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Interplay between DNA repair pathways at replication forks in mammalian cells
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular DNA is continuously being damaged by various agents of both endogenous and exogenous origin including a wide range of chemical compounds, UV-light and ionizing radiation. In order to remove lesions from the DNA and maintain genomic stability, different repair processes have evolved. If not repaired prior to DNA replication, such lesions may block the replication machinery and the capacity to rescue replication stalled at the site of a DNA lesion is thus also important for cellular survival and the maintenance of genome integrity. To date, the interplay between pathways of DNA repair and mechanisms of replication bypass in mammalian cells has not been fully elucidated.

The present thesis focuses on the roles played by base- and nucleotide-excision repair and by homologous recombination in reducing errors in connection with replication of damaged DNA in mammalian cells. We were also interested in the effects of caffeine on cellular mechanisms that facilitate replication bypass. Furthermore, we have investigated the mutagenicity of glycidamide and attempted to identify the DNA repair pathway responsible for removing lesions induced by this genotoxic metabolite of acrylamide. In all of these studies Chinese hamster cell lines harbouring mutations in genes encoding proteins associated with DNA repair were employed as the model system. Moreover, we explored the possibility of using these cell lines to develop a system for high-throughput, rapid detection of genotoxic agents.

Our results indicates that both nucleotide excision repair and homologous recombination are involved in maintaining replication fork progression on DNA damaged by UV-light or benzo(a)pyrene-7,8-diol-9,10-epoxide. In this connection homologous recombination appears to be a time-consuming process. We also found that the replication fork delay observed following exposure to UV-light is prolonged even further by subsequent treatment with caffeine, both in the case of wild-type cells and cells deficient in homologous recombination or nucleotide-excision repair. In addition, the frequency of mutations induced by UV-irradiation was attenuated, while the level of recombination was enhanced by caffeine. These results indicate that caffeine inhibits translesion DNA synthesis, thereby favouring the use of homologous recombination to bypass lesions that stall replication.

On the basis of our findings with methyl methanesulfonate (MMS) and N-methyl-N´-nitro-N-nitrosoguanidine (MNNG), we propose that O6-methylguanine is the major substrate for homologous recombination following treatment with alkylating agents. Both of these agents also stall replication forks, probably due to the presence of unrepaired intermediates in base-excision repair. Moreover, we discuss the possibility of a Rad51-mediated pathway for homologous recombination that is dependent on XRCC1.

Furthermore, glycidamide was shown to cause mutations in the hprt gene of wild-type Chinese hamster ovary cells. Our results also suggest that the DNA lesions induced by this compound are repaired by short-patch BER. However, we were unable to identify the lesion responsible for the mutations.

Finally, the Chinese hamster ovary cell lines employed here provide a useful tool for the screening of genotoxic compounds and, in addition, for obtaining mechanistic information concerning the pathways involved in the repair of DNA lesions induced by various agents.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi , 2005.
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:su:diva-689OAI: oai:DiVA.org:su-689DiVA: diva2:197276
Public defence
2005-10-27
Opponent
Supervisors
Available from: 2005-10-06 Created: 2005-10-06Bibliographically approved
List of papers
1. Screening for genotoxicity using the DRAG assay: investigation of halogenated environmental contaminants
Open this publication in new window or tab >>Screening for genotoxicity using the DRAG assay: investigation of halogenated environmental contaminants
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2004 In: Mutation Research/Genetic Toxicology and Environmental Mutagenesis, ISSN 1383-5718, Vol. 563, no 1, 35-47 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24342 (URN)
Note
Part of urn:nbn:se:su:diva-689Available from: 2005-10-06 Created: 2005-10-06Bibliographically approved
2. Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells
Open this publication in new window or tab >>Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells
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2005 In: Mutation Research/Genetic Toxicology and Environmental Mutagenesis, ISSN 1383-5718, Vol. 580, no 1-2, 81-89 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24343 (URN)
Note
Part of urn:nbn:se:su:diva-689Available from: 2005-10-06 Created: 2005-10-06Bibliographically approved
3. A method to monitor replication fork progression in mammalian cells: nucleotide excision repair enhances and homologous recombination delays elongation along damaged DNA
Open this publication in new window or tab >>A method to monitor replication fork progression in mammalian cells: nucleotide excision repair enhances and homologous recombination delays elongation along damaged DNA
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24344 (URN)
Note
Part of urn:nbn:se:su:diva-689Available from: 2005-10-06 Created: 2005-10-06 Last updated: 2010-01-13Bibliographically approved
4. Caffeine delays replication fork progression and enhances homologous recombination induced in Chinese hamster cell lines by UV-irradiation
Open this publication in new window or tab >>Caffeine delays replication fork progression and enhances homologous recombination induced in Chinese hamster cell lines by UV-irradiation
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24345 (URN)
Note
Part of urn:nbn:se:su:diva-689Available from: 2005-10-06 Created: 2005-10-06 Last updated: 2010-01-13Bibliographically approved
5. Two recombination pathways involved in repair of alkylated lesions
Open this publication in new window or tab >>Two recombination pathways involved in repair of alkylated lesions
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24346 (URN)
Note
Part of urn:nbn:se:su:diva-689Available from: 2005-10-06 Created: 2005-10-06 Last updated: 2010-01-13Bibliographically approved

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