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Role of RecQ helicases in maintenance of genomic stability during mitosis
Černoch, Marek ; Janščák, Pavel (advisor) ; Půta, František (referee)
Helicases are proteins capable of unwinding nucleic acids, their malfunction can be dangerous for genome stability of the cell. Five RecQ-family helicases identified in human cells participate in many cellular events during the whole cell cycle, including mitosis, and therefore are very important for correct functioning. The mutations in RecQ helicases can cause them to malfunction and seriously damage various cell processes, for example DNA replication, DNA damage control or sister chromatids separation. The mutations can also lead to dangerous syndromes, with the hallmark symptom of increased risk of cancer.
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The role of RECQ5 helicase in maintanance of genome stability
Urban, Václav ; Janščák, Pavel (advisor) ; Krejčí, Lumír (referee) ; Blažek, Dalibor (referee)
DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays in cells exposed to replication stress. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome stabilizing effect by acting at sites of concomitant replication and transcription. Moreover, RECQ5- deficient cells accumulate RAD51 foci that are formed in a BRCA1-dependent manner at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Importantly, BRCA1-dependent formation...
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Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I
Šišáková, Eva ; Weiserová, Marie (advisor) ; Janeček, Jiří (referee) ; Janščák, Pavel (referee)
v vuuuerJ Type I restriďion-modificationenzymeEcoRl24I is a multifunctional,hetero.oligomeric enzyme complex that cleaves DNA after extensiveATP hydrolysis coupled to processive DNA translocation.ATP hydrolysis and DNA translocationare conferredby superfamily2 (SF2) helicase motifs in the central domain of its HsdR subunit.The N-terminal domain carries a conservedregion with catalytic residuesreminiscentof the PD-@/D)xK catalytic motif of Type II restrictionenzymes. Single amino acid substitutionsin the motifs II and III reducedor removedDNA cleavage activiý of the enzymecomplexwithoutďfecting an assemblyof the complex and its DNA- binding properties.Using a combinationof bulk solution and single-moleculeassays,we investigatedthe influence of these mutationson the DNA translocationpropertiesof the enzyme,conferredby the helicase domain. Reduced ATPase activiý of the mutantswas detectedby steady-statestopped flow measurementswith the use of phosphate-binding protein.These results do not show a clear relationshipbetweenthe translocationratesand ATPase rates.Probably the broaderand bimodal distributionof hanslocationratesand the stalling eventsduring initiation revealedin single molecule experimentsall lead to a lower apparentATPase rates.We suggestanexistenceof possibleinterdomďninteractionsbetween the...
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Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I
Šišáková, Eva ; Weiserová, Marie (advisor) ; Janeček, Jiří (referee) ; Janščák, Pavel (referee)
v vuuuerJ Type I restriďion-modificationenzymeEcoRl24I is a multifunctional,hetero.oligomeric enzyme complex that cleaves DNA after extensiveATP hydrolysis coupled to processive DNA translocation.ATP hydrolysis and DNA translocationare conferredby superfamily2 (SF2) helicase motifs in the central domain of its HsdR subunit.The N-terminal domain carries a conservedregion with catalytic residuesreminiscentof the PD-@/D)xK catalytic motif of Type II restrictionenzymes. Single amino acid substitutionsin the motifs II and III reducedor removedDNA cleavage activiý of the enzymecomplexwithoutďfecting an assemblyof the complex and its DNA- binding properties.Using a combinationof bulk solution and single-moleculeassays,we investigatedthe influence of these mutationson the DNA translocationpropertiesof the enzyme,conferredby the helicase domain. Reduced ATPase activiý of the mutantswas detectedby steady-statestopped flow measurementswith the use of phosphate-binding protein.These results do not show a clear relationshipbetweenthe translocationratesand ATPase rates.Probably the broaderand bimodal distributionof hanslocationratesand the stalling eventsduring initiation revealedin single molecule experimentsall lead to a lower apparentATPase rates.We suggestanexistenceof possibleinterdomďninteractionsbetween the...
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The role of RECQ5 helicase in maintanance of genome stability
Urban, Václav ; Janščák, Pavel (advisor) ; Krejčí, Lumír (referee) ; Blažek, Dalibor (referee)
DNA replication is the most vulnerable process of DNA metabolism in proliferating cells and therefore it is tightly controlled and coordinated with processes that maintain genomic stability. Human RecQ helicases are among the most important factors involved in the maintenance of replication fork integrity, especially under conditions of replication stress. Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays in cells exposed to replication stress. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome stabilizing effect by acting at sites of concomitant replication and transcription. Moreover, RECQ5- deficient cells accumulate RAD51 foci that are formed in a BRCA1-dependent manner at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Importantly, BRCA1-dependent formation...
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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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Role of RecQ helicases in maintenance of genomic stability during mitosis
Černoch, Marek ; Janščák, Pavel (advisor) ; Půta, František (referee)
Helicases are proteins capable of unwinding nucleic acids, their malfunction can be dangerous for genome stability of the cell. Five RecQ-family helicases identified in human cells participate in many cellular events during the whole cell cycle, including mitosis, and therefore are very important for correct functioning. The mutations in RecQ helicases can cause them to malfunction and seriously damage various cell processes, for example DNA replication, DNA damage control or sister chromatids separation. The mutations can also lead to dangerous syndromes, with the hallmark symptom of increased risk of cancer.
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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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