Abstract
Little is known about the mutagenic effects of clustered DNA damage. We have designed a plasmid-based assay to determine whether closely-opposed sites of base damage lead to higher mutation frequencies than single sites of damage. The bistranded clustered damage consists of 8-oxo-7,8-dihydroguanine (8-oxoG) and dihydrothymine (DHT), with 8-oxoG placed within a restriction site on one strand and DHT positioned at sites 1, 3, 5 bp away on the opposing strand. Damaged DNA was transfected into wild type or glycosylase deficient mutant cells (fpg, mutY and fpg mutY) of E. coli, and mutations were determined by inability to cut the restriction site. Mutation frequencies were found to be significantly higher for clustered lesions than for single lesions. Mutation frequencies were similar to wild type in the fpg mutant, while a marked increase was observed in mutY mutant compared to wild type. Even higher mutation frequencies were found in fpg mutY double mutant, reaching values around 35% of the rescued plasmids. We suggest that (1) at clustered damage sites, the action of Fpg is compromised, (2) MutY has an important role for reducing the formation of mutations by 8-oxoG within a cluster, and (3) an effective back-up mechanism for excision of 8-oxoG may exist in the cell.
