|
|
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...
|
|
|
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...
|
|
|
Formation of complex chromosomal rearrangements in cancer cells and significance of these events
Rochlová, Kristina ; Zemanová, Zuzana (advisor) ; Rothová, Olga (referee)
Chromoanagenesis is a catch-all term of recently described catastrophic events that generate complex karyotypes. These events are divided according to the characteristic features and are termed chromothripsis, chromoplexis and chromoanasynthesis. Chromothripsis represents a disintegration of chromosomes or their parts into hundreds of small fragments. Those chromosome fragments are then incorrectly reassembled. Chromoplexis rearrangements are not very different from chromothripsis rearrangements. The main difference is a lower number of breakpoints and the distribution of aberrations in the whole genome. The erroneous replication processes occur during chromoanasynthesis. There are several mechanisms responsible for breakdowns of a DNA molecule. In the case of chromothripsis, micronucleus formation is probably the most important mechanism. During chromoplexis, transcriptional stress plays a major role. Replication stress is associated with chromoanasynthesis rearrangements. The result of all these processes are highly rearranged chromosomes with numerous losses or gains of genetic material. This work summarizes the current knowledge of the mechanisms that are mentioned above and the genesis of complex aberrations. At the same time, it represents the connection between complex karyotype and clonal...
|
|
| |
|
|
The evaluation of DNA oxidative damage at polytraumatic patients.
Štrofová, Marcela ; Hronek, Miloslav (advisor) ; Havel, Eduard (referee)
The aim of this study was to observe levels of oxidative DNA damage in patients with multiple injuries in correlation with the nutritional support that the patients have received during their hospital stay. Oxidative DNA damage was evaluated in two periods of time, first evaluation was performed during standard nutritional support according to the ESPEN guidelines. Second evaluation was performed after a change in nutrition according to individual parameters of metabolism and utilization of nutritional components based on indirect calorimetric measurements. This study included 6 patients with multiple injuries hospitalized in the Intensive Care Unit 1 at the Department of Surgery, University Hospital in Hradec Kralove. In this experiment DNA isolated from peripheral lymphocytes was used to evaluate oxidative DNA damage. This DNA was analyzed using the Comet Assay method. The enzymatic version of the Comet Assay was used to determine the oxidative damage of purines and pyrimidines, and the alkaline version was used for detection of single strand breaks. Mann-Whitney test was used for statistic evaluation the difference between both measuremetns, correlation analysis for relations between Comet Assay results and clinical parameters. Significant correlations between a total amount of nutrients given...
|
|
|
Intracellular signalling of Chk2 kinase and impact of its defects in oncogenesis
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Brábek, Jan (referee)
Chk2 (checkpoint kinase 2), a regulatory protein of the cell cycle checkpoints, is coded by CHEK2 gene. Chk2 belongs to serine/threonine kinase family and its dominant activity is in regulation and signal distribution of intracellular response to DNA damage. The upstream regulator of Chk2 protein is the ATM kinase that activates Chk2 by its phosphorylation on Thr68 localized in FHA domain. This in turn leads to the conformation change inducing homodimerization of Chk2 protomers and their activating phosphorylation within their kinase domains. Upon phosphorylation, catalytically active Chk2 protomers dissociate and phosphorylate various intracellular proteins (incl. p53, E2F-1, BRCA1, Cdc25A a C, BRCA2 a PLK3). By regulation of these proteins, Chk2 contributes to the cell cycle arrest, regulation of DNA repair and apoptosis. Germline mutations in CHEK2 gene were identified with the increased frequency in many human cancers, including breast and colorectal cancer. Hence, the failure of Chk2 intracellular activity contributes to the process of malignant transformation.
|
|
|
Gene PALB2 and its role in breast cancer
Zdařilová, Klára ; Janatová, Markéta (advisor) ; Žáčková Suchanová, Jiřina (referee)
Breast cancer is the most common cancer among women in the Czech Republic. Mutations in two major predisposition genes, BRCA1 and BRCA2, account only for 16 % of familial risk of breast cancer. Gene PALB2 was discovered in 2006 as a tumor suppressor. Protein product of PALB2 plays a major role in pathway of DNA repair of double-strand breaks through the homologous recombination mechanism. PALB2 links BRCA1, BRCA2 and RAD51 and is required for their recruitment to DNA damage foci and initiate homologous recombination. In a response of DNA damage PALB2 participates on regulation of the cell cycle. Protein function of PALB2 is necessary to maintain the integrity of the genome and in case of loss this function, because of the gene inactivation, it leads to genomic instability, which may be the basis for the development of tumorogenesis. Heterozygous mutations in PALB2 increase the risk of breast cancer predisposition, these mutations has been demonstrated even in pancreatic cancer and less often in ovarian cancer. Therefore, it is important to analyze truncating mutations in the PALB2 gene in BRCA1/2-negative patients from families with a strong history of hereditary breast cancer. The frequency of PALB2 mutations may be comparable to the frequency of mutations in the BRCA2 gene in Czech hereditary...
|
|
| |
|
|
Structure, function and importace of BRCA 1protein
Hojný, Jan ; Falk, Martin (referee) ; Kleibl, Zdeněk (advisor)
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...
|
guest ::
