Germline loss-of-function variants in TP53 cause Li-Fraumeni syndrome (LFS) characterized by an early onset of various cancer types including sarcomas, adrenocortical carcinoma, and breast cancer. The most common are mutations in the DNA binding domain of p53, but alterations in the oligomerization domain also cause LFS with variable level of penetrance. Here we report identification of a novel germline in-frame deletion TP53 variant c.1015_1023del p.(E339_F341del) in a family with early-onset breast cancer and other malignancies. Using functional testing, we found that a short deletion in the oligomerization domain in the p.E339_F341del variant severely impaired transcriptional activity of p53 in human cells and in a yeast model. The loss of the transactivation activity was consistent with an observed defect in formation of p53 tetramers. Finally, we found that cells expressing the p.E339_F341del variant were insensitive to inhibition of MDM2 by nutlin-3 confirming the functional defect. We conclude that the in-frame germline c.1015_1023del TP53 variant encodes a transcriptionally inactive protein and promotes LFS with a high penetrant cancer phenotype.

• Keywords
• Cancer, Li Fraumeni syndrome, TP53, p53,
• MeSH
• Adult MeSH
• Genetic Predisposition to Disease * MeSH
• Imidazoles pharmacology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Li-Fraumeni Syndrome * genetics   pathology   MeSH
• Protein Multimerization MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   chemistry   MeSH
• Breast Neoplasms * genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 metabolism   MeSH
• Pedigree MeSH
• Germ-Line Mutation * MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Imidazoles MeSH
• Tumor Suppressor Protein p53 * MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• TP53 protein, human MeSH Browser

In response to DNA replication stress, DNA damage signaling kinases inhibit origin firing and promote the remodeling and stabilization of replication forks, leading to a systemic reduction in DNA synthesis that protects genomic integrity. Little is understood about the regulatory mechanisms of replication stress recovery, including the mechanisms involved in the restart of stalled replication forks. Here, we identify the oncogenic phosphatase PPM1D/WIP1 as a critical regulator of replication fork restart. Upon recovery from replication stress, PPM1D prevents excessive MRE11- and DNA2-dependent nucleolytic degradation of stalled forks. Loss of PPM1D function leads to defects in RAD51 recruitment to chromatin and impairs RAD51-dependent fork restart. Phosphoproteomic analysis reveals that PPM1D regulates a network of ATM substrates, several of which are phosphorylated at an S/T-Q-(E/D)n motif. Strikingly, inhibition of ATM suppresses the deleterious consequences of impaired PPM1D function at replication forks, enabling timely fork restart. The dominant effect of ATM hyper-signaling in suppressing fork restart occurs, in part, through the excessive engagement of 53BP1 and consequent RAD51 antagonization. These findings uncover a new mode of ATM signaling responding to fork stalling and highlights the need for PPM1D to restrain ATM signaling and enable proper fork restart.

• Publication type
• Journal Article MeSH
• Preprint MeSH

Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

• MeSH
• Cell Death genetics   MeSH
• Humans MeSH
• Mice MeSH
• Cell Transformation, Neoplastic * genetics   MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• DNA Damage * genetics   MeSH
• Cell Proliferation genetics   MeSH
• Protein Phosphatase 2C * genetics   metabolism   MeSH
• Phosphoprotein Phosphatases genetics   metabolism   MeSH
• Cellular Senescence * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• PPM1D protein, human MeSH Browser
• Protein Phosphatase 2C * MeSH
• Phosphoprotein Phosphatases MeSH

RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.

• MeSH
• Tumor Suppressor p53-Binding Protein 1 * metabolism   genetics   MeSH
• Chromatin * metabolism   genetics   MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Histones * metabolism   MeSH
• Homologous Recombination genetics   MeSH
• Humans MeSH
• DNA End-Joining Repair MeSH
• DNA Repair MeSH
• Cell Cycle Proteins metabolism   genetics   MeSH
• Recombinational DNA Repair MeSH
• DNA Replication MeSH
• Ubiquitination * MeSH
• Ubiquitin-Protein Ligases * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 * MeSH
• Chromatin * MeSH
• DNA-Binding Proteins * MeSH
• Histones * MeSH
• Cell Cycle Proteins MeSH
• RAD18 protein, human MeSH Browser
• TP53BP1 protein, human MeSH Browser
• Ubiquitin-Protein Ligases * MeSH

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

• MeSH
• Phosphorylation MeSH
• Humans MeSH
• DNA Damage MeSH
• Telomeric Repeat Binding Protein 2 * MeSH
• Telomere-Binding Proteins * metabolism   MeSH
• Proteomics MeSH
• Shelterin Complex * MeSH
• Telomere metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Telomeric Repeat Binding Protein 2 * MeSH
• Telomere-Binding Proteins * MeSH
• Shelterin Complex * MeSH
• TINF2 protein, human MeSH Browser

Genome integrity is protected by the cell-cycle checkpoints that prevent cell proliferation in the presence of DNA damage and allow time for DNA repair. The transient checkpoint arrest together with cellular senescence represent an intrinsic barrier to tumorigenesis. Tumor suppressor p53 is an integral part of the checkpoints and its inactivating mutations promote cancer growth. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of p53. Although its loss impairs recovery from the G2 checkpoint and promotes induction of senescence, amplification of the PPM1D locus or gain-of-function truncating mutations of PPM1D occur in various cancers. Here we used a transgenic mouse model carrying a truncating mutation in exon 6 of PPM1D (Ppm1dT). As with human cell lines, we found that the truncated PPM1D was present at high levels in the mouse thymus. Truncated PPM1D did not affect differentiation of T-cells in the thymus but it impaired their response to ionizing radiation (IR). Thymocytes in Ppm1dT/+ mice did not arrest in the checkpoint and continued to proliferate despite the presence of DNA damage. In addition, we observed a decreased level of apoptosis in the thymi of Ppm1dT/+ mice. Moreover, the frequency of the IR-induced T-cell lymphomas increased in Ppm1dT/+Trp53+/- mice resulting in decreased survival. We conclude that truncated PPM1D partially suppresses the p53 pathway in the mouse thymus and potentiates tumor formation under the condition of a partial loss of p53 function.

• Keywords
• cancer, cell-cycle checkpoint, protein phosphatase, tumor suppressor p53,
• MeSH
• Apoptosis * MeSH
• Cell Cycle MeSH
• Radiation, Ionizing MeSH
• Lymphoma metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Neoplasms, Radiation-Induced metabolism   MeSH
• DNA Repair MeSH
• DNA Damage MeSH
• Cell Proliferation MeSH
• Protein Phosphatase 2C physiology   MeSH
• Thymocytes cytology   metabolism   MeSH
• Thymus Gland * cytology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• Ppm1d protein, mouse MeSH Browser
• Protein Phosphatase 2C MeSH
• Trp53 protein, mouse MeSH Browser

Polo-like kinases play essential roles in cell cycle control and mitosis. In contrast to other members of this kinase family, PLK3 has been reported to be activated upon cellular stress including DNA damage, hypoxia and osmotic stress. Here we knocked out PLK3 in human non-transformed RPE cells using CRISPR/Cas9-mediated gene editing. Surprisingly, we find that loss of PLK3 does not impair stabilization of HIF1α after hypoxia, phosphorylation of the c-Jun after osmotic stress and dynamics of DNA damage response after exposure to ionizing radiation. Similarly, RNAi-mediated depletion of PLK3 did not impair stress response in human transformed cell lines. Exposure of cells to various forms of stress also did not affect kinase activity of purified EGFP-PLK3. We conclude that PLK3 is largely dispensable for stress response in human cells. Using mass spectrometry, we identify protein phosphatase 6 as a new interacting partner of PLK3. Polo box domain of PLK3 mediates the interaction with the PP6 complex. Finally, we find that PLK3 is phosphorylated at Thr219 in the T-loop and that PP6 constantly dephosphorylates this residue. However, in contrast to PLK1, phosphorylation of Thr219 does not upregulate enzymatic activity of PLK3, suggesting that activation of both kinases is regulated by distinct mechanisms.

• Keywords
• DNA damage, Polo-like kinase 3, protein kinase, protein phosphatase, stress,
• MeSH
• Cell Line MeSH
• Phosphorylation MeSH
• Humans MeSH
• Tumor Suppressor Proteins MeSH
• DNA Damage genetics   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Phosphoprotein Phosphatases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Proteins MeSH
• PLK3 protein, human MeSH Browser
• protein phosphatase 6 MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Phosphoprotein Phosphatases MeSH