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Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicus
Černý, Zbyněk ; Španová, Alena (referee) ; Pepeliaev,, Stanislav (advisor)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
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Genes of early meiotic prophase I of spermatogenesis in house mouse
Škaloudová, Eliška ; Trachtulec, Zdeněk (advisor) ; Forman, Martin (referee)
Meiosis is an essential cellular process that is necessary for gamete formation in all sexually reproducing organisms. This work is focused on the description of the genes of early stages of meiotic division in males of a mammalian model, the house mouse. The first part summarizes meiosis focusing on prophase I, which is longer than prophase II. Prophase I is divided into five stages, namely leptotene, zygotene, pachytene, diplotene, and diakinesis. Mouse spermatogenesis and its differences from oogenesis are also briefly described. The second part provides a list of genes encoding proteins required for initiation of meiotic division, pairing and synapses of chromosomes, and initiation of the catalysis of double-strand breaks. Double-strand breaks are repaired by homologous recombination, which may result in so-called crossing-over, the major source of genetic variability. The work deals with the early stage of homologous recombination and components required for this process. Localization of meiotic double-strand breaks in the genome is not random and is under the control of the Prdm9 gene, which seems to take multiple roles, such as the formation of new subspecies of the house mouse. Knowledge of the genes controlling the early stages of meiotic division is a prerequisite to understanding some of...
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The Role of FBH1 in Maintenance of Genome Stability
Šimandlová, Jitka ; chevelev, Igor (advisor) ; Kratochvíl, Lukáš (referee)
The genome is constantly threatened by various damaging agents and maintaining its integrity is crucial for all organisms. Several repair pathways have been implicated in the removal of different types of lesions from DNA. Among them, homologous recombination (HR) plays a key role in repair of double-strand breaks. HR is a highly important repair mechanism which has to be tightly regulated to prevent excessive HR events. These events could interfere with other DNA repair pathways, generate toxic intermediates, or block the progression of the replication fork. Therefore, it is not surprising that cells have evolved mechanisms that counteract inappropriate HR events. As it has been shown recently, cells possess DNA helicases capable of preventing excessive recombination. A novel human DNA helicase, hFBH1, belonging to the superfamily I has been shown to function as pro- and anti- recombinase. Similar to the two members of RecQ family, BLM and RECQL5, FBH1 disrupts Rad51 from nucleofilament. However, FBH1 might also promote initiation of HR. The FBH1 helicase possesses additional high conserved F-box motif which allows it to act within a Skp1-Cullin-F-box, SCF, complex as ubiquitin ligase and target proteins for degradation.
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Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
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RTEL1 as a novel suppressor of homologous recombination
Žítek, Ondřej ; chevelev, Igor (advisor) ; Kratochvíl, Lukáš (referee)
Regulator of telomere elongation helicase 1 (RTEL1) is a DNA helicase crucial for regulation of telomere length in mice while its loss has been associated with shortened telomere length, chromosome breaks, and translocations. Moreover, RTEL1 is an important member of the DNA double-strand break-repair (DSBR) pathway. It maintains genome stability directly by suppressing homologous recombination through disassembling D loop recombination intermediates during DNA repair. Antirecombinase properties of RTEL1 make it the key protein required in meiosis and mitosis to execute non-crossover way of DSBR by promoting synthesis-dependent strand annealing (SDSA). Defect in any of these functions might lead to glioma predisposition in human.
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Chromosomal damage and DNA repair capacity in blood lymphocytes as transient markers in carcinogenesis.
Kroupa, Michal ; Vodička, Pavel (advisor) ; Štětina, Rudolf (referee)
Recent knowledge suggests that the onset of cancer is modulated by the interplay of internal and external environmental factors along with numerous gene variants. Structural chromsomal aberrations in peripheral blood lymphocytes are considered as biomarkers of effect of genotoxic carcinogens and reflect elevated risk of cancer. Incomplete or deficient repair of double-strand breaks in DNA underlie chromosomal aberrations and the measurement of cytogenetic alterations may reflect interindividual differences in the response towards the mutagen. In this study the expected deficiences in the DNA repair capacity have been determined in incident oncological patients with breast, colorectal and urogenital cancers. The determination of chromosomal aberrations have been supplemented by the measurement of variants in genes involved in double-strand breaks repair (XRCC3, rs861539; RAD54L, rs1048771). Methodologically, we employed conventional cytogenetic analysis, cytogenetic analysis following the induction of chromocomal damage by bleomycin ("Challenge assay"), TaqMan discrimination analysis for the detection of allelic variants and statistical analyses. By using these methods we did not observe statistically signifiant differences either in chromosomal breaks (p=0,354) or in a percentage of cells with...
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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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The role of DNA repair mechanisms in the pathogenesis of myelodysplastic syndrome.
Válka, Jan ; Čermák, Jaroslav (advisor) ; Pospíšilová, Dagmar (referee) ; Penka, Miroslav (referee)
Background: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests the involvement of DNA repair mechanism defects in the pathogenesis of this disorder. The first part of this work was focused on monitoring of gene expression of DNA repair genes in MDS patients and on their alterations during disease progression. In the second part, next generation sequencing was used to detect single nucleotide polymorphisms (SNPs) and mutations in DNA repair genes and their possible association with MDS development was evaluated. Methods: Expression profiling of 84 DNA repair genes was performed on bone marrow CD34+ cells of patients with MDS. Screening cohort consisted of 28 patients and expression of selected genes was further validated on larger cohort of 122 patients with all subtypes of MDS. Paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 recombinase protein was done on samples acquired by trephine-biopsy. Targeted enrichment resequencing of exonic parts of 84 DNA repair genes was performed on the screening cohort of MDS patients. Real-time PCR was used for genotyping of selected SNPs in the population study. Results: RAD51 and XRCC2 genes showed...
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CRISPR/Cas9-based genome editing in mice: state of the art and future perspectives
Eliáš, Jan ; Kašpárek, Petr (advisor) ; Čáp, Michal (referee)
Mutant mice are crucial tools for understanding gene functions in vivo. Recently, generation of mouse mutants was revolutionized by rapid developement of programmable nucleases, predominantly by the CRISPR/Cas9 system. Genome editing based on introduction of CRISPR/Cas9 components into early stage mouse embyros allows fast and inexpensive generation of gene-deficient animal models, especially when compared to the traditional techniques based on modification of embryonic stem cells (ESCs). The ability of CRISPR/Cas9 to induce double-strand break (DSB) at a given location of genomic DNA enables effective gene-ablation by random modification of the coding sequences or by complete ablation of the gene. However, precise modification of the gene sequences, such as incorporation of a DNA fragment into specific loci, are still difficult to make. In this work, I present a review of CRISPR/Cas9 system, its use in production of mutant mice and possible modifications of the system to increase the efficiency of precise gene-targeting. Keywords: CRISPR/Cas9, mouse, transgenesis, homologous recombination
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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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