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 containing base damage as a model of radiation induced clustered damage. One of the advantages of this strategy is that the effect of specific configurations of clustered damage could be examined in detail. Using a bacterial plasmid-based assay, we have investigated the biological consequences of bistranded clustered damage sites which consist of a single strand break (SSB) and an apurinic/apyrimidinic (AP) site. Firstly, plamids were ligated with oligonucleotides with clustered lesions. Following transformation of the ligated plasmids into wild type strain of Escherichia coli, both the transformation efficiency and the mutation frequencies were measured. We found a significantly lower transformation frequency for the clustered SSB + AP lesions than that for either a single SSB or a single AP site, which implies that a double strand break is formed during the processing of the cluster. Also, significantly higher mutation frequencies were observed for the clusters compared to those for singly damaged sites. When the two lesions were very close to each other, almost every plasmid possessed a mutation. The major types of mutation induced by the cluster were AP:C to C:G and a 1-bp deletion at the position of the AP:C pair. These results show a clear contrast to the results from clusters comprised of base lesions. We suggest that the biological consequences of clustered DNA damage strongly depends on the type of the comprising lesions.
