Apex1 can cleave complex clustered DNA lesions in cells

Current data indicate that clustered DNA damage generated by ionizing radiation contains 2-5 damages within 20 bps. The complexity of clustered damage is also believed to increase as the linear energy transfer of the radiation increases. Complex lesions are therefore biologically relevant especially with the use of carbon ion beam therapy to treat cancer. Since two closely opposed AP site analogs (furans) are converted to a double strand break (DSB) in cells, we hypothesized that breakage could be compromised by increasing the complexity of the cluster. We have examined the repair of clusters containing three and four lesions in mouse fibroblasts using a luciferase reporter plasmid. The addition of a third furan did reduce but not eliminate cleavage, while a tandem 8-oxo-7,8-dihydroguanine (8oxoG) immediately 5′ to one furan in a two or three furan cluster decreased DSB formation by a small amount. In vitro studies using nuclear extracts demonstrated that the tandem 8oxoG was not removed under conditions where the furan was cleaved, but the presence of the 8oxoG reduced cleavage at the furan. Interestingly, a cluster of an 8oxoG opposite a furan did not form a DSB in cells. We have shown that Apex1 can cleave these complex clustered lesions in cells. This therefore indicates that Apex1 can generate complex DSBs from clustered lesions consisting of base damage and AP sites. Repair of these complex DSBs may be compromised by the nearby oxidative damage resulting in potentially lethal and biologically relevant damage.