Interplay between DNA repair pathways at replication forks in mammalian cells

Detta är en avhandling från Stockholm : Institutionen för genetik, mikrobiologi och toxikologi

Sammanfattning: 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.

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