Mechanistic study of the effects of 8-oxo-7,8-dihydroguanine (8-oxoG) on reporter gene expression in mammalian cells

DNA damage causes replication errors, leading to genetic instability or cell death. Besides that, many types of DNA base modifications have been shown to interfere with transcriptional elongation if they are located in the transcribed DNA strand of active genes, acting as roadblocks for RNA polymerases. It is widely assumed that transcription blockage by endogenous DNA damage is responsible for the early cell senescence in organs and accelerated ageing observed in individuals with compromised nucleotide excision repair.rnThe aims of this work were to design new experimental systems for testing transcription blocking potentials of DNA base modifications in an individual gene and to apply these test systems to the investigation of the effects of a frequent endogenously generated base modification, namely 8-oxo-7,8-hydroxyguanine (8-oxoG), on the gene transcription in cells. Several experimental strategies were employed for this purpose. First, I constructed an episomal vector encoding for a short-lived EGFP-ODC fusion protein and measured expression of the reporter gene in permanently transfected clonal cell lines exposed to DNA damaging agents. Second, the expression of plasmid-borne EGFP gene damaged with photosensitisers to obtain one or several oxidative purine modifications per plasmid molecule was determined in transiently transfected human and mouse host cells in an approach known as “host cell reactivation”. As a prerequisite for these experiments, a robust method of precise quantitative measurement of the EGFP gene expression in transiently transfected cells by flow cytometry was developed and validated. Third, I elaborated a very efficient procedure for insertion of synthetic oligonucleotides carrying 8-oxoG into plasmid DNA, avoiding any unwanted base damage and strand breaks. The consequences of 8-oxoG placed in defined positions in opposing DNA strands of the EGFP gene for transcription were measured by host cell reactivation in cells with functional 8-oxoguanine DNA glycosylase (OGG1) gene and in OGG1 null cells.rnThe results obtained in Ogg1-/- cells demonstrated that unrepaired 8-oxoG, even if situated in the transcribed DNA strand, does not have any negative effect on the reporter gene transcription. On the other hand, as few as one 8-oxoG was sufficient to cause a significant decrease of the gene expression in OGG1-proficient cell lines, i.e. in the presence of base excision repair. For two analysed positions of 8-oxoG in the plasmid DNA, the inhibition of gene transcription by the base modification correlated with the efficiency of its excision by purified OGG1 protein under cell-free conditions. Based on these findings, it has to be concluded that the observed decrease of transcription is mediated by excision of the base modification by OGG1 and probably caused by the repair-induced single-strand breaks. The mechanism of transcription inhibition by 8-oxoG is therefore clearly distinct from stalling of elongating RNA polymerase II complexes at the modified base.