Abstract
Clustered DNA damage, defined as two or more lesions within one to two helical turns of DNA by a single radiation track, is a unique feature of ionizing radiation. We are currently focusing our interest on the biological effects of non-dsb clustered damage in vivo, whose effect has remained largely unknown. We have chosen an experimental approach that utilizes synthetic DNA containing base damage as a model of radiation induced clustered damage. One of the advantages of this strategy is that the effect of a specific type of damage could be clarified in detail. Using a bacterial plasmid-based assay, we have investigated the mutagenic potential of bistranded clustered damage sites which consist of 8-oxo-7,8-dihydroguanine (8-oxoG) and dihydrothymine (DHT) at defined separations. We found a significantly higher mutation frequency for the clustered DHT + 8-oxoG lesions than that for either a single 8-oxoG or a single DHT in wild-type and in glycosylase-deficient strains of E. coli. A marked increase in mutation frequencies 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. These results suggest that (1) Fpg activity is compromised for removal of lesions within clustered damage sites (2) the loss of the DHT strand contributes, at least partly, for the enhancement of mutation frequency by the cluster, and (3) MutY is the most important glycosylase for reducing the mutagenic potential of the cluster.
