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Unravelling molecular mechanisms of the abasic crosslink DNA repair
Hušková, Andrea ; Šilhán, Jan (advisor) ; Bařinka, Cyril (referee) ; Pavlíček, Jiří (referee)
4 Abstract DNA as the primary carrier of genetic information guarantees organisms to live, grow, develop and reproduce. However, this most vital molecule in the cell is subject to various damages every moment. If it is not repaired, the cell and the organism will eventually succumb to inevitable destruction. One of the most serious damages is abasic site interstrand crosslink (Ap-ICL). Ap-ICL is formed spontaneously when an abasic site covalently pairs with an adenine on the opposite strand. The lack of information on repair mechanisms, the influence of the local sequence and its stability leads to questions about the fate, toxicity and occurrence of these lesions in cells. During evolution, several mechanisms have evolved to repair these and other damages to ensure the organism's survival. A recently discovered pathway known to repair Ap-ICL is named after the DNA glycosylase responsible for removing Ap-ICL. NEIL3 DNA glycosylase is recruited to Ap-ICL by ubiquitylation of DNA helicase, which is part of the DNA replication complex. NEIL3 glycosylase contains several zinc-finger domains, that bind to the damaged DNA and ensure its catalytic function. The molecular mechanism of the NEIL3 glycosylase repair process is currently not known. In order to answer the aforementioned unknowns, the rate of formation,...
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Structural studies of an abasic site DNA damage repair and DNA interstrand cross-link formation
Landová, Barbora ; Bouřa, Evžen (advisor) ; Bařinka, Cyril (referee) ; Schneider, Bohdan (referee)
DNA damage refers to any alteration or modification in the DNA structure that deviates from its natural state. Abasic site (Ap site) is one of the most common DNA lesions resulting from spontaneous depurination/depyrimidination or enzymatic base excision. When left unrepaired it can lead to a cascade of genetic mutations, potentially causing diseases like cancer. Understanding DNA repair mechanisms is vital for medical research and applications. Bacterial MutM is a DNA repair glycosylase, removing DNA damage generated by oxidative stress and preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of procaryotic enzymes, sharing structural and functional similarities with their eukaryotic counterparts, such as NEIL1-NEIL3. Here, I present two crystal structures of MutM from pathogenic Neisseria meningitidis: MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. One of the DNA lesion repaired by MutM is the Ap site, which, if not repaired, may spontaneously lead to the formation of an abasic site interstrand crosslink (Ap-ICL) with an adjacent adenine in the opposite strand. NEIL3 glycosylase is known to remove Ap-ICL. With a...
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Biogenesis and function of nuclear iron-sulfur proteins
Panova, Ekaterina ; Benda, Martin (advisor) ; Smutná, Tamara (referee)
Iron-sulfur clusters are important inorganic cofactors of many cellular reactions, including those that occur in the nucleus. Nuclear iron-sulfur proteins play an important role in DNA replication, genome repair, and maintenance of genome stability. The biosynthesis of these iron-sulfur clusters is initiated in the mitochondria by the iron-sulfur cluster assembly pathway (ISC), continues in the cytosol by the cytosolic iron-sulfur cluster assembly pathway (CIA), and ends with the incorporation of the clusters into target apoproteins such as polymerases, primases, helicases, endonucleases, or glycosylases. This bachelor thesis summarizes current knowledge about the pathways of iron-sulfur cluster biosynthesis, the functions of nuclear iron-sulfur proteins, and the role of the clusters in these proteins, including the phenotypes and clinical manifestations caused by the absence of iron-sulfur clusters. Keywords: iron-sulfur clusters, metalloproteins, nucleus, DNA replication, DNA repair
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Effect of chromatin on the repair of double-strand DNA breaks after cleavage by CRISPR/Cas and other programmable nucleases in plants
Trojan, Jakub ; Přibylová, Adéla (advisor) ; Procházková, Klára (referee)
Plants are highly resistant to ionizing radiation, also thanks to a high-quality repair system for repairing double-stranded breaks. Double-strand breaks in plants are repaired by four repair pathways. Most often, double-strand breaks are repaired by non-homologous end joining (NHEJ), which joins the broken ends without further processing. More accurate but slower and more complex is repair through homologous recombination (HR), which repairs the break using a homologous sequence. HR repair takes place preferentially in a region with active transcription and during the S and G2 phases of the cell cycle. Alternatively, repair further proceeds through single-strand annealing (SSA) or Theta mediated end joining (TMEJ). Both pathways are based on short homology between the overlapping ends of the double-strand break. An often neglected part of repairs is the overcoming of repressive chromatin, which protects the genome from DNA damage and prevents access of nucleases but also acts as a barrier for repair proteins. This work summarizes the current knowledge about DNA repair in plants. Furthermore, describe the influence of chromatin not only on the repair but also on the activity of programmable nucleases used in genetic engineering, such as zinc finger nucleases (ZFNs), transcription activator-like...
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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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Genetic variability in sporadic colorectal cancer: Searching for novel risk, prognostic and predictive biomarkers.
Jirásková, Kateřina ; Vodička, Pavel (advisor) ; Machoň, Ondřej (referee) ; Eckschlager, Tomáš (referee)
Colorectal cancer (CRC) is a major public health problem worldwide. Despite improvements in the diagnostic process and advancement in the treatment methods, the prognosis remains poor. To improve survival rates, it is important to identify people with the predisposition for CRC and to detect the potentially curable early stage of the disease. Furthermore, identifying those who would have an adverse clinical outcome associated with a particular chemotherapy would help to avoid redundant chemotherapy burden in patients and contribute to enhanced therapeutic efficacy, while minimizing treatment-related toxicity. The aim of the Thesis was to search for novel promising diagnostic, prognostic and predictive DNA-based biomarkers of sporadic form of CRC. As each patient is genetically unique, these biomarkers would aid clinicians in better diagnosis and/or in the selection of an optimal type of therapy for an individual CRC patient based on their molecular profile. In order to explore this issue, we investigated several candidate genes in healthy individuals as well as in newly diagnosed cancer patients. The major outcomes of this PhD study, which were fully reported in seven publications included in the present Thesis, are 1) The observation of several candidate single nucleotide polymorphisms in microRNA...
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Implication of eukaryotic DNA repair machinery in viral replication
Hron, Tomáš ; Španielová, Hana (advisor) ; Harant, Karel (referee)
Eukaryotic DNA damage response is an important mechanism which ensures genome stability. Its components are also mobilized during viral infection as a reaction against extraneous nucleic acid. Additionally, DNA repair machinery seems to be activated by some viruses purposely to provide their replication. This activation is mediated mainly by viral proteins which are able to interact with cellular factors. In many cases, key components of DNA damage mechanisms are associated with viral replication centre and likely participate in this process. Furthermore, cellular DNA damage signaling is exploited to provide competent environment for viral reproduction. However, particular mechanisms how these cellular factors participate in viral infection are still largely unclear. In this thesis, the principles of relationship between viral infection and eukaryotic DNA damage response are summarized and main viral families which are known to activate and utilize these mechanisms for its genom replication are described.
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Posttranslational modification of the adapter protein DAXX in the cellular response to genotoxic stress
Bražina, Jan ; Anděra, Ladislav (advisor) ; Černý, Jan (referee) ; Vodička, Pavel (referee)
Maintaining the chromosome continuity and complete genetic information in human cells is crucial for cell survival and the whole organism. It prevents life-threatening pathologies and preserves genetic continuity. However, cellular DNA is exposed to both endogenous and exogenous stress damaging its content and integrity. This stress activates mechanisms involving detection and repair of these damaged sites (DDR). One of the most serious types of DNA damage double-stranded breaks (DSB) occuring when both strands are severed. DSBs trigger wave of PTMs that regulate protein interactions, nuclear localization and catalytic activity of hundreds of proteins. Such modifications include acetylation, methylation, SUMOylation, ubiquitinylation and especially phosphorylation. The most important kinases involved in DDR kinases are ATM, ATR and DNA-PK. These kinases are activated immediately after the detection of the damaged area. DAXX (Death-associated protein 6) is an adapter and predominantly nuclear protein, which is involved in chromatin remodeling, gene expression modulation, antiviral response and depositing histone H3.3 variants into chromatin or telomeres. Daxx is essential for murine embryogenesis, since the homozygous deletion is lethal in E9.5-10. In 2006 a study mapping the substrates of kinases...
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Structure, function and importace of BRCA 1protein
Hojný, Jan ; Kleibl, Zdeněk (advisor) ; Falk, Martin (referee)
Studies of factors contributed to the development of hereditary breast and ovary cancers lead to the discovery of Breast Cancer 1 gene (BRCA1). The protein product of this tumor suppressor gene is nuclear phosphoprotein that plays a critical role in DNA repair and it is required for genome integrity control. The BRCA1 protein is the key component for correct assembly of reparation complexes formed in sites of DNA double strand breaks. Furthermore, BRCA1 protein is implicated in regulation of cell cycle checkpoints and it is also involved in regulation of gene expression in response to DNA damage. These activities suggest that BRCA1 protein plays a crucial role in orchestration of intracellular response to genotoxic DNA damage. Loss of BRCA1 functions leads to the DNA-damage repair mechanisms failure resulting in genomic instability and a tolerance of genomic alterations in affected cells. The genomic instability is the initial step toward early malignant transformation of cells lacking BRCA1 proteins. The aim of this work is to summarize the information about structure, functions known and the importance of BRCA1 protein with respect to the current discoveries enabling elucidation of versatile BRCA1-containing multiprotein complexes in which BRCA1 protein acts as the multiplatform interacting...
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Mechanisms of DNA repair in the moss Physcomitrella patens
Holá, Marcela ; Angelis, Karel (advisor) ; Bříza, Jindřich (referee) ; Fajkus, Jiří (referee)
Over the course of an organism's life, its genome is exposed to endogenous and exogenous chemical, physical and biological agents - genotoxins. These genotoxins alter its basic structural components - sugar residues, phosphodiester bonds, and nitrogenous bases. Organisms have therefore evolved a plethora of different strategies to both repair DNA lesions and maintain genomic stability. These DNA repair pathways are linked with several other cell pathways, including chromatin remodelling, DNA replication, transcription, cell cycle control, apoptosis - programmed cell death (PCD), thereby providing a coordinated cellular response to DNA damage. Biochemical mechanisms of DNA repair are relatively well understood in yeast and mammals, however, far less so in plants. While these repair mechanisms are evolutionary conserved, significant differences still remain. Therefore, further investigation is required. This thesis summarises the introduction of a novel plant model - the moss, Physcomitrella patens (Physcomitrella). As a haploid gametophyte with unique characteristics of high frequency of homologous recombination (HR), and apical growth of filaments, it is an ideal organism to study DNA repair in plants. Previous research on Physcomitrella regarding mechanisms of DNA lesion repair induced by...
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