Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

The Cockayne syndrome B (CSB) gene product is involved in the repair of various types of base modifications in actively transcribed DNA sequences. To investigate its significance for the repair of endogenous oxidative DNA damage, homozygous csb 7/7 /ogg1 7/7 double knockout mice were generated. These combine the deficiency of CSB with that of OGG1, a gene coding for the mammalian repair glycosylase that initiates the base excision repair of 7,8-dihydro-8-oxoguanine (8-oxoG). Compared to ogg1 7/7 mice, csb 7/7 /ogg1 7/7 mice were found to accumulate with age severalfold higher levels of oxidited purine modifications in hepatocytes, splenocytes and kidney cells. In contrast, the basal (steady-state) levels of oxidative DNA modifications in cells from csb 7/7 mice were not different from those in wild-type mice and did not increase with age. The analysis of the repair rates of additional oxidative DNA base modifications induced by photosensitization in immortalized embryonic fibroblasts was in accordance with these findings: compared to wild-type cells, the global repair was only slightly affected in csb 7/7 cells, more compromised in ogg1 7/7 cells, but virtually absent in csb 7/7 /ogg1 7/7 cells. An inhibition of transcription by a-amanitin did not block the Csbdependent repair in ogg1 7/7 fibroblasts. The influence of Csb on the global repair of 8-oxoG was not detectable in assays with total protein extracts and in a shuttle vector system. The data indicate a role for Csb in the removal of 8-oxoG from the overall genome that is independent of both Ogg1-mediated base excision repair and regular transcription.

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