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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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Study of regulation and function of DNA repair enzymes UBE2T and FANCL
Hušková, Andrea ; Šilhán, Jan (advisor) ; Martínková, Markéta (referee)
Due to the action of endogenous and exogenous agents, DNA is subject up to 70,000 lesions per day, thus the existence of repair mechanisms and enzymes is more than necessary. We know basic mechanisms of several specific DNA repair pathways, of which the Fanconi anaemia (FA) repair pathway is one of the least explored. FA is a rare, autosomal recessive disorder characterized by early onset bone marrow failure, developmental defects, genomic instability and predisposition to acute myeloid leukaemia and solid tumours. The primary diagnosis of FA is a hypersensitivity to cross-linking agents of DNA due to inactivation of one of the 21 genes from the FA repair pathway, the so-called FANC genes (FA complementation group). The molecular defect in FA is an impaired repair of DNA interstrand cross-links (ICLs). The ICLs are cytotoxic lesions that inhibit the process of DNA replication and transcription. A crucial step in the FA pathway that initiates ICL repair is a monoubiquitination of FANCD2. FANCD2 monoubiquitination is a base for the recruitment of additional proteins that coordinate DNA repair. Ubiquitin is recruited via activating enzyme E1 (UBA1), ubiquitin-conjugating enzyme E2T (UBE2T) and transferred onto FANCD2 by multisubunit E3 ligase (FA core complex). There are up to 11 different proteins...
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