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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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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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Potenciální využití WIP1 fosfatasy v terapii nádorového onemocnění prsu
Pecháčková, Soňa ; Macůrek, Libor (advisor) ; Souček, Pavel (referee) ; Krejčí, Lumír (referee)
Cells in our body respond to genotoxic stress by activation of a conserved DNA damage response pathway (DDR). Depending on the level DNA damage, DDR signaling promotes temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). Checkpoints prevent progression through the cell cycle and facilitate repair of damaged DNA. DDR represents an intrinsic barrier preventing genome instability to protect cells against cancer development. WIP1 (encoded by PPM1D) phosphatase is a major negative regulator of DDR pathway and is essential for checkpoint recovery. This thesis contributed to the understanding of molecular mechanisms of WIP1 function and revealed how WIP1 can be involved in tumorigenesis. Firstly, we described that WIP1 protein levels decline during mitosis by APC-Cdc20 dependent proteasomal degradation. WIP1 is phosphorylated at multiple residues which inhibit its enzymatic activity. We propose that inhibition of WIP1 in mitosis allows sensing of low levels of DNA damage that appear during unperturbed mitosis. Further, we identified novel gain-of-function mutations of PPM1D which result in expression of C-terminally truncated WIP1. These truncated WIP1 variants are enzymatically active and exhibit increased protein stability. As result, cells...
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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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