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Structural studies of an abasic site DNA damage repair and DNA interstrand cross-link formation
Landová, Barbora
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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Tolerance of DNA damage by novel biologically active platinum complexes
Vystrčilová, Jana ; Vrána, Oldřich (referee) ; Nováková,, Olga (advisor)
The anti-tumor activity of platinum based drugs is mediated by their ability to attack DNA. Platinum complexes can alter the structure of DNA by modifying the bases, mainly guanines. The biological consequnces of such interactions are compromising replication and transcription. RNA polymerase complex can stall at a damaged site in DNA and mark the lesion for repair by proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Main goal of this thesis was to study RNA polymerases tolerance of DNA damage by novel, biologically active platinum (II) complexes involving derivatives of aromatic cytokinines as the ligands; cis-[Pt(2-chloro-6-(4-methoxybenzylamino)-9-isopropylpurin)2Cl2](PR-001), cis-[Pt(2-chloro-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-002 )and cis-[Pt(2-(3-hydroxypropylamino)-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-005). DNA templates (constructs) that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II and bacteriophage T7 RNA polymerase were prepared. The method is making use of polymerase chain reaction (PCR) and biotin-streptavidin interactions and paramagnetic particles to purify the final product. Synthetic oligomers duplexes (75-mer, 56-mer and 15-mer) are ligated to 5´-biotin pCI-neo-G-lessT7 PCR fragment, the 15-mer is either unmodified or modified with a site-specific lesion of PR-005 and cisplatin. We also studied the inhibition of RNA polymerases activity on globally modified plasmid pCI-neo and pUC 19 by novel platinum complexes and cisplatin. We found that bifunctional adducts of complex PR-005 contrary to adducts of PR-001 and PR-002 effectively decrease amount of full lenght transcripts produced by both, human and bacterial RNA polymerases. This result can be explained by a sterical block, induced to DNA by intrastrand cross-link of PR-005 with bulky aromatic ligands.
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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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Mechanistic insights into alcohol-induced interstrand crosslink repair by the nuclease SLX4-XPF-ERCC1
Havlíková, Jana ; Šilhán, Jan (advisor) ; Lux, Vanda (referee)
Alcohol ranks among the most widely used recreational drugs in the world, even though it is considered a risk factor for more than 200 diseases. The primary negative impact of alcohol lies in its metabolite, acetaldehyde, which, as a highly reactive compound, can form mutagenic adducts and interstrand crosslinks (ICLs) in DNA. The formation of ICLs, which have a covalent nature and block the separation of the two DNA strands during replication, is one of the important causes of mutagenesis and carcinogenesis. To maintain genomic stability, repair mechanisms have evolved. One of them is a pathway that uses proteins encoded by Fanconi anaemia genes, whose defects lead to the disease of the same name. Defects in repair pathways can be particularly dangerous in individuals with impaired functionality in other metabolic pathways, such as alcoholics and individuals with mutations in genes that result in the accumulation of toxic acetaldehyde. The theoretical part of this thesis deals with alcohol metabolism, in vivo acetaldehyde formation, and its interactions with DNA. The ICL and their repair pathways are characterized in more detail. A separate chapter is dedicated to Fanconi anaemia. The practical part of this work focuses on the preparation of site-specific acetaldehyde- induced ICL (AA-ICLs) and the study...
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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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The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
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The impact of mutant huntingtin on oxidative stress in primary fibroblasts isolated from a new Huntington's disease knock in porcine model
Sekáč, Dávid ; Ellederová, Zdeňka (advisor) ; Hanzlíková, Hana (referee)
Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...
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The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
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Použití spektroskopických technik pro studium interakcí platinových komplexů a nanočástic s nukleovými kyselinami =: Interactions study of nanoparticles and platinum complexes with nucleic acids assessed by spectroscopic techniques /
Nejdl, Lukáš
The ability of the metal ions to form a covalent bond with the nucleic acids (DNA or RNA) is critical for their structural properties and functions. In the 60s of the last century the potential of platinum complexes in anticancer therapy was revealed. The success of these complexes in anticancer treatment is given by their ability to bind to the DNA bases to form different types of coordination covalent bonds. The formation of these bonds results in an interference of the DNA secondary structure and thereby blocking of important cellular processes such as replication or transcription. Presented thesis examines the ability of metal and semimetal ions (Zn(III), As(III) and As(V)), platinum nanoparticles (PtNPs) and cadmium-based quantum dots (QDs-CdS) to influence the DNA secondary structure. In this work the interactions of metal ions with DNA were firstly investigated due to the implementation and verification of instrumental methods. Results of these studies served as the basis for subsequent experiments dealing with the effects of nanoparticles on eukaryotic cells with regard to DNA damage. In this work we demonstrated that PtNPs show higher affinity for DNA polymerases than to DNA. For this reason, PtNPs can arrest the cell cycle and trigger apoptosis. The affinity rate of nanoparticle binding to DNA is determined by its size, as was shown by the experiments with variously sized CdS-QDs.
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Effect of selected antineoplastic drugs on the gene expression of DNA repair proteins
Klčová, Silvia ; Jirkovská, Anna (advisor) ; Suchá, Simona (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Silvia Klčová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: Effect of selected antineoplastic drugs on the gene expression of DNA repair proteins. Every single cell of the human body is continuously exposed to a wide range of stress factors, with the consequence of damage to the DNA molecule. The resulting changes represent variety of alterations - from simple alkylation modifications of bases to the most unfavorable double-strand breaks (DSBs). However, the effect of cellular stress and subsequent genotoxic DNA damage is a double-edged sword. On the one hand, typical alterations in the genome can be triggered by mutagenic agents (such as components of tobacco smoke or ionizing radiation). Consequences of their action can accumulate and trigger loss of control over various steps of the cell cycle, which results in tumor cell transformation. On the other hand, however, inducing detrimental impact affecting the genome of tumor cells is one of the fundamental approaches in cytostatic treatment of cancer. Therefore, we focused our research on several antineoplastic drugs widely used in clinical practice (etoposide, daunorubicin, dexrazoxane) or undergoing clinical...
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