Hypomorphic PCNA mutation underlies a human DNA repair disorder
Emma L. Baple, … , Catherine M. Green, Andrew H. Crosby
Emma L. Baple, … , Catherine M. Green, Andrew H. Crosby
Published June 9, 2014
Citation Information: J Clin Invest. 2014;124(7):3137-3146. https://doi.org/10.1172/JCI74593.
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Research Article Genetics

Hypomorphic PCNA mutation underlies a human DNA repair disorder

Abstract

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Authors

Emma L. Baple, Helen Chambers, Harold E. Cross, Heather Fawcett, Yuka Nakazawa, Barry A. Chioza, Gaurav V. Harlalka, Sahar Mansour, Ajith Sreekantan-Nair, Michael A. Patton, Martina Muggenthaler, Phillip Rich, Karin Wagner, Roselyn Coblentz, Constance K. Stein, James I. Last, A. Malcolm R. Taylor, Andrew P. Jackson, Tomoo Ogi, Alan R. Lehmann, Catherine M. Green, Andrew H. Crosby

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Figure 4

Perturbed PCNA interactions resulting from the p.Ser228Ile mutation.

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Perturbed PCNA interactions resulting from the p.Ser228Ile mutation. (A)...
(A) Front and side views of the PCNA homotrimer, with Ser228 highlighted in magenta and the IDCL highlighted in cyan. Image was generated using POLYVIEW-3D (40), based on the 1YM crystal structure, rendered using PyMol. (B) Graphical representation of SILAC-based comparative PCNA-interaction analyses. Lysine [13C6/15N2] and arginine [13C6/15N4] labeled (“heavy”; H) or unlabeled (“light”; L) cell extracts were purified on PCNA WT or p.Ser228Ile affinity columns, and eluted proteins were analyzed by mass spectrometry. Each point represents the observed heavy/light ratios of a protein present in 3 of 3 experiments. Known PCNA-interacting proteins are shown in blue (see Supplemental Table 1). Proteins of particular interest are identified by name. (C) Recombinant His-PCNA (WT or p.Ser228Ile) was added to HeLa cell extracts and proteins bound to Ni-NTA. Proteins eluted at 1M NaCl or retained on the beads were analyzed by Western blot for Fen1. (D) Anti-Fen1 immunoprecipitates, from extracts made from lymphoblastoid lines derived from affected individuals or family members, were analyzed by Western blot for the presence of PCNA, Fen1, and actin. PCNA was almost undetectable in the precipitation from cell extracts derived from affected individuals. (E) E. coli lysates expressing empty vector (EV) or PCNA (WT or p.Ser228Ile) were mixed with lysates expressing GST or GST fused to the PIP box of Fen1. Inputs and glutathione-purified proteins were analyzed by SDS-PAGE and Coomassie staining. (F) Similar to E, except GST fusions comprising the PIP box of Lig1 and XPG were used.