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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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New Strategies in Electrochemical Detection of DNA Damage Induced by Interaction with UV radiation
Přibylová, Monika ; Vyskočil, Vlastimil (advisor) ; Fischer, Jan (referee)
V této bakalářské práci byl zkoumán netradiční redoxní indikátor 5-nitro-1,10-fenantrolin jako možný ukazatel poškození DNA. Měření probíhalo elektrochemicky technikou diferenční pulzní voltametrie v tříelektrodovém zapojení. Jako pracovní elektroda byla použita elektroda z ultračistého grafitu, která byla modifikována nízkomolekulární DNA z lososích spermií. Poškození DNA probíhalo pomocí UVC záření o vlnové délce 254 nm. Byl studován rozdíl výšky voltametrických signálů 5-nitro-1,10-fenantrolinu mezi nepoškozenou a poškozenou DNA. Nově vyvinuté metody se ukázaly jako slibné pro detekci míry poškození DNA v závislosti na čase ozařování a v závislosti na vzdálenosti od zdroje ozařování. Poškození DNA bylo rozsáhlejší u obou studovaných faktorů v závislosti na jejich rostoucích hodnotách. Klíčová slova Elektrochemie DNA biosenzory Netradiční elektrodové materiály Poškození DNA Biologicky aktivní látky Záření
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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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DNA damage response in mammalian oocytes
Vachová, Veronika ; Šolc, Petr (advisor) ; Nevoral, Jan (referee)
During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...
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Ddi1-like proteins: a novel family of retroviral-like aspartyl proteases
Šmilauerová, Kristýna ; Grantz Šašková, Klára (advisor) ; Šmahel, Michal (referee)
Ubiquitin-proteasome system is one of the key pathways which maintain cell homeostasis. Its purpose is to degrade damaged, misfolded or unnecessary proteins. It is also involved in multiple other processes such as DNA damage repair, cell cycle control or signaling. The entire system consists of multiple components, which are mutually strictly regulated. Important part of this system is group of so called proteasome adaptor proteins. Their role is to recognize and bind targeted substrates and transport them to the proteasome for degradation. Ddi1-like (abbrev. from DNA damage-inducible protein 1) protein family, a group of proteins with retroviral aspartyl protease-like domain, belongs to proteasome adaptor proteins. Global biological role of this protein family is only partially understood the most studied member is Ddi1 protein from Saccharomyces cerevisiae, and it is thus a subject of active research. This thesis summarizes published information about this protein family, describes its general characteristics and known functions, situates them in the context of cell processes and thereby might suggest the course of further study.
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The Role of DNA Repair in the Onset and Therapy of Ovarian Cancer
Tomášová, Kristýna ; Vodička, Pavel (advisor) ; Čáp, Michal (referee)
DNA repair and DNA damage response are very important biological systems, inevitable to maintain genomic stability and fidelity of the genetic information, for the onset of ovarian cancer. Further, DNA repair is also substantially involved in the response to the therapy, since many chemotherapeutics act as DNA damaging agents. This literary analysis is intended to survay the relevance of DNA repair to ovarian carcinogenesis. Special emphasis is placed on repair defects, as it is inextricably associated with the onset of cancer and treatment outcome. Apart from well-known alternations in ovarian cancer susceptibility genes, such as BRCA1 and BRCA2 involved in homologous recombination repair, ample space will be dedicated to less common gene mutations across different repair pathways. Research confirms that abnormalities in the proteins responsible for homologous recombination repair are the leading cause of ovarian cancer. The majority of authors also suggested that targeting DNA repair pathways, especially base excision repair, can improve chemotherapy efficiency in a synergic manner. The same applies to nucleotide excision repair, which repairs platinum-DNA adducts and thus contibutes to platinum drugs resistance emerging. By way of contrast, mismatch repair in ovarian cancer is rather poorly...
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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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Mercury Electrodes as Tools for Voltammetric Determination of Biologically Active Organic Compounds and for Detection of Their Interaction with DNA
Horáková, Eva ; Vyskočil, Vlastimil (advisor) ; Ludvík, Jiří (referee) ; Vytřas, Karel (referee)
The main aim of this work was to use traditional mercury electrodes for the development of voltammetric methods of determination of organic xenobiotics and for the electrochemical study of the interaction between double-stranded deoxyribonucleic acid (DNA) and these compounds. In relation to my previous research work (conducted in the framework of my diploma thesis), firstly, 4-nitrobiphenyl (4-NBP), the suspected carcinogen, was studied. Interaction of DNA with 4-NBP was studied using differential pulse voltammetry (DPV), cyclic voltammetry (CV), and chronocoulometry at a hanging mercury drop electrode (HMDE), and using CV and alternating current voltammetry at a DNA modified HMDE. Using CV, the reduction mechanism was investigated. The interaction of DNA with 4-aminobiphenyl (4-ABP), a metabolite of 4-NBP, and 4-NBP reduction intermediates was studied. It was found that the interaction of DNA with 4-NBP or 4-ABP results in a formation of a DNA aggregate with these analytes. The second studied analyte was methyl violet 2B (MV). For determination of MV in a buffered solution were used: direct current tast polarography and differential pulse polarography at a dropping mercury electrode, and direct current voltammetry, DPV, and differential pulse adsorptive stripping voltammetry (DPAdSV) at HMDE. The...
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Novel Approaches in Electrochemical Determination of Xenobiotic Compounds and in Study of Their Interaction with DNA
Hájková, Andrea ; Vyskočil, Vlastimil (advisor) ; Trnková, Libuše (referee) ; Labuda, Ján (referee)
Presented Ph.D. Thesis is focused on the development of analytical methods applicable for determination of selected xenobiotic compounds and for monitoring DNA damage they can induce. The main attention has been paid to the development and testing of non-toxic electrode materials for preparation of miniaturized electrochemical devices and novel electrochemical DNA biosensors. 2-Aminofluoren-9-one (2-AFN) was selected as a model environmental pollutant, which belongs to the group of hazardous genotoxic substances. Its carcinogenic and mutagenic effects may represent a risk to living and working environment. 2-AFN has one oxo group, where the cathodic reduction occurs, and one amino group, where the anodic oxidation occurs. The voltammetric behavior of 2-AFN in the negative potential region was investigated at a mercury meniscus modified silver solid amalgam electrode (m-AgSAE) representing a non-toxic and more mechanically robust alternative to mercury electrodes. This working electrode was subsequently used for the development of a newly designed miniaturized electrode system (MES), which has many benefits as the possibility of simple field measurements, easy portability, and the measurement in sample volume 100 µL. Moreover, a glassy carbon electrode (GCE) was used for further investigation of...
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Structure determination of helical domain of DNA damage-inducible protein 2
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human Ddi2 and his homologues are scarcely explored family of ubiquitin- like/ubiquitin-associated domain proteins (UBL/UBA proteins), containing domain which is structurally related to dimeric aspartyl proteases from retroviruses. The most studied of this family is Ddi1 protein from Saccharomyces cerevisiae, which functions within the ubiquitin- proteasome system. This key cellular system exploits tightly regulated enzymatic apparatus for highly selective posttranslational modifications of proteins with molecules of ubiquitin, which serves as intracellular signal determining the proteins fate, importantly its degradation in many cases. Ddi1 plays a role of proteasome adaptor within this context, helping the recognition of ubiquitylated proteins by the proteasome. Even though the function as a proteasome receptor is possible for human Ddi2 protein as well, its cellular role might have been altered during the evolution. One of the important steps in decoding its purpose in cell is exploration of function of its individual domains. This work focuses on structural study of neighbouring region of this protease domain, where the helix-rich region called HDD domain is located. This region of Ddi2 was cloned, expressed and subjected to the NMR measurements. Structural information based on the NMR data was...
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