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Molecular mechanisms of Wip1 phosphatase function in DNA damage response
Štorchová, Radka ; Macůrek, Libor (advisor) ; Fajkus, Jiří (referee) ; Líčeníková Hořejší, Zuzana (referee)
Human cells are constantly exposed to diverse factors causing DNA lesions, which activate the DNA damage response (DDR). Depending on the severity of DNA damage, DDR can promote temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). DDR signalling is regulated by a cascade of post- translational modifications, where key mediators are represented by protein kinases ATM, ATR and DNA-PK. Wip1 phosphatase (encoded by PPM1D gene) plays an important role in DDR termination by dephosphorylation of many targets of these kinases. In this thesis, we investigated checkpoint-independent functions of PPM1D in cells and described several new substrates. We discovered, that PPM1D interacts with the shelterin complex and localizes at telomeres. PPM1D dephosphorylates the shelterin component TRF2 at S410. TRF2 S410 phosphorylation enhanced TRF2 interaction with TIN2, indirectly also increasing recruitment of TPP1 to telomeres. Importantly, cells over expressing PPM1D showed increased number of telomeric fusions. These findings might be very relevant for some cancer types, in particular those expressing high levels of PPM1D or carrying C-terminally truncated mutations in PPM1D. To validate the published substrates and to detect possible new targets of...
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Role of the WIP1 phosphatase in the nucleolus
Palková, Natálie ; Macůrek, Libor (advisor) ; Sztacho, Martin (referee)
Protein phosphatase 2C delta (known as WIP1) is an important negative regulator of the DNA damage response (DDR) signalling. As a chromatin-bound protein, it dephosphorylates and thus inactivates ATM kinase and the transcription factor p53. Increased expression of WIP1 suppresses the function of the tumour suppressor p53 and contributes to the development of several cancer types, including breast and brain tumours. In recent years, it has been shown that WIP1 can also regulate cellular processes that are not directly linked to DDR, such as ensuring telomere stability. However, alternative functions of the WIP1 protein have not yet been fully understood. In this work, we described a novel role of the WIP1 phosphatase in the nucleolus, the organelle responsible for ribosome biogenesis. We found that WIP1 associates with many nuclear proteins, and using deletion mutants, we identified a nucleolar localisation sequence (NoLS) at the C-terminus of the protein. Using super-resolution microscopy, we detected the localisation of WIP1 phosphatase in the fibrillar centres of the nucleolus. We employed an inducible Cas9 system for generating double-strand breaks in ribosomal DNA and found that WIP1 has an impact on recruitment of DNA repair factors to the nucleolar caps. Analysis based on quantitative...
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The role of truncated PPM1D/Wip1 phosphatase in cancer
Martiníková, Andra-Stefania ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Mistrík, Martin (referee)
When encountering damage, the cells activate the DNA Damage Response (DDR) pathway and stop the cell cycle until the DNA is repaired. PPM1D/WIP1 phosphatase resumes the cell cycle after the damage has been repaired, by directly dephosphorylating DNA damage markers. The DDR pathway prevents genome instability or cancer development. Mutations in the Ppm1d gene encoding PPM1D result in an overstable and truncated protein observed both in cancer patients and in cancer cell lines. In this thesis, we used an "in-house" transgenic mouse model in which mutations in the exon 6 of the Ppm1d gene resulted in a truncated PPM1D protein. First, we observed high PPM1D levels and impaired DDR to gamma ionizing radiation (IR) in the mouse thymi having truncated PPM1D (Ppm1dT/+ ). We then bred the Ppm1dT/+ mice with the Trp53+/- heterozygote knock-out mice which are prone to thymic lymphoma. The Ppm1dT/+ Trp53+/- double-mutants had a higher frequency of developing IR-induced T-cell lymphomas, compared to the single Trp53+/- mutants. Moreover, truncated PPM1D leads to a defective cell cycle checkpoint activation in human non-transformed RPE cells (RPE1), which then proliferate despite the presence of DNA damage. RPE1 cells also display increased proliferation after replication stress. RPE1 or U2OS cells with...
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DNA repair in oocytes and early embryos
Koubovská, Markéta ; Ďuríček, Tomáš (advisor) ; Macůrek, Libor (referee)
Our diploma thesis focused on the effect of hormonal stimulation on DNA integrity of mammals' oocytes. Later, we also focused on its effect on DNA integrity on mammals' embryos and their developmental potential. Maintaining DNA integrity in germ cells is essential for their quality during fertilization and further development. DNA integrity is disrupted by different types of damage, especially by generating DNA double-strand breaks. We examined the DNA damage in mouse oocytes and embryos, dependent on the dose of hormonal stimulation by PMSG hormone. Immunofluorescence detection γ-H2AX, marker of DNA double-strand breaks, on immature GV oocytes confirmed our primary hypothesis that increasing hormonal stimulation dose increases the number of DNA double-strand breaks. We performed the same experimental design in old mice, but there was no significant difference between groups as it was between groups in young mice. We observed a slight delay in meiotic maturation in hyperstimulated mice using live-cell microscopy compared to the control. In mature MII oocytes, we detected a 3-fold increase in γ-H2AX fluorescence intensity. This difference disappeared in fertilized zygotes, where all γ-H2AX foci were present only in the male pronucleus in both, control and hyperstimulated groups. In terms of...
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New molecular mechanisms involved in cell cycle control
Aquino, Cecilia ; Macůrek, Libor (advisor) ; Anger, Martin (referee) ; Braun, Marcus (referee)
Cecilia Aquino Perez, M. Sc. Doctoral thesis abstract In this doctoral, thesis we aimed to find and study novel mechanisms regulating cell cycle phase transitions in non-stressed conditions and in context of the cell response to various types of stress. First, we focused on studying Polo-like kinase 3 that has previously been implicated in activation of the cell cycle checkpoint after DNA damage. For this, we employed CRISPR/Cas9- mediated gene editing to knock-out PLK3 in RPE cells while in parallel performing RNA interference assays and submitting the cells to different types of stress. The main observation was that in both systems PLK3 was disposable for response to DNA damage, hypoxia and osmotic stress. Through mass spectrometry analysis of purified EGFP-PLK3 we identified PP6 and its regulatory subunits PPP6R1 and PPP6R3 as novel PLK3 interactors. We observed that PLK3 is phosphorylated in its conserved residue Thr-219 and that PP6 depletion boosted PLK3 phosphorylation status but did not affect its kinase activity. The possible regulation of PLK3 trough PP6 is interesting and its biological relevance will be addressed by future research. Next, we performed a transcriptomic analysis in human RPE-FUCCI cells aiming to identify new regulators of the cell cycle. We selected Family with sequence...
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Molecular mechanisms of checkpoint signalling and termination
Benada, Jan ; Macůrek, Libor (advisor) ; Brábek, Jan (referee) ; Truksa, Jaroslav (referee)
Cells employ an extensive signalling network to protect their genome integrity, termed DNA damage response (DDR). The DDR can trigger cell cycle checkpoints which prevent cell cycle progression and allow repair of DNA damage. The failures in these safeguarding mechanism are represented by serious human malignancies, most predominantly by cancer development. This work aims to contribute to the understanding of how do the cells negatively regulate DDR and cell cycle checkpoint signalling. We focused mainly on Wip1 (PPM1D) phosphatase, which is a major negative regulator of DDR and is indispensable for checkpoint recovery. Firstly, we have shown that Wip1 is degraded during mitosis in APC-Cdc20 dependent manner. Moreover, Wip1 is phosphorylated at multiple residues during mitosis, resulting in inhibition of its enzymatic activity. We suggest that the abrogation of Wip1 activity enables cells to react adequately even to low levels of DNA damage encountered during unperturbed mitosis. In the following publication, we have investigated why the mitotic cells trigger only early events of DDR and do not proceed to the recruitment of DNA repair factors such as 53BP1. We showed that 53BP1 is phosphorylated within its ubiquitination-dependent recruitment domain by CDK1 and Plk1. These phosphorylations prevents...
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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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The role of 53BP1 in the cellular response to double-strand DNA breaks
Liďák, Tomáš ; Macůrek, Libor (advisor) ; Rösel, Daniel (referee)
DNA damage may result in various pathological conditions and contributes to aging and development of cancer. Evolutionarily conserved DNA damage response prevents the acumulation of mutations and protects against genomic instability. Tumor suppressor p53-binding protein 1 (53BP1) is an important regulator of the cellular response to DNA double-strand breaks (DSB) and is a canonical component of ionizing radiation-induced foci which are formed at DNA DSB following radiation exposure. Recently, new insights have been gained into its functions in the DNA damage response. Apart from its subtle role in the DNA damage checkpoints signaling, 53BP1 is a well established player in the DNA DSB repair pathway choice. The outcome of DNA repair is influenced by 53BP1 in several contexts. 53BP1 controls 5' end resection at DNA ends, improves DSB repair in heterochromatin, promotes the mobility of uncapped telomeres and mediates synapsis of DNA ends during V(D)J and class switch recombination. 53BP1 contributes to repair defect in BRCA1 (breast cancer type 1 susceptibility protein)-deficient cells, which may have an impact on the treatment of some types of breast cancer. The aim of this bachelor's thesis is to summarize new findings about the role of 53BP1 in the cellular response to DNA DSB. Powered by TCPDF (www.tcpdf.org)
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Role of RAD18 in ubiquitin signaling at DNA double-strand breaks
Palek, Matouš ; Macůrek, Libor (advisor) ; Čermák, Lukáš (referee)
RAD18 is an E3 ubiquitin ligase that prevents the replication forks from collapsing caused by damaged DNA. As an important factor controlling replication, its dysregulation was shown to be associated with some human tumours. However, the clinical relevance of this finding is unknown. The aim of the thesis was evaluation of selected RAD18 variants that had been identified in breast and ovarian cancer patients. This work revealed functional defects of RAD18 variants not only in replication fork protection but also in repair of DNA double-strand breaks. This unconventional role of RAD18 is known to be dependent on upstream ubiquitination events, however, its contribution to the repair per se is not understood. This work aimed to elucidate the function of RAD18 in DNA double-strand break repair by homologous recombination focusing especially on its relationship with 53BP1. Data presented here show that RAD18 effectively disrupts 53BP1 accumulation in the repair foci by competition for the same binding partner and thus promotes resection of DNA ends. This antagonistic function of RAD18 is restricted both spatially (to the vicinity of the repair centre) and temporarily (to S phase). Moreover, it seems to be regulated by existence of RAD18 in two distinct complexes. Potential models for this regulation...
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Role of genetic factors in the prognosis and prediction of efficacy of chemotherapy in breast carcinoma patients
Brynychová, Veronika ; Souček, Pavel (advisor) ; Macůrek, Libor (referee) ; Stružinská, Ivana (referee)
Changes in the regulation of apoptosis and cell cycle are involved in tumor development, progression, and resistance to antitumor therapy. The aim of this work was to evaluate the importance of apoptotic caspases and regulators of cytokineses as possible prognostic and predictive markers in breast carcinoma patients. In addition to determining the transcript levels of selected genes in tumor and control tissues obtained from breast carcinoma patients, we have also focused on the importance of alternative splice variants of caspases and their potential genetially determined regulation. We analysed the obtained data in relation to the clinical-pathological characteristics of the tumors, the progression-free survival of patients and to the response of the patients to the neoadjuvant chemotherapeutic treatment. Part of the work was determination of protein expression levels and verification of the importance of selected candidates for the effect of chemotherapy by functional study. The transcript levels of caspase 2, 3, 7, 8, 9, 10, the specifically detected splice variants caspase 2S, 2L, 3A and B, 3S, 9A, 9B, 8L, and the transcript levels of KIF14 and CIT in breast carcinomas were unrelated to the progression-free survival of patients, or to the response of patients to neoadjuvant treatment. The...
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