Demethylation Enhances Removal of Pyrimidine Dimers from the Overall Genome and from Specific DNA Sequences in Chinese Hamster Ovary Cells

Abstract

We have examined the effects of changes in cytosine methylation on DNA repair in UV-irradiated Chinese hamster ovary (CHO) cells. A hypomethylated derivative of the CHO K₁B₁₁ line, B₁₁aza, was established by passaging B₁₁ cells over several months in increasing concentrations of 5-azacytidine; greater than 60% demethylation was consistently demonstrated in these conditioned cells. Following a UV dose of 10 J/m², the amount of repair replication performed within 24 h was approximately twofold higher in B₁₁aza cells than in control B₁₁ cells. Removal of T4 endonuclease V-sensitive sites (ESS) from specific restriction fragments within and around the dihydrofolate reductase (DHFR) gene was then examined in B₁₁aza cells and compared with that in B₁₁ cells. Although demethylation had little or no effect on repair in the 5′ half of the DHFR gene, within a nontranscribed sequence immediately downstream from the gene, or within an extragenic region further downstream from the DHFR gene, significant increases in repair were observed at the 3′ end of the DHFR gene and within an extragenic region upstream of the DHFR gene. However, the increases in DNA repair were not accompanied by any changes in overall cellular resistance to UV when colony-forming ability was assayed. We suggest that the level of DNA methylation may play an indirect role in the regulation of DNA repair, perhaps through an effect on chromatin structure or transcriptional activity.