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Dynamics of the DYNLL1–MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs

Nature Structural & Molecular Biology volume 30, pages 1456–1467 (2023)Cite this article

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Abstract

The extent and efficacy of DNA end resection at DNA double-strand breaks (DSB) determine the repair pathway choice. Here we describe how the 53BP1-associated protein DYNLL1 works in tandem with the Shieldin complex to protect DNA ends. DYNLL1 is recruited to DSBs by 53BP1, where it limits end resection by binding and disrupting the MRE11 dimer. The Shieldin complex is recruited to a fraction of 53BP1-positive DSBs hours after DYNLL1, predominantly in G1 cells. Shieldin localization to DSBs depends on MRE11 activity and is regulated by the interaction of DYNLL1 with MRE11. BRCA1-deficient cells rendered resistant to PARP inhibitors by the loss of Shieldin proteins can be resensitized by the constitutive association of DYNLL1 with MRE11. These results define the temporal and functional dynamics of the 53BP1-centric DNA end resection factors in cells.

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Fig. 1: DYNLL1 recruitment to DSBs is dependent on 53BP1 but independent of other 53BP1-associated factors.
Fig. 2: DYNLL1 regulates MRE11 activity independent of 53BP1.
Fig. 3: Functional effect of DYNLL1 at DSBs in 53BP1-deficient cells.
Fig. 4: DYNLL1 disrupts MRE11 dimerization to impair its retention on chromatin.
Fig. 5: Functional comparison of DYNLL1 and the Shieldin complex.
Fig. 6: Kinetics and dependencies of Shieldin complex recruitment to DSBs.
Fig. 7: DYNLL1 is required for Shieldin loading to DSBs.
Fig. 8: Phosphorylated DYNLL1 negatively regulates end resection and Shieldin complex recruitment at DSBs.

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Data availability

Numerical source data are provided with this paper. A description of the AlphaFold modeling can be found in the Methods. All other data reported in this paper will be shared by the lead contact upon request.

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Acknowledgements

D.C. is supported by grants R01 CA208244 and R01 CA264900, Gray Foundation Team Science Award, DOD Ovarian Cancer Award W81XWH-15-0564/OC140632, Tina’s Wish Foundation, Detect Me If You Can, V Foundation Award and the Claudia Adams Barr Program in Innovative Basic Cancer Research. M.L.S. is supported by National Institutes of Health (NIH) grant F32 GM149115. J.A.T. and A.S. were partly supported by NIH grants P01 CA092548 and R35 CA220430, plus Cancer Prevention Research Institute of Texas grant RP180813 and an endowed Robert A. Welch Chemistry Chair supported by the Welch Foundation. B.T. was supported by the Polish National Agency for Academic Exchange (grant PPN/WAL/2019/1/00018) and by the Foundation for Polish Science (START Program). We thank M. Hammel and K. H. Burnett for their help in SEC–SAXS data collection at the SIBYLS beamline. The SEC–SAXS data collection at SIBYLS is supported in part by the NIH-NIGMS grant P30 GM124169-01 (ALS-ENABLE). We thank the Center for Macromolecular Interactions at Harvard Medical School for access to their Monolith NT.115Pico (NanoTemper) system. We also thank J. Newman for providing the MRE11 expression construct to produce the recombinant protein. The model in Fig. 8 was created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Michelle L. Swift, Rui Zhou.

Authors and Affiliations

  1. Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Michelle L. Swift, Rui Zhou, Aleem Syed, Lisa A. Moreau, Alan D. D’Andrea, Yizhou Joseph He & Dipanjan Chowdhury

  2. Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Rui Zhou

  3. Institute of Radiation Oncology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Rui Zhou

  4. Center for DNA Damage and Repair, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Lisa A. Moreau & Alan D. D’Andrea

  5. Department of Biostatistics and Translational Medicine, Medical University of Łódź, Łódź, Poland

    Bartłomiej Tomasik

  6. Department of Oncology and Radiotherapy, Medical University of Gdańsk, Faculty of Medicine, Gdańsk, Poland

    Bartłomiej Tomasik

  7. Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    John A. Tainer

  8. Department of Molecular and Cellular Oncology and Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    John A. Tainer

  9. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Panagiotis A. Konstantinopoulos

  10. Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Dipanjan Chowdhury

  11. Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    Dipanjan Chowdhury

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Contributions

M.L.S., R.Z. and Y.J.H. performed most of the experiments. A.S. purified recombinant proteins, designed and performed MST assays, and performed SEC–SAXS analyses and structural modeling. L.A.M. performed metaphase spreads and radial formation assay. B.T. generated plasmids and created cell lines. Y.J.H., J.A.T., A.D.D., P.A.K. and D.C. conceived the study. D.C. and M.L.S. wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Yizhou Joseph He or Dipanjan Chowdhury.

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Competing interests

A.D.D. reports consulting for AstraZeneca, Bayer AG, Blacksmith/Lightstone Ventures, Bristol Myers Squibb, Cyteir Therapeutics, EMD Serono, Impact Therapeutics, PrimeFour Therapeutics, Pfizer, Tango Therapeutics and Zentalis Pharmaceuticals/Zeno Management; is an Advisory Board member for Cyteir and Impact Therapeutics; a stockholder in Cedilla Therapeutics, Cyteir, Impact Therapeutics and PrimeFour Therapeutics, and reports receiving commercial research grants from Bristol Myers Squibb, EMD Serono, Moderna and Tango Therapeutics. The remaining authors declare no competing interests.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Dimitris Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 53BP1 is necessary for chromatin localization of DYNLL1. Related to Fig. 1.

(a) Representative immunofluorescence images of RPE1 cells subjected to laser microirradiation. Cells were fixed at indicated time points post laser microirradiation and processed for immunofluorescence with DYNLL1 and 53BP1 antibodies. (b) Representative images of RPE1 wild-type or 53BP1−/− cells 2 h after exposure to 2 Gy irradiation or laser microirradiation. Cells were fixed and processed for immunofluorescence using antibodies against 53BP1, GFP (DYNLL1), and γH2AX. (c) RPE1 cells depleted of p53, 53BP1, or DYNLL1 using CRISPR/Cas9 were exposed to 10 Gy irradiation. Protein was collected after 3 h. Localization of DYNLL1 to chromatin was evaluated by subcellular fractionation followed by immunoblotting for DYNLL1. (d–f) Representative images of COV362 cells (D) and COV362 wild-type or 53BP1−/− cells (E, F) exposed to 2 Gy irradiation (D, E) or laser microirradiation (F). 2 h post-recovery cells were fixed and processed for immunofluorescence using antibodies against 53BP1, GFP (DYNLL1), and γH2AX. Box plots show mean and center, quartiles (boxes), and range (whiskers)(D). (g) Representative immunofluorescence images of RPE1 cells depleted of DYNLL1 using CRISPR/Cas9 and exposed to 2 Gy irradiation. 2 h post-irradiation, cells were fixed and processed for immunofluorescence using antibodies against 53BP1 and γH2AX. (A-G) n = 3 biologically independent experiments, counting ≥ 100 cells per experiment. Error bars represent the mean±s.e.m. P-values for foci quantification and "laser positive" cell analysis were calculated using two-sided unpaired t-tests. P values are indicated by nonsignificant (P >0.05), *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P<0.0001). Black line in dot plots represent median. Scale bar = 20μm.

Source data

Extended Data Fig. 2 Force tethering DYNLL1 to chromatin inhibits MRE11 foci formation. Related to Fig. 2.

(a, b) RPE1 53BP1−/− cells were transfected with EGFP-tagged DYNLL1 or DYNLL1-FHA constructs. Cells were subjected to 2 Gy irradiation (A) or laser microirradiation (B). 2 h later cells were fixed and processed for immunofluorescence using antibodies against GFP (DYNLL1) and γH2AX. (c, d) COV362 53BP1−/− cells were transfected with a EGFP-tagged DYNLL1 and DYNLL1-FHA constructs. Cells were subjected to 2 Gy irradiation (C) or laser microirradiation (D). 2 h later cells were fixed and processed for immunofluorescence using antibodies against GFP (DYNLL1) and γH2AX. (e) COV362 53BP1−/− cells were transfected with EGFP-tagged DYNLL1 and DYNLL1-FHA constructs. Cells were exposed to 2 Gy irradiation. 2 h post-irradiation cells were fixed and processed for immunofluorescence using antibodies against MRE11, GFP (DYNLL1) and γH2AX. (A-E) n = 3 biologically independent experiments, counting ≥ 100 cells per experiment. Error bars represent the mean±s.e.m. P-values for foci quantification and "laser positive" cell analysis were calculated using two-sided unpaired t-tests. P values are indicated by nonsignificant (P >0.05), *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P<0.0001). Black line in dot plots represent median. Scale bar = 20μm.

Source data

Extended Data Fig. 3 DYNLL1 chromatin binding suppresses 53BP1 loss-induced restoration of HR in BRCA1 deficient cells. Related to Fig. 3.

(a) MEFs expressing EGFP-tagged DYNLL1, and EGFP-tagged DYNLL1-FHA domains constructs were exposed to 2 Gy irradiation. 2 h after irradiation cells were fixed and processed for immunofluorescence using a GFP (DYNLL1) antibody. (b, c) MEF p53−/− BRCA1−/− 53BP1−/− cells (B) and COV362 53BP1−/− cells (C) were transfected with EGFP-tagged DYNLL, or EGFP-tagged DYNNL1-FHA constructs. Cells were exposed to 2 Gy irradiation. 2 h later, cells were fixed and processed for immunofluorescence using antibodies against GFP (DYNLL1), RAD51, and γH2AX. (d) COV362 53BP1−/− cells were transfected EGFP-tagged DYNLL, or EGFP-tagged DYNNL1-FHA constructs. Cells were treated with indicated concentrations of Olaparib for 6 days. Percent survival was determined via a cell viability assay. (A-D) n = 3 biologically independent experiments, counting ≥ 100 cells per experiment. Error bars represent the mean±s.e.m. P-values for foci quantification were calculated using two-sided unpaired t-tests. P-value measurements for cell survival curves were assessed by non-regression curve analysis. P values are indicated by nonsignificant (P >0.05), *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P<0.0001). Black line in dot plots represent median. Scale bar = 20μm.

Source data

Extended Data Fig. 4 DYNLL1 interferes with MRE11 dimerization. Related to Fig. 4.

(a) Predicted structure of full-length MRE11 created by AlphaFold Monomer V2 for Uniprot Accession number P49959. The structured catalytic domain of MRE11 is highlighted with a red circle. The model is color coded in terms of confidence in prediction and respective color schemes for the confidence is given in the figure. In general, disordered regions have less confidence in model prediction, thus indicating the unstructured regions of MRE11 beyond capping domain. (b) Coomassie-stained protein gels indicating the quality of the recombinant protein used in the current study. Left: DYNLL1 mutants after cleaving the His-tag with TEV protease. Right: MRE11 catalytic domain after the gel-filtration purification step. The red rectangle indicates the fractions that are combined. M indicates the protein standards and * indicates the MRE11 degradation bands. (C) Average MST response (n = 3) measured from labeled MRE11 in the MST buffer or MST buffer with 5 μM DYNLL1-S88A or DYNLL1-S88D mutant. (d) Change in the normalized fluorescence (ΔFnorm) as result of thermophoresis in the MST experiment plotted as a function of concentration of unlabeled MRE11. The resulting curves represents MRE11 dimerization in the absence of any DYNLL1 (black circles), in the presence of 500 nM DYNLL1-S88D (red triangles) or in the presence of 5 μM DYNLL1-S88D (blue squares). The Kd values are measured by fitting the curves with Kd model in the analysis software. (B-D) The data points represent average of three independent measurements and error bars represents standard deviation.

Source data

Extended Data Fig. 5 Solution structures of DYNLL1-WT and mutants.

(a) AlphaFold2 predicted models of DYNLL1 dimer (left) and monomer (right). (b) Size exclusion chromatography elution profiles for DYNLL1-WT, S88A and S88D mutants. For clarity, initial 8 mL (pre-void volume with no peaks) were omitted from the chromatograms. (c–e) FoXS fitting of experimental X-ray scattering data (red dots) to theoretical SAXS profiles (solid lines) derived from structural models of DYNLL1 monomer (M) and dimer (D) or mixture of monomer and dimer state. The goodness of the fit is evaluated by χ2. Guinier plots from the measured scattering intensity (I(q)) as a function of scattering vector (q) in the low q region shown as insets for WT and mutant proteins.

Source data

Extended Data Fig. 6 Depletion of the Shieldin complex does not affect MRE11 recruitment. Related to Fig. 5.

(a) Protein expression from lysates collected from RPE1 wild-type or DYNLL1−/− cells 2 h after 2 Gy irradiation. (b) COV362 cells overexpressing EGFP-DYNLL1 and transfected with siRNA targeting either 53BP1, or SHLD1 were exposed to 2 Gy irradiation. Cells were fixed 2 h post exposure and processed for immunofluorescence using antibodies against GFP (DYNLL1), γH2AX, and MRE11. (A,B) n = 3 biologically independent experiments, counting ≥ 100 cells per experiment. P-values for foci quantification were calculated using two-sided unpaired t-tests. P values are indicated by nonsignificant (P >0.05), *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P<0.0001). Black line in dot plots represent median. Scale bar = 20μm.

Source data

Extended Data Fig. 7 Shieldin is recruited to DSBs later than DYNLL1 and in G1 phase only. Related to Fig. 6.

(a) Representative images from Fig. 6a. (b) Quantification of GFP positive stripes for cells expressing DYNLL1-EGFP or SHLD1-EGFP after exposure to laser microirradiation and fixed at indicated time points. (c) Quantification of number of DYNLL1 foci for RPE1 cells transduced with lentivirus comprised of the Fucci system reporter assay. Cells were exposed to 10 Gy irradiation, fixed 6 h later, and processed using antibodies against Geminin, Cdt1, and DYNLL1. (d) Representative images for Fig. 6c and Extended Data Fig. 6c. (e) Representative western blots showing knockdown of indicated proteins for Fig. 6d. (f) RPE1 BRCA1−/− were subjected to 10 Gy IR and 4 h later were fixed and processed for immunofluorescence using antibodies against SHLD1, Cyclin A, and γH2AX. (A-F) n = 3 biologically independent experiments, counting ≥ 100 cells per experiment. Error bars represent the mean±s.e.m. P-values for foci quantification and ‘laser positive’ cell analysis was calculated using two-sided unpaired t-tests. P values are indicated by nonsignificant (P >0.05), *(P < 0.05), **(P < 0.01), ***(P < 0.001), ****(P<0.0001). Black line in dot plots represent median. Scale bar = 20μm.

Source data

Extended Data Fig. 8 Shieldin functions downstream of DYNLL1, but is not dependent on DYNLL1 for its localization to chromatin. Related to Fig. 7.

(a) RPE1 p53−/− and RPE1 p53−/− 53BP1−/− cells were transfected with EGFP-tagged DYNLL1 or EGFP-tagged DYNLL1-FHA. Cells were then transfected with mCherry-SHLD1 and subjected to laser microirradiation. 2 h after laser microirradiation cells were fixed and processed for immunofluorescence using antibodies against GFP (DYNLL1), mCherry (SHLD1), and γH2AX. n = 3 biologically independent experiments. Scale bar = 20μm.

Supplementary information

Source data

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Statistical source data for Fig. 1.

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Statistical source data for Extended Data Fig. 1.

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Uncropped western blots for Extended Data Fig. 2.

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Statistical source data for Extended Data Fig. 2.

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Statistical source data for Extended Data Fig. 3.

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Statistical source data for Extended Data Fig. 4.

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Uncropped gels for Extended Data Fig. 4.

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Statistical source data for Extended Data Fig. 5.

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Statistical source data for Extended Data Fig. 6.

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Uncropped western blots for Extended Data Fig. 6.

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Statistical source data for Extended Data Fig. 7.

Source Data Extended Data Fig. 7

Uncropped western blots for Extended Data Fig. 7.

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Swift, M.L., Zhou, R., Syed, A. et al. Dynamics of the DYNLL1–MRE11 complex regulate DNA end resection and recruitment of Shieldin to DSBs. Nat Struct Mol Biol 30, 1456–1467 (2023). https://doi.org/10.1038/s41594-023-01074-9

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