAD51AF488 signal (blue) in the absence or presence of BCDX2. Each line represents 1 min timepoint. Nucleation rate was calculated for each time frame of the smoothed kymograph by detecting peaks in the AF488 intensity profile. e, Kymographs of RAD51 filament growth (by movement of ssDNA into protein + ATP channel) and subsequent disassembly (movement into buffer only channel containing no ATP). Growth and disassembly rates were measured as a slope of the border of the RAD51AF488 signal. f, Scatter plot (median and IQR) of RAD51 disassembly rates in the absence (n = 47 filaments) and presence (n = 50 filaments) of BCDX2. Two-sided Mann-Whitney test. g, Force measured between the traps as a function of time absence (n = 6) or presence of BCDX2 (n = 6), BE144ACDX2 (n = 6), BCE161ACDX2 (n = 7) and BE144ACE161ACDX2 (n = 6). n values are independent experiments. Shaded area represents SEM.

Extended Data Fig. 7 ATP hydrolysis is required for ssDNA binding.

a, Changes in fluorescence anisotropy upon binding of WT BCDX2 to FAM-dN15nt ssDNA in the presence of ATPγS (n = 3, magenta), AMP-PNP (n = 3, turquoise), ADP.BeFx (n = 3, navy) and ADP.Vanadate (n = 3, brown). Lines denote quadratic curve fits. Each point and error bar denotes mean + s.d. n values are independent experiments. b, Calculated ssDNA binding affinity constants (centre = KD values, error bars = 95% CI lower and upper limits) of wild-type BCDX2 in the presence of ATP, ADP, ADP.AlFx, ATPγS, AMP-PNP, ADP.BeFx and ADP.Vanadate. X axis = log10 scale. c, SDS-PAGE of dual labelled BCDX2, BE144ACDX2 and BCE161ADX2. Left = Coomassie stain. Right = AF555 and AF647 fluorescence. For gel source data, see Supplementary Fig. 1. d, Bar chart (mean + s.d.) of binding frequencies of BCDX2, BE144ACDX2 and BCE161ADX2 to ssDNA in the absence (n = 3, n = 5, n = 5 independent experiments, respectively) or presence (n = 4, n = 5, n = 5 independent experiments, respectively) of RAD51 in the first 30 s window. Unpaired two-tailed t-test. e and f, Normalized fluorescence intensity for AF555 signal for fluorescently labelled BCDX2, BE144ACDX2 or BCE161ADX2 over time in the absence (n = 3, n = 4, n = 4 independent experiments, respectively) or presence (n = 4, n = 5, n = 5 independent experiments, respectively) of unlabelled RAD51. Shaded area represents SEM. g, Force measured between the traps as a function of time of RAD51 in the absence (n = 5) and presence of fluorescently labelled BCDX2 (n = 7), BE144ACDX2 (n = 6) or BCE161ADX2 (n = 5). n values are independent experiments. Shaded area represents SEM. h, Kymographs of RAD51AF555/BCDX2AF647 FRET. 1 = FRET between RAD51 and BCDX2. 2 = BCDX2 dissociates or RAD51 binding. 3 = RAD51 dissociates. i, Scatter plot of AF647 intensity values for RAD51 alone (bleed-through) (n = 39), BAF647CDX2 (n = 48) and BCDX2ybbr-AF647 (n = 61). Median + IQR. Two-sided Mann-Whitney statistical test.

Extended Data Fig. 8 Mechanism of ssDNA binding by BCDX2.

a, Cryo-EM model of BCDX2 bound to ssDNA. Unmodelled density of ssDNA is labelled (red) and an additional density, thought to be due to a protein conformation change, is indicated (blue). b, Difference plot between BCDX2-ADP.AlFx and BCDX2-ADP.AlFx-ssDNA showing level of deuterium uptake after 3 (orange), 30 (red), 300 (blue) and 3000 (black) seconds. Positive values = exposure (red); negative values = protection (blue). Purple text = L1 loops. Orange text = L2 loops. RAD51B: 110 peptides, 97.7% coverage, 3.38 redundancy. RAD51C: 113 peptides, 98.1% coverage, 3.32 redundancy. RAD51D: 108 peptides, 98.5% coverage, 3.88 redundancy. XRCC2: 89 peptides, 97.9% coverage, 3.27 redundancy. c, Conservation of putative ssDNA binding arginine residues in the L1 loops of RAD51B, RAD51C, RAD51D and XRCC2. d, SDS-PAGE of mutant BCDX2 proteins. For gel source data, see Supplementary Fig. 1. Fluorescence anisotropy ssDNA binding curves in the absence of ATP (blue) or ADP (red) for e, BR217ACDX2 (n = 3) and BR231ACDX2 (n = 3), f, BCR258ADX2 (n = 3) and BCR258HDX2 (n = 3), g, BCDR221AX2 (n = 3), and h, BCDX2R159A (n = 3). n values are independent experiments. Lines denote quadratic curve fits. Each point and error bar denotes mean + s.d. i, Calculated ssDNA binding affinity constants (centre = KD values, error bars = 95% CI lower and upper limits) of BCDX2 arginine mutants. X axis = log10 scale. j, Bar chart (mean + s.d.) of ATP hydrolysis rate. Unpaired two-tailed t-test. All n=3, except wt which is n = 6. n values represent independent experiments. k, Correlation of ssDNA binding affinity (errors = 95% CI) against fold increase in ssDNA ATPase stimulation (mean + s.d.). Curve fitting by exponential decay curve. l, Bar chart (mean + s.d.) of FRET fluorescence ratio (IA/ID) between 5′-Cy3-dN15nt or dN15nt-Cy3-3′ and BCDX2, BAF647CDX2 and BCDX2AF647. All n = 3 independent experiments. Two tailed unpaired t-test.

Extended Data Fig. 9 Interplay between BCDX2 and RAD51 filaments.

a, RAD51B binds to ssDNA and RAD51. b, Structure of the RAD51 filament (PDB = 5H1B) bound to ssDNA (left). Modelling of RAD51BCTD on RAD51-1 (rmsd = 1.024 Å) (right). Zoomed view (upper panels) showing engagement of RAD51R229/R241 and RAD51BR217/R231 with ssDNA. c, RAD51B interacts RAD51C during ATP hydrolysis, promoting high affinity ssDNA binding by the BCDX2 complex. d, Structural modelling of RAD51C binding a triplet of nucleotides in both RAD51C ground and active intermediate conformations.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Greenhough, L.A., Liang, CC., Belan, O. et al. Structure and function of the RAD51B–RAD51C–RAD51D–XRCC2 tumour suppressor. Nature 619, 650–657 (2023). https://doi.org/10.1038/s41586-023-06179-1

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