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 on the biological effects of non-dsb type of clustered damage in vivo, whose effect has remained largely unknown. We have chosen an experimental approach that utilizes synthetic DNA damage as a model of radiation induced clustered damage. One of the advantages of this strategy is that the effect of clustered damage with defined configurations could be examined in detail. Using a bacterial plasmid-based assay, we have investigated the biological consequences of bistranded clustered damage sites which are comprised of combinations of base lesions, apurinic/apyrimidinic (AP) sites, and single strand breaks (SSBs). Plamids were ligated with oligonucleotides with clustered lesions. Following transformation of the ligated plasmids into wild type strain of Escherichia coli, clustered damage-induced mutations were characterized. Although the transformation efficiencies were low, the major types of mutation induced by the AP + AP or AP + SSB cluster were G insertion opposite AP and a 1-bp deletion at the base pair with AP. On the other hand, mutations were occasionally found at base pairs where lesions had not been placed with clusters such as dihydrothymine (DHT) + 7,8-dihydro-8-oxoguanine (8-oxoG), and DHT + SSB. From these results, we suggest that, in a bistranded base cluster, 1) either base lesion is remained unrepaired and 2) some mutations arise not at but very close to the base pair where base lesions were originally placed.
