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Molecular mechanisms underlying maintenance of genome stability
Burdová, Kamila ; Janščák, Pavel (advisor) ; Cséfalvay, Eva (referee) ; Krejčí, Lumír (referee)
Cells in our body are challenged every day by DNA damage arising as a result of both endogenous and exogenous insults. The ability of cells to repair DNA lesions is essential for correct propagation of genetic information. The most cytotoxic DNA lesion is DNA double-strand break (DSB) while oxidative DNA damage is one of the most frequent lesions. The aim of this thesis was to improve current the knowledge of the molecular mechanisms underlying the repair of DSBs and oxidative DNA damage. The major source of oxidative damage in cells are reactive oxygen species that are constantly generated as by-products of cell metabolism. One of the most frequent lesions is 7,8- dihydro-8-oxo-guanine (8-oxo-G) that gives rise to 8-oxo-G:A mispairs during DNA replication and if left unrepaired, results in accumulation of DNA mutations. We found that Werner helicase (WRN) physically interacts with DNA polymerase λ (Polλ) and stimulates DNA gap-filling by Polλ opposite to 8-oxo-G followed by strand displacement synthesis in MutY DNA glycosylase homolog (MUTYH) initiated base excision repair (BER) of 8-oxo-G:A mispairs. There are two major pathways involved in repair of DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). NHEJ is highly error-prone while HR is error-free. There are two...
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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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