ro-19-8022 and 8-hydroxyguanosine

In mammalian cells, 7,8-dihydro-8-oxoguanine (8-oxoG) and some other oxidative guanine modifications are removed from the DNA by base excision repair, which is initiated by OGG1 protein. We have tested whether this repair is inducible in mouse embryonic fibroblasts (MEFs), MCF-7 breast cancer cells and primary human fibroblasts by a pretreatment with the photosensitizer Ro19-8022 plus light, which generates predominantly 8-oxoG, or with methyl methanesulfonate (MMS), which generates alkylated bases and abasic sites (AP sites). The results indicate that the repair rate of the oxidative guanine modifications induced by the photosensitizer was not increased if a priming dose of the oxidative or alkylating agent was applied 6 or 18h prior to a challenging dose, although pretreatments with both agents resulted in two-fold elevated glutathione levels as an indication for an adaptive response. Similarly, the activity of total protein extracts of the cells to incise at a single 8-oxoG residue in an oligonucleotide was unchanged. It has to be concluded that the repair of 8-oxoG is not inducible by oxidative or alkylation damage.

Topics: Animals; Antineoplastic Agents, Alkylating; Base Pairing; DNA; DNA Damage; DNA Repair; Female; Fibroblasts; Glutathione; Guanosine; Humans; Methyl Methanesulfonate; Mice; Oxidation-Reduction; Oxidative Stress; Photosensitizing Agents; Pyrrolidines; Quinolizines; Time Factors; Tumor Cells, Cultured

Cockayne syndrome (CS) is an autosomal recessive human disease characterized by UV-sensitivity as well as neurological and developmental abnormalities. Two complementation groups have been established, designated CS-A and CS-B. Traditionally, CSA and CSB have been ascribed a function in the transcription-coupled repair (TCR) pathway of nucleotide excision repair (NER) that efficiently removes bulky lesions from the transcribed strand of RNA polymerase II transcribed genes. To assess the role of the CSB protein in the repair of the highly mutagenic base lesion 7,8-dihydro-8-oxoguanine (8-oxoG), we have investigated the removal of this lesion using an in vitro incision approach with cell extracts as well as an in vivo approach with a modified protocol of the gene-specific repair assay, which allows the measurement of base lesion repair in intragenomic sequences. Our results demonstrate that the integrity of the CSB protein is pivotal for processes leading to incision at the site of 8-oxoG and that the global genome repair (GGR) of this lesion requires a functional CSB gene product in vivo.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Animals; Cell Line; Cell Survival; Cricetinae; DNA Helicases; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Genome; Guanosine; Light; Molecular Sequence Data; Mutation; N-Glycosyl Hydrolases; Photosensitizing Agents; Protein Structure, Tertiary; Pyrrolidines; Quinolizines; Sequence Alignment; Tetrahydrofolate Dehydrogenase