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The Repair of Oxidative DNA Damage in the Chinese Hamster Cell Line Deficient in Nucleotide Excision Repair
Strejčková, Lada ; Trejtnar, František (advisor) ; Pávek, Petr (referee)
The repair of oxidative DNA damage in the Chinese hamster cell line deficient in nucleotide excision repair Abstract The aim of our work was to specify DNA oxidative damage repair in the Chinese hamster ovary cell line. Comet assay was used to detect the damage consequences - DNA breaks. We were capable of achieving high method sensitivity due to application of specific endonucleases and specific inhibitors of excision repair - inhibitor of poly(ADP- ribose)polymerase PARP 3-aminobenzamide (3-AB) and cytosine arabinoside with hydroxyurea (AraC/HU). PARP-1 is an important factor in the base excision repair (BER) process. PARP-1 deficient cells show recovery impairment both in LP-BER and SP-BER way. AraC/HU is known as the long-patch excision repair inhibitor. The results of our experiments showed the inhibiting effect of 3-AB on the repair of hydrogen peroxide-induced DNA damage. But in the same setting the AraC/HU effect on repair was not observed. To make the value of experimental results more significant and clear, we decided for additional experiments with monofunctional alkylating agent methyl methanesulfonate (MMS). Damage caused by MMS showed slower repair both when 3-AB or AraC/HU were present during the repair period. Based on all available results we suppose that the MMS and also hydrogen peroxide...
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The repair of the oxidative DNA damage in Chinese hamster cells deficient in nucleotide excision DNA repair.
Strejčková, Lada ; Hochmann, Jiří (advisor) ; Polívková, Zdeňka (referee)
Nucleotide excision repair (NER) is one of the mechanisms of how to repair DNA damaged by the external influences. First of all, it plays the role when removing the results caused by UV radiation. The damaged bases in the form of pyrimidine dimers are enzymatically splitted out from DNA as part of oligonucleotides. The effect of the UV light on DNA is however manifested also indirectly, by oxidation of the bases. In our experiment we tried to find out whether the cells deficient in nucleotide excision repair are able to repair the damages induced only by oxidative agent. We stated the extent of DNA damages in the cells in vitro and the ability of their re- paration after treatment with the dilution of hydrogen peroxide in different concentrations. During the experiments we used the cell lines of a Chinese hamster, namely the parental cell line AA8 and their derivative UV-20, which is defective in gene ERCC1. This gene takes part in creation of nucleases participating in NER. For measuring of DNA lesions we used the method of alkaline comet assay, the ba- sis of which is alkaline single-cell gel electrophoresis. The principle of this method consists in the detection of DNA strand breaks originating in the damaged alkali-labile sites. We evaluated the number of DNA single-strand breaks. We detected...
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The repair of the oxidative DNA damage in Chinese hamster cells deficient in nucleotide excision DNA repair.
Strejčková, Lada ; Hochmann, Jiří (advisor) ; Polívková, Zdeňka (referee)
Nucleotide excision repair (NER) is one of the mechanisms of how to repair DNA damaged by the external influences. First of all, it plays the role when removing the results caused by UV radiation. The damaged bases in the form of pyrimidine dimers are enzymatically splitted out from DNA as part of oligonucleotides. The effect of the UV light on DNA is however manifested also indirectly, by oxidation of the bases. In our experiment we tried to find out whether the cells deficient in nucleotide excision repair are able to repair the damages induced only by oxidative agent. We stated the extent of DNA damages in the cells in vitro and the ability of their re- paration after treatment with the dilution of hydrogen peroxide in different concentrations. During the experiments we used the cell lines of a Chinese hamster, namely the parental cell line AA8 and their derivative UV-20, which is defective in gene ERCC1. This gene takes part in creation of nucleases participating in NER. For measuring of DNA lesions we used the method of alkaline comet assay, the ba- sis of which is alkaline single-cell gel electrophoresis. The principle of this method consists in the detection of DNA strand breaks originating in the damaged alkali-labile sites. We evaluated the number of DNA single-strand breaks. We detected...
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The Repair of Oxidative DNA Damage in the Chinese Hamster Cell Line Deficient in Nucleotide Excision Repair
Strejčková, Lada ; Trejtnar, František (advisor) ; Pávek, Petr (referee)
The repair of oxidative DNA damage in the Chinese hamster cell line deficient in nucleotide excision repair Abstract The aim of our work was to specify DNA oxidative damage repair in the Chinese hamster ovary cell line. Comet assay was used to detect the damage consequences - DNA breaks. We were capable of achieving high method sensitivity due to application of specific endonucleases and specific inhibitors of excision repair - inhibitor of poly(ADP- ribose)polymerase PARP 3-aminobenzamide (3-AB) and cytosine arabinoside with hydroxyurea (AraC/HU). PARP-1 is an important factor in the base excision repair (BER) process. PARP-1 deficient cells show recovery impairment both in LP-BER and SP-BER way. AraC/HU is known as the long-patch excision repair inhibitor. The results of our experiments showed the inhibiting effect of 3-AB on the repair of hydrogen peroxide-induced DNA damage. But in the same setting the AraC/HU effect on repair was not observed. To make the value of experimental results more significant and clear, we decided for additional experiments with monofunctional alkylating agent methyl methanesulfonate (MMS). Damage caused by MMS showed slower repair both when 3-AB or AraC/HU were present during the repair period. Based on all available results we suppose that the MMS and also hydrogen peroxide...
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