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Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

Abstract

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

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Figure 1: DONSON mutations cause severe microcephaly and short stature.
Figure 2: Mutations in DONSON affect DONSON protein levels.
Figure 3: DONSON loss results in replication fork stalling and increased genome instability.
Figure 4: DONSON localizes to replication forks.
Figure 5: Depletion of DONSON compromises activation of cell cycle checkpoints.
Figure 6: Increased spontaneous chromosome breakage and fragmentation of mitotic chromosomes in DONSON-depleted cells.
Figure 7: Cells from patients with DONSON mutations have spontaneous defects in replication fork progression that result in DNA damage.

References

  1. Klingseisen, A. & Jackson, A.P. Mechanisms and pathways of growth failure in primordial dwarfism. Genes Dev. 25, 2011–2024 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. O'Driscoll, M., Ruiz-Perez, V.L., Woods, C.G., Jeggo, P.A. & Goodship, J.A. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat. Genet. 33, 497–501 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Ogi, T. et al. Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR–ATRIP Seckel syndrome. PLoS Genet. 8, e1002945 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. German, J. Bloom's syndrome. I. Genetical and clinical observations in the first twenty-seven patients. Am. J. Hum. Genet. 21, 196–227 (1969).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Harley, M.E. et al. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism. Nat. Genet. 48, 36–43 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Qvist, P. et al. CtIP mutations cause Seckel and Jawad syndromes. PLoS Genet. 7, e1002310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rosin, N. et al. Mutations in XRCC4 cause primary microcephaly, short stature and increased genomic instability. Hum. Mol. Genet. 24, 3708–3717 (2015).

    CAS  PubMed  Google Scholar 

  8. Bicknell, L.S. et al. Mutations in the pre-replication complex cause Meier–Gorlin syndrome. Nat. Genet. 43, 356–359 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bicknell, L.S. et al. Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier–Gorlin syndrome. Nat. Genet. 43, 350–355 (2011).

    Article  CAS  PubMed  Google Scholar 

  10. Guernsey, D.L. et al. Mutations in origin recognition complex gene ORC4 cause Meier–Gorlin syndrome. Nat. Genet. 43, 360–364 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Fenwick, A.L. et al. Mutations in CDC45, encoding an essential component of the pre-initiation complex, cause Meier–Gorlin syndrome and craniosynostosis. Am. J. Hum. Genet. 99, 125–138 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Murray, J.E. et al. Extreme growth failure is a common presentation of ligase IV deficiency. Hum. Mutat. 35, 76–85 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Murray, J.E. et al. Mutations in the NHEJ component XRCC4 cause primordial dwarfism. Am. J. Hum. Genet. 96, 412–424 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shaheen, R. et al. Genomic analysis of primordial dwarfism reveals novel disease genes. Genome Res. 24, 291–299 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zeman, M.K. & Cimprich, K.A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zou, L. & Elledge, S.J. Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. MacDougall, C.A., Byun, T.S., Van, C., Yee, M.C. & Cimprich, K.A. The structural determinants of checkpoint activation. Genes Dev. 21, 898–903 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nam, E.A. & Cortez, D. ATR signaling: more than meeting at the fork. Biochem. J. 436, 527–536 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Chou, D.M. & Elledge, S.J. Tipin and Timeless form a mutually protective complex required for genotoxic stress resistance and checkpoint function. Proc. Natl. Acad. Sci. USA 103, 18143–18147 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kemp, M.G. et al. Tipin–replication protein A interaction mediates Chk1 phosphorylation by ATR in response to genotoxic stress. J. Biol. Chem. 285, 16562–16571 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Milner, R.D., Khallouf, K.A., Gibson, R., Hajianpour, A. & Mathew, C.G. A new autosomal recessive anomaly mimicking Fanconi's anemia phenotype. Arch. Dis. Child. 68, 101–103 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Germanaud, D. et al. Simplified gyral pattern in severe developmental microcephalies? New insights from allometric modeling for spatial and spectral analysis of gyrification. Neuroimage 102, 317–331 (2014).

    Article  PubMed  Google Scholar 

  24. Martin, C.A. et al. Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy. Nat. Genet. 46, 1283–1292 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Trimborn, M. et al. Mutations in microcephalin cause aberrant regulation of chromosome condensation. Am. J. Hum. Genet. 75, 261–266 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Griffith, E. et al. Mutations in pericentrin cause Seckel syndrome with defective ATR-dependent DNA damage signaling. Nat. Genet. 40, 232–236 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Yeo, G. & Burge, C.B. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol. 11, 377–394 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Bandura, J.L. et al. humpty dumpty is required for developmental DNA amplification and cell proliferation in Drosophila. Curr. Biol. 15, 755–759 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Cortez, D. Preventing replication fork collapse to maintain genome integrity. DNA Repair (Amst.) 32, 149–157 (2015).

    Article  CAS  Google Scholar 

  30. Errico, A. & Costanzo, V. Mechanisms of replication fork protection: a safeguard for genome stability. Crit. Rev. Biochem. Mol. Biol. 47, 222–235 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Fuchs, F. et al. Clustering phenotype populations by genome-wide RNAi and multiparametric imaging. Mol. Syst. Biol. 6, 370 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Papadopoulos, D.K. et al. Probing the kinetic landscape of Hox transcription factor–DNA binding in live cells by massively parallel fluorescence correlation spectroscopy. Mech. Dev. 138, 218–225 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. Bacia, K. & Schwille, P. Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nat. Protoc. 2, 2842–2856 (2007).

    Article  CAS  PubMed  Google Scholar 

  34. Sirbu, B.M. et al. Analysis of protein dynamics at active, stalled and collapsed replication forks. Genes Dev. 25, 1320–1327 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Liu, S. et al. ATR autophosphorylation as a molecular switch for checkpoint activation. Mol. Cell 43, 192–202 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Durkin, S.G., Arlt, M.F., Howlett, N.G. & Glover, T.W. Depletion of CHK1, but not CHK2, induces chromosomal instability and breaks at common fragile sites. Oncogene 25, 4381–4388 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. Ozeri-Galai, E., Schwartz, M., Rahat, A. & Kerem, B. Interplay between ATM and ATR in the regulation of common fragile site stability. Oncogene 27, 2109–2117 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Toledo, L.I. et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).

    Article  CAS  PubMed  Google Scholar 

  39. Brown, E.J. & Baltimore, D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 14, 397–402 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Brown, E.J. & Baltimore, D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev. 17, 615–628 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Forment, J.V., Blasius, M., Guerini, I. & Jackson, S.P. Structure-specific DNA endonuclease Mus81–Eme1 generates DNA damage caused by Chk1 inactivation. PLoS One 6, e23517 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Couch, F.B. et al. ATR phosphorylates SMARCAL1 to prevent replication fork collapse. Genes Dev. 27, 1610–1623 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ragland, R.L. et al. RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells. Genes Dev. 27, 2259–2273 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hodskinson, M.R. et al. Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF–ERCC1 in DNA cross-link repair. Mol. Cell 54, 472–484 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Svendsen, J.M. et al. Mammalian BTBD12 (SLX4) assembles a Holliday junction resolvase and is required for DNA repair. Cell 138, 63–77 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Takahashi, T., Nowakowski, R.S. & Caviness, V.S. Jr. The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J. Neurosci. 15, 6046–6057 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Snow, M.H.L. Gastrulation in the mouse: growth and regionalization of epiblast. J. Embryol. Exp. Morphol. 42, 293–303 (1977).

    Google Scholar 

  48. Murga, M. et al. A mouse model of ATR-Seckel shows embryonic replicative stress and accelerated aging. Nat. Genet. 41, 891–898 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Despras, E., Daboussi, F., Hyrien, O., Marheineke, K. & Kannouche, P.L. ATR–Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells. Hum. Mol. Genet. 19, 1690–1701 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Kawabata, T. et al. Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression. Mol. Cell 41, 543–553 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kumagai, A., Lee, J., Yoo, H.Y. & Dunphy, W.G. TopBP1 activates the ATR–ATRIP complex. Cell 124, 943–955 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Bass, T.E. et al. ETAA1 acts at stalled replication forks to maintain genome integrity. Nat. Cell Biol. 18, 1185–1195 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Haahr, P. et al. Activation of the ATR kinase by the RPA-binding protein ETAA1. Nat. Cell Biol. 18, 1196–1207 (2016).

    Article  CAS  PubMed  Google Scholar 

  54. Duursma, A.M., Driscoll, R., Elias, J.E. & Cimprich, K.A. A role for the MRN complex in ATR activation via TOPBP1 recruitment. Mol. Cell 50, 116–122 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Higgs, M.R. et al. BOD1L is required to suppress deleterious resection of stressed replication forks. Mol. Cell 59, 462–477 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Singh, G. & Cooper, T.A. Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing. Biotechniques 41, 177–181 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Sirbu, B.M., Couch, F.B. & Cortez, D. Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA. Nat. Protoc. 7, 594–605 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Conti, C. et al. Replication-fork velocities at adjacent replication origins are coordinately modified during DNA replication in human cells. Mol. Biol. Cell 18, 3059–3067 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Turriziani, B. et al. On-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics. Biology (Basel) 3, 320–332 (2014).

    CAS  Google Scholar 

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Acknowledgements

We would like to thank the families and clinicians for their involvement and participation. We are grateful to R.S. Taylor (University of Manchester), D.-J. Kleinjan (University of Edinburgh), and J. Lukas and C. Lukas (University of Copenhagen) for their kind gifts of reagents. We thank E. Freyer, J. Wills, J. Ding, A. Fluteau, C. Keith, D. Longman, and the IGMM FACS, core sequencing and mass spectrometry facilities for technical assistance and advice. The Walking With Giants Foundation and Potentials Foundation supported the Primordial Dwarfism Registry (M.B.B.). This work is supported by funding from Cancer Research UK (C17183/A13030) (G.S.S., M.R.H. and A.V.), the Medical Research Council (MR/M009882/1) (J.J.R.), Worldwide Cancer Research (13-1012) (A.Z.), the Birmingham Children's Hospital Research Foundation (BCHRF400) (R.M.A.M.), the University of Birmingham (J.J.R., R.M.A.M. and A.B.), Newlife—the Charity for Disabled Children (P.C. and L.S.B.), Medical Research Scotland (L.S.B.) and the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St. Thomas' NHS Foundation Trust and King's College London (H.B., A. Amar, N.J.P., M.A.S. and C.G.M.), the German Federal Ministry of Education and Research (BMBF) (1GM1404; E-RARE network EuroMicro) (G. Yigit), KSCDR funding and KACST grant 09-MED941-20 (F.S.A.), an EMBO Long-Term Fellowship (ALTF 7-2015) the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) and the Swiss National Science Foundation (P2ZHP3_158709) (O.M.). A.P.J. was supported by the Medical Research Council UK, the Lister Institute for Preventative Medicine and the European Research Council (ERC; award 281847).

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Authors

Contributions

J.J.R., M.R.H., P.C., O.M., A.Z., A.L., R.M.A.M., A.B. and G.S.S. designed and performed the cell biology experiments; J.E.M., L.S.B., R.S, C.V.L., F.S.A., M.A.S., C.G.M., Y.L., S.M. and G. Yigit performed next-generation sequencing and analysis; L.S.B., P.C., R.C.C., R.S., A.V., J.E.M., M.A.S., C.V.L., Z.T., M.A.M.R., H.B., A. Amar., S.M., A. Almoisheer, H.S.A. and N.J.P. performed sequencing, genotyping, linkage analysis, analysis of splicing and other molecular genetics experiments; D.C. and S.R.W. performed the iPOND experiments; P.T., D.K.P. and K.S. performed FCCS analysis; A.v.K. performed mass spectrometry analysis; E.F., M.Z.S., S.A.T., A. Alswaid, S.A., J.Y.A.-A., M.A.B., A.F.B., L.C., H.C., A.D., R.F., E.H., E.F.P., A.P., L.S., S.T., G. Yoon., J.A., P.N., A.J.Q., B.D.H., M.A. and R.H. contributed clinical cases and clinical data, and analysis for the study; M.B.B., C.A.W., J.E.M., L.S.B., A.M.R.T., F.S.A., C.G.M. and A.P.J. recruited study cohorts and performed a review of phenotypes and sample collection; J.J.R., M.R.H., L.S.B., A.P.J. and G.S.S. wrote the manuscript; and G.S.S., C.G.M., F.S.A. and A.P.J. planned and supervised the study.

Corresponding authors

Correspondence to Fowzan S Alkuraya, Christopher G Mathew, Andrew P Jackson or Grant S Stewart.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22, Supplementary Table 2 and Supplementary Note (PDF 3380 kb)

Supplementary Table 1

Clinical phenotype data of individuals with DONSON mutations (XLSX 17 kb)

Supplementary Table 3

Proteomic mass spectrometry screen for GFP-DONSON interactors. (XLSX 262 kb)

Supplementary Table 4

DONSON primer sequences used in this study (XLSX 13 kb)

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Reynolds, J., Bicknell, L., Carroll, P. et al. Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism. Nat Genet 49, 537–549 (2017). https://doi.org/10.1038/ng.3790

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