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HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis

Nature volume 502, pages 381–384 (2013)Cite this article

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Abstract

Repair of interstrand crosslinks (ICLs) requires the coordinated action of the intra-S-phase checkpoint and the Fanconi anaemia pathway, which promote ICL incision, translesion synthesis and homologous recombination (reviewed in refs 1, 2). Previous studies have implicated the 3′–5′ superfamily 2 helicase HELQ in ICL repair in Drosophila melanogaster (MUS301 (ref. 3)) and Caenorhabditis elegans (HELQ-1 (ref. 4)). Although in vitro analysis suggests that HELQ preferentially unwinds synthetic replication fork substrates with 3′ single-stranded DNA overhangs and also disrupts protein–DNA interactions while translocating along DNA5,6, little is known regarding its functions in mammalian organisms. Here we report that HELQ helicase-deficient mice exhibit subfertility, germ cell attrition, ICL sensitivity and tumour predisposition, with Helq heterozygous mice exhibiting a similar, albeit less severe, phenotype than the null, indicative of haploinsufficiency. We establish that HELQ interacts directly with the RAD51 paralogue complex BCDX2 and functions in parallel to the Fanconi anaemia pathway to promote efficient homologous recombination at damaged replication forks. Thus, our results reveal a critical role for HELQ in replication-coupled DNA repair, germ cell maintenance and tumour suppression in mammals.

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Figure 1: A mouse model of HELQ deficiency.
Figure 2: Helq damage sensitivity and Helq Fancd2 double-mutant phenotypes.
Figure 3: HELQ interacts with DNA replication stress response factors.
Figure 4: HELQ influences DNA repair and homologous recombination efficiency.

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Acknowledgements

We wish to thank S. West for purified BCDX2 complexes and antibody reagents; Ó. Fernández-Capetillo for ATR inhibitors; D. Cox, G. Martin and H. Chapman for assistance with mouse breeding and maintenance; E. Nye, T. Bunting and B. Spencer-Dene for histopathology services; I. Rosewell for transgenic services; H. Flynn for mass spectrometry services; and M. Petalcorin for assistance with BAC recombineering. O.M. and A.A.S. are supported by grants of the Swiss National Science Foundation (PDFMP3_127523) and the Vontobel Foundation. The laboratory of A.D. is supported by National Institutes of Health grant R01-DK43889. The laboratories of S.J.B. and C.S. are funded by Cancer Research UK. The S.J.B. laboratory is also funded by a European Research Council advanced investigator grant (RecMitMei). S.J.B. is a Royal Society Wolfson Research Merit Award holder.

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  1. J. Mark Skehel

    Present address: Present address: Mass Spectrometry, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.,

Authors and Affiliations

  1. DNA Damage Response Laboratory, London Research Institute, Cancer Research UK, Clare Hall, South Mimms EN6 3LD, UK,

    Carrie A. Adelman, Rafal L. Lolo, Kenichiro Matsuzaki, Zuzana Horejsi, Valérie Borel & Simon J. Boulton

  2. Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby 2800, Denmark,

    Nicolai J. Birkbak

  3. Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland,

    Olga Murina & Alessandro A. Sartori

  4. Department of Radiation Oncology, Dana Farber Cancer Institute, Boston, 02215, Massachusetts, USA

    Kalindi Parmar & Alan D’Andrea

  5. Protein Analysis and Proteomics Laboratory, London Research Institute, Cancer Research UK, Clare Hall, South Mimms EN6 3LD, UK,

    J. Mark Skehel

  6. Experimental Histopathology Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK,

    Gordon Stamp

  7. Translational Cancer Therapeutics Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK,

    Charles Swanton

  8. UCL Cancer Institute, Huntley Street, London WC1E 6DD, UK,

    Charles Swanton

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Contributions

C.A.A., R.L.L. and S.J.B. designed the study, performed experiments and wrote the manuscript unless otherwise stated, G.S. performed mouse post-mortem analyses and advised on histopathology, O.M. and A.A.S. performed and supervised DR-GFP homologous recombination assays, K.M. performed RAD51 foci experiments, Z.H. performed human clonogenic survival assays, K.P. and A.D. performed and supervised mouse bone marrow experiments, V.B. assisted with mouse tumour watch monitoring, J.M.S. supervised mass spectrometry, N.J.B. and C.S. advised on experiments and manuscript revisions, and all authors contributed to revision of the manuscript.

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Correspondence to Simon J. Boulton.

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Extended data figures and tables

Extended Data Figure 1 Allele and subfertility of HELQ deficiency.

a, Schematic of the β-geo gene trap and its approximate location within the Helq genomic locus. b, Sequence and traces showing the exact location of the gene trap insertion as determined by sequencing of splinkerette PCR products. c, HELQ western blot from wild-type (+/+) and Helq mutant (ΔC/ΔC) mouse cells, showing loss of HELQ and absence of a HELQ–βgal fusion protein (which, if present, would be evident in the region of the blot marked with the red bar). d, Table of observed and expected Mendelian ratios calculated from heterozygous matings. Chi-squared analysis was used to test for deviation of observed from expected. e, Average weights of Helq mice tracked between 2 and 12 weeks of age. Means of 5–13 mice for each group are shown, and for clarity, s.d. is not plotted. Differences are not significant. f, Table of ovarian pathology in 30-week-old Helq control and mutant females (black text) and heterozygous females within the tumour watch study (blue text, 17–21 months old). g–l, Histological sections of testes from HelqΔC/ΔC males showing various degrees of atrophy, including: normal tubules (g), mild atrophy (h, i), pockets of atrophy (arrows), pyknotic nuclei (asterisks); moderate atrophy (j, k), missing spermatogenic layer (arrowheads) and severe atrophy (l), with only Sertoli cells present.

Extended Data Figure 2 HELQ-deficient germ cell and tumour phenotypes.

a, Immunohistochemical analysis of adult testes labelled with the stem cell marker c-KIT+ (brown) to highlight spermatogonia, and counterstained with haematoxylin to visualize remaining cells in the tubule (blue). Two representative images from wild-type (left) and mutant (right) mice are shown, with red circles indicating c-KIT+ cells. The number of c-KIT+ cells for each panel is indicated in the bottom right corner. Boxed regions in top panels are magnified in bottom panels to demonstrate staining. S, Sertoli cells; SG, c-KIT+ spermatogonia. b, Tabulation of average c-KIT+ cells per tubule normalized to the number of Sertoli cells. c, Testis weights plotted by mouse age in days. Linear regression used to generate slope of best-fit line; tested best-fit line for deviation of slope from 0: R2 and P values are indicated, revealing no correlation between age and testes weight for Helq mutants. d, Table of tumour frequency and tumour spectrum of Helq mutant and control mice showing data for all mice, females, and males in the tumour watch cohort. 129/B6 background phenomena are coloured in grey text, Helq mutant-specific effects are in black, and female-specific pathology is highlighted in pink.

Extended Data Figure 3 Tumour histology of HELQ deficiency.

a, The frequency of liver steatosis in all mice, and inflammation and activated mammary tissue in female mice. b–f, Ovary sections showing normal wild-type ovary (b) and common ovarian pathology in mutant animals (c–f). Low-magnification (c) and high-magnification (d) images of dysgenic ovary from a Helq mutant exhibiting a sex cord stromal tumour containing tubular-like structures. Low-magnification (e) and high-magnification (f) images of large nodular granulosa cell tumour from a Helq mutant. Arrowheads indicate mitotic figures (f). g–j, Pituitary sections showing low-magnification (g) and high-magnification (h) images of normal wild-type pituitary. Low-magnification (i) and high-magnification (j) images of pituitary tumour from a Helq mutant mouse. Arrows indicate boundary where large, haemorrhagic pituitary adenoma compresses overlying brain (h).

Extended Data Figure 4 Characterization of HelqΔC/ΔC bone marrow and generation of Helq Fancd2 double-mutant offspring.

a, HelqΔC/ΔC and control bone marrow cells were isolated and exposed to MMC at the indicated doses and clonogenic survival of haematopoietic progenitors was plotted as percentage of surviving cells relative to untreated. Means ± s.e.m. for three mice per genotype are shown. b–i, Bone marrow (BM) from mutant and control mice was isolated and subjected to various haematopoietic stem and progenitor cell analyses: tabulation of bone marrow LSK (lineage− Sca-1+ c-Kit+) cell populations (b, c); bone marrow c.f.u.-c (colony-forming units in culture) assays (d); bone marrow day-28 cobblestone area-forming cells (CAFCs; e); and total donor-derived leukocyte (f), myeloid (g) and lymphoid (h, i) engraftment upon bone marrow transplantation. Raw data (symbols) and means (horizontal lines) from three mice are plotted (b, c, e); means ± s.e.m. for three mice per genotype (d); and means ± s.e.m. for 6–10 recipients for each genotype (f–i). j, siRNA-treated U2OS cells were plated for clonogenic survival and treated with the indicated reagents. k, Observed and expected Mendelian ratios calculated from Helq+/ΔC Fancd2+/− double heterozygous matings. Chi-square analysis was used to test for deviation of observed from expected.

Extended Data Figure 5 HELQ mass spectrometry, its relationship with the RAD51 paralogues, ATR, and overexpression.

a, HELQ purification scheme and SDS–PAGE gel showing proteins co-purified with HELQ–Flag and control Flag immunoprecipitates. b, Cells treated with the indicated siRNAs were collected and probed for HELQ and the RAD51 paralogues. c, XRCC3 immunoprecipitated from HELQ–Flag and Flag control cell lysates and probed for Flag, XRCC3 and RAD51C (positive XRCC3 interacting protein control). IgG was used as a negative control. d, HeLa cells transiently expressing recombinant HELQ–GFP (green panels) fixed and stained with DAPI (blue panels) to identify nuclei. Two examples of spontaneous nuclear aggregation patterns are shown: small focal aggregates (right) and large filamentous aggregates (left). e, Chromatin fractions of HELQ–GFP cells treated with or without 2 μM APH for 24 h. f, Cells treated as in e, with or without 3 mM ATR inhibitor (ATRi). Quantification of HELQ in chromatin fractions normalized to H3 (right). g, Cells treated with or without 100 ng ml−1 MMC for 24 h and fractionated as in e.

Extended Data Figure 6 Spontaneous defects, checkpoint indices, damage foci and clonogenic survival of HELQ-deficient cells.

a, Primary Helq mutant and control cell lines were grown in physiological O2 for the indicated number of days and passaged regularly to generate a cumulative population doubling (CPD) curve. Means ± s.d. of triplicate replicas are shown. b, c, Helq mutant and control cells grown on coverslips were formaldehyde fixed and DAPI stained to determine levels of spontaneous micronuclei formation: percentage of 100 cells exhibiting 1 or more micronucleus (b); representative images (c), micronuclei (arrows). d, e, DNA combing used to calculate replication fork rates of primary cells grown under atmospheric (d) or physiological (e) O2. Cells in d were treated with or without 2.5 μM CPT for 15 min during labelling. f, Examples of origin containing IdU (green)- and CldU (red)-labelled fibres. g, Right versus left replication tract lengths to determine fork asymmetry (defined as tracts falling outside the interquartile lines).

Extended Data Figure 7 Checkpoint and double-strand break repair function.

a, b, Immortalized HelqΔC/ΔC and Helq+/+ cells treated with or without 500 ng ml−1 MMC (a) or 50 nM CPT (b) for 20 h and probed for the indicated checkpoint indices. c, Primary wild-type (WT), HELQ-deficient (HQ) and FANCD2-deficient (D2) cells were left untreated (−), or exposed to 5 Gy irradiation and collected 30 min later, or 3 μM APH for 16 h and harvested 10 min later, and lysates were probed for the indicated checkpoint indices. d, Phospho-Ser 345 CHK1 levels in U2OS cells subjected to 1 µM MMC for 24 h. e, Immortalized mouse cells were treated with 1 µM MMC for 24 h, allowed to recover for the indicated times (in hours), and stained for γH2AX. f, Quantification of percentage of positive cells from e. g, RPA32 and RAD51 foci formation in U2OS cells ± 1 μM MMC for 24 h subjected to control and HELQ siRNA.

Extended Data Figure 8 Homologous recombination dynamics and PARP inhibitor sensitivity.

a, U2OS cells treated with or without 100 ng ml−1 MMC for 24 h, allowed to recover for the indicated times (in hours), fractionated and probed for RAD51. H4 and α-tubulin are shown as controls for chromatin fractionation. b, DR–GFP reporter cells treated with the indicated siRNAs were probed for BRCA2, HELQ and RAD51; transcription factor IIH (TFIIH) is shown as a loading control. c, siRNA-treated U2OS cells were plated for clonogenic survival and treated with the indicated reagents.

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Adelman, C., Lolo, R., Birkbak, N. et al. HELQ promotes RAD51 paralogue-dependent repair to avert germ cell loss and tumorigenesis. Nature 502, 381–384 (2013). https://doi.org/10.1038/nature12565

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