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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.

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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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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 or Christopher G Mathew or Andrew P Jackson or Grant S Stewart.

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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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