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EXD2 promotes homologous recombination by facilitating DNA end resection

Nature Cell Biology volume 18, pages 271–280 (2016)Cite this article

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Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN–CtIP-dependent end resection, which generates the 3′ single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3′–5′ exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.

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Figure 1: EXD2 is a CtIP interactor and its depletion sensitizes cells to DNA damage.
Figure 2: EXD2 depletion impairs DNA end resection following DSB induction.
Figure 3: EXD2 promotes HR and suppresses genome instability.
Figure 4: EXD2 displays 3′–5′ exonuclease activity in vitro.
Figure 5: Nuclease activity of EXD2 is required for DSB repair in vivo.
Figure 6: EXD2 promotes resection through a common mechanism with MRE11.
Figure 7: EXD2 is required for efficient HR.

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Acknowledgements

We thank S. Jackson, M. de Bruijn, J. Riepsaame, V. Macaulay, G. Legube, G. Stewart, F. Esashi, C. Green and R. Chapman for cell lines, plasmids and antibodies; J. A. Tainer for the provision of MRE11 exo- and endonuclease inhibitors; the Mass Spectrometry Laboratory (IBB PAS, Warsaw, Poland) for their work on analyses of GFP–CtIP IP experiments; J. A. Newman for advice on protein purification and D. Waithe for help with image analysis. We also thank G. Ira for critical reading of the manuscript. R.B., J.N. and W.N. are funded by Worldwide Cancer Research and MRC Senior Non-Clinical Fellowships awarded to W.N., P.J.M. and O.G. are funded by an MRC Grant, H.T.B. is funded by a Cancer Research UK Studentship and R.A.D. and T.T.P. are funded by the Cancer Research and Prevention Institute of Texas (CPRIT) grant RP110465-P4.

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Author notes

  1. Ronan Broderick and Jadwiga Nieminuszczy: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK

    Ronan Broderick, Jadwiga Nieminuszczy, Peter J. McHugh & Wojciech Niedzwiedz

  2. Structural Genomics Consortium, Old Road Campus Research Building, Roosevelt Drive, University of Oxford, Oxford OX3 7DQ, UK

    Hannah T. Baddock & Opher Gileadi

  3. The Howard Hughes Medical Institute and Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA

    Rajashree A. Deshpande & Tanya T. Paull

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Contributions

R.B. and J.N. carried out the majority of experimental work with contributions from W.N.; H.T.B., P.J.M. and O.G. contributed to the purification and analysis of the biochemical activities of EXD2. R.A.D. and T.T.P. purified the MRN complex. W.N. conceived the project and wrote the manuscript with editing contributions from R.B., J.N., T.T.P. and P.J.M.

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Correspondence to Wojciech Niedzwiedz.

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Integrated supplementary information

Supplementary Figure 5

Cartoon illustrating the phylogenetic tree for the EXD2 gene.

Supplementary Figure 6 Additional characterisation of resection defect in EXD2-depleted cells.

(a,b) Western blotting confirming depletion of endogenous EXD2 72 h post-transfection with either control siRNA (siControl) or siRNAs targeting EXD2 (siEXD2-1 and 2). α-Tubulin acts as a loading control. These experiments were carried out three times independently. (c) RPA foci in U2OS cells 72 h post-transfection with control siRNA (siControl) or an siRNA oligo targeting EXD2 (siEXD2). Cells were either untreated or were exposed to 8 Gy IR and left to recover for 1 h before fixation and then stained for RPA and DAPI as indicated. Scale bar = 20 μm. (d) Quantification of the percentage of cells treated as in (c), exhibiting greater than 15 RPA foci per nucleus. n = 300 cells (siControl untreated), 390 cells (siEXD2 untreated), 355 cells (siControl 8Gy IR) and 403 cells (siEXD2 8gy IR) respectively, pooled from three independent experiments. Bars represent mean values ± s.e.m. The Chi-square test was used to determine statistical significance. (e) Western blotting of various DDR proteins in U2OS cells 72 h post-transfection with control siRNA (siControl) or an siRNA oligo targeting EXD2 (siEXD2) with cells treated with 8 Gy IR. Samples were acquired at the indicated time points post-IR treatment. Chk2-p T68 acts as a control for ATM activation, RPA2 pS4/S8 acts to indicate resection efficiency, γH2AX serves to indicate DSB induction with RPA and histone H3 serving as loading controls. This experiment was carried out two times independently. (f) Quantification of the percentage of G1, S or G2/M U2OS cells as analysed by propidium iodide staining and FACS analysis. Cells were analysed 72 h post-transfection with control siRNA (siControl) or siRNA targeting EXD2 (siEXD2) (mean values ± s.e.m., n = 5 independent experiments).

Supplementary Figure 7 Additional characterisation of purified EXD2.

(a) Alignment of the partial amino acid sequences of EXD2 proteins from various vertebrate organisms (human, mouse, chicken and xenopus are depicted) with a partial amino acid sequence of the exonuclease domain of human Werner helicase protein (WRN). Highly conserved residues shown to mediate the exonuclease activity of WRN protein which are also conserved in human EXD2 and its vertebrate homologues are indicated with red arrows. (b) Coomassie stained SDS-PAGE gel of EXD2 WT or D108A E110A mutant protein ectopically expressed in E. coli and purified to homogeneity for use in in vitro biochemistry experiments. (c) InstantBlue stained SDS-PAGE gel of the truncated EXD2 proteins (WT and D108A E110A mutant) used in this study. (d) Mass spectrum of truncated EXD2 (K76–V564) WT protein confirming sample purity. (e) Mass spectrum of truncated EXD2 (K76–V564) D108A E110A protein confirming sample purity. (f) 5′ radiolabeled ssDNA or dsDNA substrate (10 nM molecules) was incubated for indicated amounts of time with EXD2 WT (K76–V564) or EXD2 (K76–V564) D108A E110A (EXD2 mut) protein (100 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. This experiment was carried out two times independently.

Supplementary Figure 8 Western blots confirming EXD2 knockdown efficiency and additional characterisation of EXD2 in vivo and in vitro.

(a) Western blotting determining the relative levels of expression of Flag–HA–EXD2 WT or D108A E110A mutant proteins in U2OS cells stably expressing these fusion proteins. Two independent clones for each construct are shown. Cells were transfected with control siRNA (siControl) or siRNA targeting endogenous EXD2 (siEXD2 3′UTR) as indicated and collected for western blotting. MCM2 serves as a loading control. This experiment was carried out three times independently. (b) Quantification of the frequency of RAD51 focus-positive U2OS cells 72 h post-transfection with the indicated siRNA. Cells were either untreated or exposed to 8 Gy IR and left to recover for 6 h before fixation. Cells were stained with DAPI and RAD51 as indicated. The percentage of cells exhibiting RAD51 foci was quantified. n = 366 cells (siControl 0 min), 386 cells (siEXD2 0 min), 337 cells (siMRE11 0 min), 315 cells (siEXD2/siMRE11, 0 min), n = 308 cells (siControl 360 min), 321 cells (siEXD2 360 min), 337 cells (siMRE11 360 min), 374 cells (siEXD2/siMRE11, 346 min), respectively, pooled from three independent experiments. Bars represent mean values ± s.e.m. The Chi-square test was used to determine statistical significance. (c) Western blotting confirming the depletion of EXD2 and MRE11 in U2OS cells 72 h post-transfection with control siRNA (siControl) or siRNA targeting EXD2 or MRE11 as indicated. MCM2 serves as a loading control. This experiment was carried out three times independently. (d,e) 5′ radiolabeled ssDNA or dsDNA 50-mer substrates (1 nM molecules) (d) or 5′ radiolabeled dsDNA substrates (1 nM molecules) containing a nick or 1 nucleotide gap (e) were incubated for the indicated amounts of time with EXD2 WT protein (70 nM). Samples were resolved on a 20% TBE-Urea polyacrylamide gel and visualised by phosphorimaging. These experiments were carried out once. (f,g) Western blotting confirming the depletion of EXD2 and MRE11 in ER-AsiSI U2OS cells (f) and DR-GFP U2OS cells (g) 72 h post-transfection with control siRNA (siControl) or siRNA targeting EXD2 or MRE11 as indicated. MCM2 serves as a loading control. These experiments were carried out three times independently.

Supplementary Figure 9 Generation and characterisation of EXD2 knockout cell lines.

(a) EXD2 knockout generation strategy using the CRISPR-Cas9 nickase. Schematic representation of the human EXD2 genomic locus with guide RNAs sequences highlighted in green and predicted cut sites marked by red arrows. (b) Representative images of RPA foci in HeLa control cells and in two independent clones of HeLa EXD2−/− cells treated with 1 μM CPT for 1 h. Scale bar = 5 μm. (c) Quantification of the percentage of HeLa or HeLa EXD2−/− cells treated as in b. exhibiting greater than 15 RPA foci per nucleus. Data from two independent HeLa EXD2−/− clones are represented. n = 617 cells (HeLa), 433 cells (HeLa EXD2−/− cl.1) and 429 (HeLa EXD2−/− cl.2) respectively, pooled from three independent experiments. Bars represent mean values ± s.e.m. The Chi-square test was used to determine statistical significance. (d) Western blot of HeLa EXD2−/− clones and parental cells treated with 1 μM CPT for 1 h. RPA2 pS4/S8 acts as an indicator of resection, MCM2 acts as a loading control. This experiment was carried out two times independently. (e) Survival of HeLa control cells and HeLa EXD2−/− cells treated with the indicated doses of CPT. Survival data from two independent EXD2−/− clones is depicted. Survival data represent mean ± s.e.m., (n = 3 independent experiments). (f) Western blot of HeLa EXD2−/− clones and parental cells probed with antibodies against MRE11 and CtIP. α-Tubulin acts as a loading control. This experiment was carried out three times independently.

Supplementary Figure 10 EXD2 is not required for CtIP or MRE11 recruitment to site of DNA damage.

(a) Immunofluorescence microscopy of U2OS cells stably expressing GFP-CtIP treated with control siRNA (siControl) or siRNA targeting EXD2 (siEXD2) following the induction of localized DSBs by microirradiation. DAPI serves as a marker for the cell nucleus and γH2AX serves as a marker for DSB induction. Scale bar = 20 μm. This experiment was carried out three times independently. (b) Western blotting confirming EXD2 knock down efficacy in samples from (a). α-Tubulin serves as a loading control. This experiment was carried out three times independently. (c) Immunofluorescence microscopy of WT HeLa and HeLa EXD2−/− clones stained for MRE11 using an antibody recognising the endogenous protein following the induction of localized DSBs by microirradiation. γH2AX serves as a marker for DSB induction. Scale bar = 20 μm. This experiment was carried out three times independently.

Supplementary Figure 11

Original uncropped images of western blots.

Supplementary Table 1 Antibodies.
Full size table
Supplementary Table 2 siRNA sequences.
Full size table
Supplementary Table 3 DNA oligonucleotides used in in vitro nuclease assays.
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Supplementary Table 4 qPCR primer sequences used in ER-AsiSI resection assay.
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Broderick, R., Nieminuszczy, J., Baddock, H. et al. EXD2 promotes homologous recombination by facilitating DNA end resection. Nat Cell Biol 18, 271–280 (2016). https://doi.org/10.1038/ncb3303

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