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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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Cell cycle regulation and genome integrity protection in the early mammalian embryos
Knoblochová, Lucie ; Drutovič, David (advisor) ; Carr, Antony M. (referee) ; Fulková, Helena (referee)
(English version) Infertility is a major health problem, as it affects one in every six people worldwide (Njagi et al., 2023). One of the major reasons for infertility are aneuploidies, additions or losses of an entire or partial chromosome during cell division. Aneuploidies thus negatively influence cellular processes and potentially lead to developmental problems or embryo loss. It has been thought for a long time that aneuploidies arise mostly during oocyte development, and these mechanisms have been well studied. However, recent evidence has shown that aneuploidies arise also de novo after fertilisation and during the early embryonic development; but the molecular mechanisms of these abnormalities still remains elusive. Aneuploidies often originate during cell cycle division from unrepaired DNA damage in mitosis. DNA damage is sensed by DNA damage response (DDR) signalling pathways, which slow down or arrest cell cycle progression until it is resolved. An essential DDR factor during typical cell cycle progression is checkpoint kinase 1 (CHK1). However, the role of DDR factors in the early embryos, and especially CHK1, have not been well studied. Early embryonic development is regulated by maternal factors stored in the oocyte until the transcription of the embryonic genome begins. To study...
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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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ADP-ribosylation in ARH3-deficient cells and its impact on cellular functions
Kuttichová, Barbora ; Hanzlíková, Hana (advisor) ; Valihrach, Lukáš (referee)
ADP-ribosylation is a crucial post-translational modification that regulates various cellular processes, including DNA repair. It is catalysed by poly-ADP-ribose polymerases (PARPs) and involves the transfer of ADP-ribose moieties from the redox cofactor NAD+ to proteins, including histones. To maintain cellular homeostasis, ADP-ribose chains need to be rapidly degraded by ADP-ribosyl glycohydrolases. While poly-ADP-ribose glycohydrolase (PARG) is highly efficient, it cannot cleave the terminal ADP-ribose moiety. For the removal of the terminal mono-ADP-ribose, two glycohydrolases, TARG1 and ARH3, are involved. This removal process is necessary because it enables DNA repair factors to access the site of DNA damage. The primary goal of this thesis is to characterise cells derived from patients with homozygous ARH3 mutations and to develop appropriate tools to improve our understanding of the molecular mechanism by which ARH3 mutations affect ADP-ribosylation and how it contributes to the onset of the associated neurological disease. To achieve this, I measured the levels of ARH3 protein and detected increased mono-ADP-ribosylation in ARH3-mutated patient-derived fibroblasts. Furthermore, I assessed the sensitivity of these cells to different PARP inhibitors, which hold potential for the therapeutic...
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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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Males-females differences in the spectrum of chromosomal aberrations in the group of nanocomposites production workers
Rössnerová, Andrea ; Pelcová, D. ; Ždímal, Vladimír ; Elzeinova, Fatima ; Margaryan, Hasmik ; Chvojková, Irena ; Topinka, Jan ; Schwarz, Jaroslav ; Ondráček, Jakub ; Koštejn, Martin ; Komarc, M. ; Vlčková, Š. ; Fenclová, Z. ; Lischková, L. ; Dvořáčková, Š. ; Rössner ml., Pavel
An increase in the use of nanomaterials (NM) has been witnessed in many areas of human life. Therefore, assessment of genotoxicity of NM and nanoparticles (NP) is one of the main objectives of genetic toxicology. Despite this fact, human cytogenetic studies following the exposure to NP are still rare. Moreover, no relevant information on possible differences in sensitivity to NP related to gender is available.\n\nIn this study we periodically (in September 2016, 2017 and 2018; pre-shift and post-shift each year) analyzed a group of workers (both genders), working long time in nanocomposites research, and matched controls. Aerosol exposure monitoring of particulate matter including nano-sized fractions was carried out during working shift. Micronucleus assay using Human Pan Centromeric probes, was applied to distinguish, besides the frequency of total MN in binucleated cells (BNC), also other types of chromosomal damage (losses and breaks). Moreover, whole-chromosome painting (WCP) for autosome #1 and both gonosomes (X and Y) were applied in third sampling period (2018) with the aim to identify the particular structural and numerical chromosomal aberrations.\n\nObtained results showed: (i) differences in the risk of exposure to NP related to individual working processes (welding, smelting and machining); (ii) differences in chemical composition of nano-fraction; (iii) no effect of chronic exposure of NP (total MN) opposite to significant effect of acute exposure; (iv) gender-related DNA damage differences (females seem to be more sensitive to chromosomal losses). Additional data from WCP suggested increased frequency of numerical aberrations in gonosomes.
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Using PCR to study the DNA damage
Jansová, Adéla ; Jirkovská, Anna (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Bc. Adéla Jansová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: Using PCR to study the DNA damage Key words: functions of DNA, DNA damage, PCR, SINE elements The transmission of genetic information to future generations is possible thanks to DNA replication using enzymes, mainly DNA polymerase. The most important function of DNA is the biosynthesis of proteins that perform specific functions throughout the cell. The coding DNA sequences are the source for protein synthesis. These are produced by transcription of a DNA sequence using RNA polymerase and then translated into amino acids by translation. Non-coding sequences have mainly regulatory functions, they are functional DNA molecules (rRNA, tRNA, snRNA) and regulatory regions (promoters, enhancers and silencers) as well as transposons (SINE, LINE) and pseudogenes. DNA damage is caused by UV radiation, ionizing radiation, chemicals (cisplatin-based drugs, alkylating agents, etc.), reactive oxygen species, and DNA damage by base deamination. When damage occurs, repair processes are activated to remove mismatches, adducts and breaks. If the damage is not repaired by repair processes, the damage leads to mutation formation, senescence...
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