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Characterization of Antirecombinase Activity of Human FBH1 Helicase
Šimandlová, Jitka ; Janščák, Pavel (advisor) ; Cséfalvay, Eva (referee)
Homologous recombination (HR) is an essential mechanism for accurate repair of DNA double-strand breaks (DSBs). However, HR must be tightly controlled because excessive or unwanted HR events can lead to genome instability, which is a prerequisite for premature aging and cancer development. A critical step of HR is the loading of RAD51 molecules onto single-stranded DNA regions generated in the vicinity of the DSB, leading to the formation of a nucleoprotein filament. Several DNA helicases have been involved in the regulation of the HR process. One of these is human FBH1 (F-box DNA helicase 1) that is a member of SF1 superfamily of helicases. As a unique DNA helicase, FBH1 additionally possesses a conserved F-box motif that allows it to assemble into an SCF complex, an E3 ubiquitin ligase that targets proteins for degradation. FBH1 has been implicated in the restriction of nucleoprotein filament stability. However, the exact mechanism of how FBH1 controls the RAD51 action is still not certain. In this work, we revealed that FBH1 actively disassembles RAD51 nucleoprotein filament. We also show that FBH1 interacts with RAD51 and RPA physically in vitro. Based on these data, we propose a potential mechanism of FBH1 antirecombinase function.
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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 ; Kratochvíl, Lukáš (referee) ; chevelev, Igor (advisor)
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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The Role of FBH1 in Maintenance of Genome Stability
Šimandlová, Jitka ; Kratochvíl, Lukáš (referee) ; chevelev, Igor (advisor)
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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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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